xnf7 functions in dorsal-ventral patterning of the Xenopus embryo.
El-Hodiri H M,Shou W,Etkin L D
Xenopus nuclear factor 7 (xnf7) is a maternally expressed nuclear protein that is retained in the cytoplasm from oocyte maturation until the midblastula transition (MBT). Mutations of the xnf7 phosphorylation sites to glutamic acids (dnxnf7) resulted in the retention of the endogenous protein in the cytoplasm past the MBT, indicating that cytoplasmic retention is a phosphorylation dependent process. In addition, dnxnf7 acted as a dominant negative mutant by keeping the endogenous xnf7 protein in the cytoplasm past the MBT. Overexpression of dnxnf7 in future dorsal blastomeres resulted in a ventralized or posteriorized phenotype in which the embryos lacked anterior structures, while overexpression in ventral blastomeres resulted in dorsalized embryos. dnxnf7 also affected the expression of both dorsal and ventral mesodermal markers. These data suggest that xnf7 functions in dorsal/ventral patterning and that the movement of the protein from the cytoplasm to the nucleus at the MBT is critical for the execution of a genetic program conferring a dorsal or ventral identity to the mesoderm.
The human homolog of Sex comb on midleg (SCMH1) maps to chromosome 1p34.
Berger J,Kurahashi H,Takihara Y,Shimada K,Brock H W,Randazzo F
Polycomb group genes were originally identified in Drosophila as repressors required to maintain the silenced state of homeotic loci. About ten Polycomb group genes have been cloned in Drosophila, and mammalian homologs have been identified for most of these. Here, we isolate cDNAs encoding two isoforms of a human homolog of Drosophila Sex comb on midleg (Scm), named Sex comb on midleg homolog-1 (SCMH1). Overall, SCMH1 has 94% identity to its mouse counterpart Scmh1, and 41% identity to Scm, and contains two 1(3)mbt domains, and the SPM domain that are characteristic of Scm. SCMH1 is widely expressed in adult tissues, and maps to chromosome 1p34.
DNA methylation at promoter regions regulates the timing of gene activation in Xenopus laevis embryos.
Stancheva Irina,El-Maarri Osman,Walter Joern,Niveleau Alain,Meehan Richard R
The levels of genomic DNA methylation in vertebrate species display a wide range of developmental dynamics. Here, we show that in contrast to mice, the paternal genome of the amphibian, Xenopus laevis, is not subjected to active demethylation of 5-methyl cytosine immediately after fertilization. High levels of methylation in the DNA of both oocyte and sperm are maintained in the early embryo but progressively decline during the cleavage stages. As a result, the Xenopus genome has its lowest methylation content at the midblastula transition (MBT) and during subsequent gastrulation. Between blastula and gastrula stages, we detect a loss of methylation at individual Xenopus gene promoters (TFIIIA, Xbra, and c-Myc II) that are activated at MBT. No changes are observed in the methylation patterns of repeated sequences, genes that are inactive at MBT, or in the coding regions of individual genes. In embryos that are depleted of the maintenance methyltransferase enzyme (xDnmt1), these developmentally programmed changes in promoter methylation are disrupted, which may account for the altered patterns of gene expression that occur in these embryos. Our results suggest that DNA methylation has a role in regulating the timing of gene activation at MBT in Xenopus laevis embryos.
Polycomb-like genes are necessary for specification of dopaminergic and serotonergic neurons in Caenorhabditis elegans.
Yang Yong,Sun Yinyan,Luo Xin,Zhang Yuxia,Chen Yaoyao,Tian E,Lints Robyn,Zhang Hong
Proceedings of the National Academy of Sciences of the United States of America
The molecular mechanisms underlying the formation of neurons with defined neurotransmitters are not well understood. In this study, we demonstrate that the PcG-like genes in Caenorhabditis elegans, sop-2 and sor-3, regulate the formation of dopaminergic and serotonergic neurons and several other neuronal properties. sor-3 encodes a novel protein containing an MBT repeat, a domain that contains histone-binding activity and is present in PcG proteins SCM and Sfmbt in other organisms. We further show that mutations in sor-3 lead to ectopic expression of Hox genes and cause homeotic transformations. Specification of certain neuronal identities by these PcG-like genes appears to involve regulation of non-Hox gene targets. Our studies revealed that the PcG-like genes are crucial for coordinately regulating the expression of discrete aspects of neuronal identities in C. elegans.
Brd4 associates with mitotic chromosomes throughout early zebrafish embryogenesis.
Toyama Reiko,Rebbert Martha L,Dey Anup,Ozato Keiko,Dawid Igor B
Developmental dynamics : an official publication of the American Association of Anatomists
Brd4 is a member of the BET (bromodomains and extraterminal) subfamily of bromodomain proteins that includes chromatin-modifying proteins and transcriptional regulators. Brd4 has a role in cell cycle progression, making it indispensable in mouse embryos and cultured cells. The N-terminal domain of Brd4 participates in a fusion oncogene. Brd4 associates with acetylated histones in chromatin, and this association persists during mitosis implicating Brd4 in epigenetic memory. Brd4 sequence, particularly the bromodomains and ET domain, is conserved in the zebrafish and Xenopus laevis proteins reported here. Brd4 is expressed and localized on mitotic chromosomes in early zebrafish embryos before and after the midblastula transition (MBT), indicating that the Brd4-chromosome association is a conserved property that is maintained even before zygotic transcription. The association of Brd4 with acetylated histones may also be conserved in early embryos as we found that histones H3 and H4 are already acetylated during pre-MBT stages.
SUMO-modified Sp3 represses transcription by provoking local heterochromatic gene silencing.
Stielow Bastian,Sapetschnig Alexandra,Wink Christina,Krüger Imme,Suske Guntram
Modification of many transcription factors including Sp3 and steroidogenic factor 1 with the small ubiquitin-like modifier (SUMO) is associated with transcriptional repression. Here, we show that SUMOylation of transcription factors bound to DNA provokes the establishment of compacted repressive chromatin with characteristics of heterochromatin. Chromatin immunoprecipitation experiments revealed SUMO-dependent recruitment of the chromatin remodeller Mi-2, MBT-domain proteins, heterochromatic protein 1, and the histone methyltransferases SETDB1 and SUV4-20H, concomitant with the establishment of histone modifications associated with repressed genes, including H3K9 and H4K20 trimethylation. These results indicate that SUMOylation has a crucial role in regulating gene expression by initiating chromatin structure changes that render DNA inaccessible to the transcription machinery.
Screening for inhibitors of low-affinity epigenetic peptide-protein interactions: an AlphaScreen-based assay for antagonists of methyl-lysine binding proteins.
Wigle Tim J,Herold J Martin,Senisterra Guillermo A,Vedadi Masoud,Kireev Dmitri B,Arrowsmith Cheryl H,Frye Stephen V,Janzen William P
Journal of biomolecular screening
The histone code comprises many posttranslational modifications that occur mainly in histone tail peptides. The identity and location of these marks are read by a variety of histone-binding proteins that are emerging as important regulators of cellular differentiation and development and are increasingly being implicated in numerous disease states. The authors describe the development of the first high-throughput screening assay for the discovery of inhibitors of methyl-lysine binding proteins that will be used to initiate a full-scale discovery effort for this broad target class. They focus on the development of an AlphaScreen-based assay for malignant brain tumor (MBT) domain-containing proteins, which bind to the lower methylation states of lysine residues present in histone tail peptides. This assay takes advantage of the avidity of the AlphaScreen beads to clear the hurdle to assay development presented by the low micromolar binding constants of the histone binding proteins for their cognate peptides. The assay is applicable to other families of methyl-lysine binding proteins, and it has the potential to be used in screening efforts toward the discovery of novel small molecules with utility as research tools for cellular reprogramming and ultimately drug discovery.
A novel member of murine Polycomb-group proteins, Sex comb on midleg homolog protein, is highly conserved, and interacts with RAE28/mph1 in vitro.
Tomotsune D,Takihara Y,Berger J,Duhl D,Joo S,Kyba M,Shirai M,Ohta H,Matsuda Y,Honda B M,Simon J,Shimada K,Brock H W,Randazzo F
Differentiation; research in biological diversity
The Polycomb group of (PcG) genes were originally described in Drosophila, but many PcG genes have mammalian homologs. Genetic studies in flies and mice show that mutations in PcG genes cause posterior transformations caused by failure to maintain repression of homeotic loci, suggesting that PcG proteins have conserved functions. The Drosophila gene Sex comb on midleg (Scm) encodes an unusual PcG protein that shares motifs with the PcG protein polyhomeotic, and with a Drosophila tumor suppressor, lethal(3)malignant brain tumor (l(3)mbt). Expressed sequence tag (EST) databases were searched to recover putative mammalian Scm homologs, which were used to screen murine cDNA libraries. The recovered cDNA encodes two mbt repeats and the SPM domain that characterize Scm, but lacks the cysteine clusters and the serine/threonine-rich region found at the amino terminus of Scm. Accordingly, we have named the gene Sex comb on midleg homolog 1 (Scmh1). Like their Drosophila counterparts, Scmh1 and the mammalian polyhomeotic homolog RAE28/mph1 interact in vitro via their SPM domains. We analyzed the expression of Scmh1 and rae28/mph1 using northern analysis of embryos and adult tissues, and in situ hybridization to embryos. The expression of Scmh1 and rae28/mph1 is well correlated in most tissues of embryos. However, in adults, Scmh1 expression was detected in most tissues, whereas mph1/rae28 expression was restricted to the gonads. Scmh1 is strongly induced by retinoic acid in F9 and P19 embryonal carcinoma cells. Scmh1 maps to 4D1-D2.1 in mice. These data suggest that Scmh1 will have an important role in regulation of homeotic genes in embryogenesis and that the interaction with RAE28/mph1 is important in vivo.
Human SFMBT is a transcriptional repressor protein that selectively binds the N-terminal tail of histone H3.
Wu Shumin,Trievel Raymond C,Rice Judd C
Human SFMBT (hSFMBT) is postulated to be a Polycomb (PcG) protein. Similar to other PcG proteins, we found that hSFMBT displays robust transcriptional repressor activity. In addition, hSFMBT localized to the nucleus where it strongly associates with chromatin by directly and selectively binding the N-terminal tail of histone H3. Importantly, we discovered that the four tandem MBT repeats of hSFMBT were sufficient for nuclear matrix-association, N-terminal tail H3 binding, and required for transcriptional repression. These findings indicate that the tandem MBT repeats form a functional structure required for biological activity of hSFMBT and predict similar properties for other MBT domain-containing proteins.
The malignant brain tumor repeats of human SCML2 bind to peptides containing monomethylated lysine.
Santiveri Clara M,Lechtenberg Bernhard C,Allen Mark D,Sathyamurthy Aruna,Jaulent Agnès M,Freund Stefan M V,Bycroft Mark
Journal of molecular biology
SCML2 (sex comb on midleg-like 2) is a constituent of the Polycomb repressive complex 1, a large multiprotein assembly required for the repression of developmental control genes. It contains two MBT (malignant brain tumor) repeats; the MBT is a protein module structurally similar to domains that bind to methylated histones. We have used NMR spectroscopy to examine the binding specificity of these repeats. Our data show that they preferentially bind histone peptides monomethylated at lysine residues with no apparent sequence specificity. The crystal structure of the complex between the protein and monomethyllysine reveals that the modified amino acid binds to an aromatic rich pocket at one end of the beta-barrel of the second repeat.
Structure and function of histone methylation binding proteins.
Adams-Cioaba Melanie A,Min Jinrong
Biochemistry and cell biology = Biochimie et biologie cellulaire
Chromatin structure is regulated by chromatin remodeling factors, histone exchange, linker histone association, and histone modification. Covalent modification of histones is an important factor in the regulation of the associated processes. The implementation and removal of various histone modifications have been implicated in DNA replication, repair, recombination, and transcription, and in RNA processing. In recent years, histone methylation has emerged as one of the key modifications regulating chromatin function. However, the mechanisms involved are complex and not well understood. A large volume of structural and biochemical information has been recently amassed for the Tudor, plant homeodomain (PHD), and malignant brain tumor (MBT) protein families. This review summarizes current knowledge of the structures and modes of recognition employed by the PHD, Tudor, and MBT domains in their interactions with target histone peptides.
Repression of zygotic gene expression in the Xenopus germline.
Venkatarama Thiagarajan,Lai Fangfang,Luo Xueting,Zhou Yi,Newman Karen,King Mary Lou
Development (Cambridge, England)
Primordial germ cells (PGCs) in Xenopus are specified through the inheritance of germ plasm. During gastrulation, PGCs remain totipotent while surrounding cells in the vegetal mass become committed to endoderm through the action of the vegetal localized maternal transcription factor VegT. We find that although PGCs contain maternal VegT RNA, they do not express its downstream targets at the mid-blastula transition (MBT). Transcriptional repression in PGCs correlates with the failure to phosphorylate serine 2 in the carboxy-terminal domain (CTD) of the large subunit of RNA polymerase II (RNAPII). As serine 5 is phosphorylated, these results are consistent with a block after the initiation step but before the elongation step of RNAPII-based transcription. Repression of PGC gene expression occurs despite an apparently permissive chromatin environment. Phosphorylation of CTD-serine 2 and expression of zygotic mRNAs in PGCs are first detected at neurula, some 10 hours after MBT, indicating that transcription is significantly delayed in the germ cell lineage. Significantly, Oct-91, a POU subclass V transcription factor related to mammalian Oct3/4, is among the earliest zygotic transcripts detected in PGCs and is a likely mediator of pluripotency. Our findings suggest that PGCs are unable to respond to maternally inherited endoderm determinants because RNAPII activity is transiently blocked while these determinants are present. Our results in a vertebrate system further support the concept that one strategy used repeatedly during evolution for preserving the germline is RNAPII repression.
Histone deacetylase 3 is selectively involved in L3MBTL2-mediated transcriptional repression.
Yoo Jung-Yoon,Choi Kyung-Chul,Kang HeeBum,Kim Young Jun,Lee Jeongmin,Jun Woo Jin,Kim Mi-Jeong,Lee Yoo-Hyun,Lee Ok-Hee,Yoon Ho-Geun
This is the first report that L(3)mbt-like 2 (L3MBTL2) specifically interacts with the histone deacetylase domain of histone deacetylase 3 (HDAC3) via its MBT domain. Here, we show that L3MBTL2 selectively interacts with HDAC3, but not other class I HDACs. An in vitro peptide-binding assay demonstrated the specific association of HDAC3 with methylated histone-K20 tail and L3MBTL2. Furthermore, depletion of HDAC3 resulted in a decrease of methylated K20-H4, as well as an increase in acetylated histone H3. Consequently, HDAC3 knock-down selectively suppressed L3MBTL2-mediated transcriptional repression. Taken together, our results reveal the concerted action of both HDAC3 and L3MBTL2 in histone deacetylation and methylation-dependent transcriptional repression.
Chromatin protein L3MBTL1 is dispensable for development and tumor suppression in mice.
Qin Jinzhong,Van Buren Denille,Huang Hsien-Sung,Zhong Lei,Mostoslavsky Raul,Akbarian Schahram,Hock Hanno
The Journal of biological chemistry
L3MBTL1, a paralogue of Drosophila tumor suppressor lethal(3)malignant brain tumor (l(3)mbt), binds histones in a methylation state-dependent manner and contributes to higher order chromatin structure and transcriptional repression. It is the founding member of a family of MBT domain-containing proteins that has three members in Drosophila and nine in mice and humans. Knockdown experiments in cell lines suggested that L3MBTL1 has non-redundant roles in the suppression of oncogene expression. We generated a mutant mouse strain that lacks exons 13-20 of L3mbtl1. Markedly reduced levels of a mutant mRNA with an out-of-frame fusion of exons 12 and 21 were expressed, but a mutant protein was undetectable by Western blot analysis. L3MBTL1(-/-) mice developed and reproduced normally. The highest expression of L3MBTL1 was detected in the brain, but its disruption did not affect brain development, spontaneous movement, and motor coordination. Despite previous implications of L3mbtl1 in the biology of hematopoietic transcriptional regulators, lack of L3MBTL1 did not result in deficiencies in lymphopoiesis or hematopoiesis. In contrast with its demonstrated biochemical activities, embryonic stem (ES) cells lacking L3MBTL1 displayed no abnormalities in H4 lysine 20 (H4K20) mono-, di-, or trimethylation; had normal global chromatin density as assessed by micrococcal nuclease digests; and expressed normal levels of c-myc. Embryonic fibroblasts lacking L3MBTL1 displayed unaltered cell cycle arrest and down-regulation of cyclin E expression after irradiation. In cohorts of mice followed for more than 2 years, lack of L3MBTL1 did not alter normal lifespan or survival with or without sublethal irradiation.
A general molecular affinity strategy for global detection and proteomic analysis of lysine methylation.
Moore Kaitlyn E,Carlson Scott M,Camp Nathan D,Cheung Peggie,James Richard G,Chua Katrin F,Wolf-Yadlin Alejandro,Gozani Or
Lysine methylation of histone proteins regulates chromatin dynamics and plays important roles in diverse physiological and pathological processes. However, beyond histone proteins, the proteome-wide extent of lysine methylation remains largely unknown. We have engineered the naturally occurring MBT domain repeats of L3MBTL1 to serve as a universal affinity reagent for detecting, enriching, and identifying proteins carrying a mono- or dimethylated lysine. The domain is broadly specific for methylated lysine ("pan-specific") and can be applied to any biological system. We have used our approach to demonstrate that SIRT1 is a substrate of the methyltransferase G9a both in vitro and in cells, to perform proteome-wide detection and enrichment of methylated proteins, and to identify candidate in-cell substrates of G9a and the related methyltransferase GLP. Together, our results demonstrate a powerful new approach for global and quantitative analysis of methylated lysine, and they represent the first systems biology understanding of lysine methylation.
The structure-activity relationships of L3MBTL3 inhibitors: flexibility of the dimer interface.
Camerino Michelle A,Zhong Nan,Dong Aiping,Dickson Bradley M,James Lindsey I,Baughman Brandi M,Norris Jacqueline L,Kireev Dmitri B,Janzen William P,Arrowsmith Cheryl H,Frye Stephen V
We recently reported the discovery of UNC1215, a potent and selective chemical probe for the L3MBTL3 methyllysine reader domain. In this article, we describe the development of structure-activity relationships (SAR) of a second series of potent L3MBTL3 antagonists which evolved from the structure of the chemical probe UNC1215. These compounds are selective for L3MBTL3 against a panel of methyllysine reader proteins, particularly the related MBT family proteins, L3MBTL1 and MBTD1. A co-crystal structure of L3MBTL3 and one of the most potent compounds suggests that the L3MBTL3 dimer rotates about the dimer interface to accommodate ligand binding.
TALE-light imaging reveals maternally guided, H3K9me2/3-independent emergence of functional heterochromatin in Drosophila embryos.
Yuan Kai,O'Farrell Patrick H
Genes & development
Metazoans start embryogenesis with a relatively naïve genome. The transcriptionally inert, late-replicating heterochromatic regions, including the constitutive heterochromatin on repetitive sequences near centromeres and telomeres, need to be re-established during development. To explore the events initiating heterochromatin formation and examine their temporal control, sequence specificity, and immediate regulatory consequence, we established a live imaging approach that enabled visualization of steps in heterochromatin emergence on specific satellite sequences during the mid-blastula transition (MBT) in Drosophila. Unexpectedly, only a subset of satellite sequences, including the 359-base-pair (bp) repeat sequence, recruited HP1a at the MBT. The recruitment of HP1a to the 359-bp repeat was dependent on HP1a's chromoshadow domain but not its chromodomain and was guided by maternally provided signals. HP1a recruitment to the 359-bp repeat was required for its programmed shift to later replication, and ectopic recruitment of HP1a was sufficient to delay replication timing of a different repeat. Our results reveal that emergence of constitutive heterochromatin follows a stereotyped developmental program in which different repetitive sequences use distinct interactions and independent pathways to arrive at a heterochromatic state. This differential emergence of heterochromatin on various repetitive sequences changes their replication order and remodels the DNA replication schedule during embryonic development.
Proteolysis of methylated SOX2 protein is regulated by L3MBTL3 and CRL4 ubiquitin ligase.
Zhang Chunxiao,Leng Feng,Saxena Lovely,Hoang Nam,Yu Jiekai,Alejo Salvador,Lee Logan,Qi Dandan,Lu Fei,Sun Hong,Zhang Hui
The Journal of biological chemistry
SOX2 is a dose-dependent master stem cell protein that controls the self-renewal and pluripotency or multipotency of embryonic stem (ES) cells and many adult stem cells. We have previously found that SOX2 protein is monomethylated at lysine residues 42 and 117 by SET7 methyltransferase to promote SOX2 proteolysis, whereas LSD1 and PHF20L1 act on both methylated Lys-42 and Lys-117 to prevent SOX2 proteolysis. However, the mechanism by which the methylated SOX2 protein is degraded remains unclear. Here, we report that L3MBTL3, a protein with the alignant-rain-umor (MBT) methylation-binding domain, is required for SOX2 proteolysis. Our studies showed that L3MBTL3 preferentially binds to the methylated Lys-42 in SOX2, although mutation of Lys-117 also partially reduces the interaction between SOX2 and L3MBTL3. The direct binding of L3MBTL3 to the methylated SOX2 protein leads to the recruitment of the CRL4 ubiquitin E3 ligase to target SOX2 protein for ubiquitin-dependent proteolysis. Whereas loss of either LSD1 or PHF20L1 destabilizes SOX2 protein and impairs the self-renewal and pluripotency of mouse ES cells, knockdown of L3MBTL3 or DCAF5 is sufficient to restore the protein levels of SOX2 and rescue the defects of mouse ES cells caused by LSD1 or PHF20L1 deficiency. We also found that retinoic acid-induced differentiation of mouse ES cells is accompanied by the enhanced degradation of the methylated SOX2 protein at both Lys-42 and Lys-117. Our studies provide novel insights into the mechanism by which the methylation-dependent degradation of SOX2 protein is controlled by the L3MBTL3-CRL4 ubiquitin ligase complex.
L3MBTL2 regulates chromatin remodeling during spermatogenesis.
Meng Chenling,Liao Jinyue,Zhao Danfeng,Huang Huihui,Qin Jinzhong,Lee Tin-Lap,Chen Degui,Chan Wai-Yee,Xia Yin
Cell death and differentiation
Lethal (3) malignant brain tumor like 2 (L3MBTL2) is a member of the MBT-domain proteins, which are involved in transcriptional repression and implicated in chromatin compaction. Our previous study has shown that L3MBTL2 is highly expressed in the testis, but its role in spermatogenesis remains unclear. In the present study, we found that L3MBTL2 was most highly expressed in pachytene spermatocytes within the testis. Germ cell-specific ablation of L3mbtl2 in the testis led to increased abnormal spermatozoa, progressive decrease of sperm counts and premature testicular failure in mice. RNA-sequencing analysis on L3mbtl2 deficient testes confirmed that L3MBTL2 was a transcriptional repressor but failed to reveal any significant changes in spermatogenesis-associated genes. Interestingly, L3mbtl2 deficiency resulted in increased γH2AX deposition in the leptotene spermatocytes, subsequent inappropriate retention of γH2AX on autosomes, and defective crossing-over and synapsis during the pachytene stage of meiosis I, and more germ cell apoptosis and degeneration in aging mice. L3MBTL2 interacted with the histone ubiquitin ligase RNF8. Inhibition of L3MBTL2 reduced nuclear RNF8 and ubH2A levels in GC2 cells. L3mbtl2 deficiency led to decreases in the levels of the RNF8 and ubH2A pathway and in histone acetylation in elongating spermatids, and in protamine 1 deposition and chromatin condensation in sperm. These results suggest that L3MBTL2 plays important roles in chromatin remodeling during meiosis and spermiogenesis.
The Drosophila melanogaster tumor suppressor gene lethal(3)malignant brain tumor encodes a proline-rich protein with a novel zinc finger.
Wismar J,Löffler T,Habtemichael N,Vef O,Geissen M,Zirwes R,Altmeyer W,Sass H,Gateff E
Mechanisms of development
The lethal(3)malignant brain tumor [t(3)mbt] gene causes, when mutated, malignant growth of the adult optic neuroblasts and ganglion mother cells in the larval brain and imaginal disc overgrowth. Via overlapping deficiencies a genomic region of approximately 6.0 kb was identified, containing l(3)mbt+ gene sequences. The l(3)mbt+ gene encodes seven transcripts of 5.8 kb, 5.65 kb, 5.35 kb, 5.25 kb, 5.0 kb, 4.4 kb and 1.8 kb. The putative MBT163 protein, encompassing 1477 amino acids, is proline-rich and contains a novel zinc finger. In situ hybridizations of whole mount embryos and larval tissues revealed l(3)mbt+ RNA ubiquitously present in stage 1 embryos and throughout embryonic development in most tissues. In third instar larvae l(3)mbt+ RNA is detected in the adult optic anlagen and the imaginal discs, the tissues directly affected by l(3)mbt mutations, but also in tissues, showing normal development in the mutant, such as the gut, the goblet cells and the hematopoietic organs.
Expression and properties of wild-type and mutant forms of the Drosophila sex comb on midleg (SCM) repressor protein.
Bornemann D,Miller E,Simon J
The Sex comb on midleg (Scm) gene encodes a transcriptional repressor of the Polycomb group (PcG). Here we show that SCM protein is nuclear and that its expression is widespread during fly development. SCM protein contains a C-terminal domain, termed the SPM domain, which mediates protein-protein interactions. The biochemical function of another domain consisting of two 100-amino-acid-long repeats, termed "mbt" repeats, is unknown. We have determined the molecular lesions of nine Scm mutant alleles, which identify functional requirements for specific domains. The Scm alleles were tested for genetic interactions with mutations in other PcG genes. Intriguingly, three hypomorphic Scm mutations, which map within an mbt repeat, interact with PcG mutations more strongly than do Scm null alleles. The strongest interactions produce partial synthetic lethality that affects doubly heterozygous females more severely than males. We show that mbt repeat alleles produce stable SCM proteins that associate with normal sites in polytene chromosomes. We also analyzed progeny from Scm mutant germline clones to compare the effects of an mbt repeat mutation during embryonic vs. pupal development. We suggest that the mbt repeat alleles produce altered SCM proteins that incorporate into and impair function of PcG protein complexes.
Structural and functional analyses of methyl-lysine binding by the malignant brain tumour repeat protein Sex comb on midleg.
Grimm Clemens,de Ayala Alonso Andres Gaytan,Rybin Vladimir,Steuerwald Ulrich,Ly-Hartig Nga,Fischle Wolfgang,Müller Jürg,Müller Christoph W
Sex comb on midleg (Scm) is a member of the Polycomb group of proteins involved in the maintenance of repression of Hox and other developmental control genes in Drosophila. The two malignant brain tumour (MBT) repeats of Scm form a domain that preferentially binds to monomethylated lysine residues either as a free amino acid or in the context of peptides, while unmodified or di- or trimethylated lysine residues are bound with significantly lower affinity. The crystal structure of a monomethyl-lysine-containing histone tail peptide bound to the MBT repeat domain shows that the methyl-lysine side chain occupies a binding pocket in the second MBT repeat formed by three conserved aromatic residues and one aspartate. Insertion of the monomethylated side chain into this pocket seems to be the main contributor to the binding affinity. Functional analyses in Drosophila show that the MBT domain of Scm and its methyl-lysine-binding activity are required for repression of Hox genes.
L3MBTL2 protein acts in concert with PcG protein-mediated monoubiquitination of H2A to establish a repressive chromatin structure.
Trojer Patrick,Cao Alina R,Gao Zhonghua,Li Yan,Zhang Jin,Xu Xiaoqin,Li Guohong,Losson Regine,Erdjument-Bromage Hediye,Tempst Paul,Farnham Peggy J,Reinberg Danny
We have identified human MBT domain-containing protein L3MBTL2 as an integral component of a protein complex that we termed Polycomb repressive complex 1 (PRC1)-like 4 (PRC1L4), given the copresence of PcG proteins RING1, RING2, and PCGF6/MBLR. PRC1L4 also contained E2F6 and CBX3/HP1γ, known to function in transcriptional repression. PRC1L4-mediated repression necessitated L3MBTL2 that compacted chromatin in a histone modification-independent manner. Genome-wide location analyses identified several hundred genes simultaneously bound by L3MBTL2 and E2F6, preferentially around transcriptional start sites that exhibited little overlap with those targeted by other E2Fs or by L3MBTL1, another MBT domain-containing protein that interacts with RB1. L3MBTL2-specific RNAi resulted in increased expression of target genes that exhibited a significant reduction in H2A lysine 119 monoubiquitination. Our findings highlight a PcG/MBT collaboration that attains repressive chromatin without entailing histone lysine methylation marks.
Geminin is required for zygotic gene expression at the Xenopus mid-blastula transition.
Kerns Sarah L,Schultz Kathryn M,Barry Kelly A,Thorne Tina M,McGarry Thomas J
In many organisms early development is under control of the maternal genome and zygotic gene expression is delayed until the mid-blastula transition (MBT). As zygotic transcription initiates, cell cycle checkpoints become activated and the tempo of cell division slows. The mechanisms that activate zygotic transcription at the MBT are incompletely understood, but they are of interest because they may resemble mechanisms that cause stem cells to stop dividing and terminally differentiate. The unstable regulatory protein Geminin is thought to coordinate cell division with cell differentiation. Geminin is a bi-functional protein. It prevents a second round of DNA replication during S and G2 phase by binding and inhibiting the essential replication factor Cdt1. Geminin also binds and inhibits a number of transcription factors and chromatin remodeling proteins and is thought to keep dividing cells in an undifferentiated state. We previously found that the cells of Geminin-deficient Xenopus embryos arrest in G2 phase just after the MBT then disintegrate at the onset of gastrulation. Here we report that they also fail to express most zygotic genes. The gene expression defect is cell-autonomous and is reproduced by over-expressing Cdt1 or by incubating the embryos in hydroxyurea. Geminin deficient and hydroxyurea-treated blastomeres accumulate DNA damage in the form of double stranded breaks. Bypassing the Chk1 pathway overcomes the cell cycle arrest caused by Geminin depletion but does not restore zygotic gene expression. In fact, bypassing the Chk1 pathway by itself induces double stranded breaks and abolishes zygotic transcription. We did not find evidence that Geminin has a replication-independent effect on transcription. We conclude that Geminin is required to maintain genome integrity during the rapid cleavage divisions, and that DNA damage disrupts zygotic gene transcription at the MBT, probably through activation of DNA damage checkpoint pathways.
MBTD1 is associated with Pr-Set7 to stabilize H4K20me1 in mouse oocyte meiotic maturation.
Luo Yi-Bo,Ma Jun-Yu,Zhang Qing-Hua,Lin Fei,Wang Zhong-Wei,Huang Lin,Schatten Heide,Sun Qing-Yuan
Cell cycle (Georgetown, Tex.)
H4K20me1 is a critical histone lysine methyl modification in eukaryotes. It is recognized and "read" by various histone lysine methyl modification binding proteins. In this study, the function of MBTD1, a member of the Polycomb protein family containing four MBT domains, was comprehensively studied in mouse oocyte meiotic maturation. The results showed that depletion of MBTD1 caused reduced expression of histone lysine methyl transferase Pr-Set7 and H4K20me1 as well as increased oocyte arrest at the GV stage. Increased γH2AX foci were formed, and DNA damage repair checkpoint protein 53BP1 was downregulated. Furthermore, depletion of MBTD1 activated the cell cycle checkpoint protein Chk1 and downregulated the expression of cyclin B1 and cdc2. MBTD1 knockdown also affected chromosome configuration in GV stage oocytes and chromosome alignment at the MII stage. All these phenotypes were reproduced when the H4K20 methyl transferase Pr-Set7 was depleted. Co-IP demonstrated that MBTD1 was correlated with Pr-Set7 in mouse oocytes. Our results demonstrate that MBTD1 is associated with Pr-Set7 to stabilize H4K20me1 in mouse oocyte meiotic maturation.
Cognition and mood-related behaviors in L3mbtl1 null mutant mice.
Shen Erica Y,Jiang Yan,Mao Wenjie,Futai Kensuke,Hock Hanno,Akbarian Schahram
Alterations in histone lysine methylation and epigenetic regulators of gene expression could play a role in the neurobiology and treatment of patients diagnosed with mood spectrum disorder, including depression and anxiety. Mutations and altered expression of various lysine methyltransferases (KMTs) and demethylases (KDMs) have been linked to changes in motivational and emotional behaviors in preclinical model systems. However, it is not known whether regulators operating downstream of histone lysine methylation could affect mood-related behavior. Malignant Brain Tumor (MBT) domain 'chromatin reader' proteins bind to methylated histone lysine residues and associate with chromatin remodeling complexes to facilitate or repress gene expression. MBT proteins, including the founding member, L3mbtl1, maintain high levels of expression in neurons of the mature brain. Here, we exposed L3mbtl1 null mutant mice to a wide range of tests exploring cognition and mood-relevant behaviors at baseline and in the context of social isolation, as a stressor to elicit depression-related behavior in susceptible mice. L3mbtl1 loss-of-function was associated with significant decreases in depression and and anxiety in some of the behavioral paradigms. This was not associated with a more generalized neurological dysfunction because cognition and memory remained unaltered in comparison to controls. These findings warrant further investigations on the role of MBT chromatin reader proteins in the context of emotional and affective behaviors.
Genome-wide copy number variation analysis identified deletions in SFMBT1 associated with fasting plasma glucose in a Han Chinese population.
Chung Ren-Hua,Chiu Yen-Feng,Hung Yi-Jen,Lee Wen-Jane,Wu Kwan-Dun,Chen Hui-Ling,Lin Ming-Wei,Chen Yii-Der I,Quertermous Thomas,Hsiung Chao A
BACKGROUND:Fasting glucose and fasting insulin are glycemic traits closely related to diabetes, and understanding the role of genetic factors in these traits can help reveal the etiology of type 2 diabetes. Although single nucleotide polymorphisms (SNPs) in several candidate genes have been found to be associated with fasting glucose and fasting insulin, copy number variations (CNVs), which have been reported to be associated with several complex traits, have not been reported for association with these two traits. We aimed to identify CNVs associated with fasting glucose and fasting insulin. RESULTS:We conducted a genome-wide CNV association analysis for fasting plasma glucose (FPG) and fasting plasma insulin (FPI) using a family-based genome-wide association study sample from a Han Chinese population in Taiwan. A family-based CNV association test was developed in this study to identify common CNVs (i.e., CNVs with frequencies ≥ 5%), and a generalized estimating equation approach was used to test the associations between the traits and counts of global rare CNVs (i.e., CNVs with frequencies <5%). We found a significant genome-wide association for common deletions with a frequency of 5.2% in the Scm-like with four mbt domains 1 (SFMBT1) gene with FPG (association p-value = 2×10 and an adjusted p-value = 0.0478 for multiple testing). No significant association was observed between global rare CNVs and FPG or FPI. The deletions in 20 individuals with DNA samples available were successfully validated using PCR-based amplification. The association of the deletions in SFMBT1 with FPG was further evaluated using an independent population-based replication sample obtained from the Taiwan Biobank. An association p-value of 0.065, which was close to the significance level of 0.05, for FPG was obtained by testing 9 individuals with CNVs in the SFMBT1 gene region and 11,692 individuals with normal copies in the replication cohort. CONCLUSIONS:Previous studies have found that SNPs in SFMBT1 are associated with blood pressure and serum urate concentration, suggesting that SFMBT1 may have functional implications in some metabolic-related traits.
Arsenic Induces Members of the mmu-miR-466-669 Cluster Which Reduces NeuroD1 Expression.
Liu Jui-Tung,Bain Lisa J
Toxicological sciences : an official journal of the Society of Toxicology
Chronic arsenic exposure can result in adverse development effects including decreased intellectual function, reduced birth weight, and altered locomotor activity. Previous in vitro studies have shown that arsenic inhibits stem cell differentiation. MicroRNAs (miRNAs) are small noncoding RNAs that regulate multiple cellular processes including embryonic development and cell differentiation. The purpose of this study was to examine whether altered miRNA expression was a mechanism by which arsenic inhibited cellular differentiation. The pluripotent P19 mouse embryonal carcinoma cells were exposed to 0 or 0.5 μM sodium arsenite for 9 days during cell differentiation, and changes in miRNA expression was analyzed using microarrays. We found that the expression of several miRNAs important in cellular differentiation, such as miR-9 and miR-199 were decreased by 1.9- and 1.6-fold, respectively, following arsenic exposure, while miR-92a, miR-291a, and miR-709 were increased by 3-, 3.7-, and 1.6-fold, respectively. The members of the miR-466-669 cluster and its host gene, Scm-like with 4 Mbt domains 2 (Sfmbt2), were significantly induced by arsenic from 1.5- to 4-fold in a time-dependent manner. Multiple miRNA target prediction programs revealed that several neurogenic transcription factors appear to be targets of the cluster. When consensus anti-miRNAs targeting the miR-466-669 cluster were transfected into P19 cells, arsenic-exposed cells were able to more effectively differentiate. The consensus anti-miRNAs appeared to rescue the inhibitory effects of arsenic on cell differentiation due to an increased expression of NeuroD1. Taken together, we conclude that arsenic induces the miR-466-669 cluster, and that this induction acts to inhibit cellular differentiation in part due to a repression of NeuroD1.
PHF20L1 antagonizes SOX2 proteolysis triggered by the MLL1/WDR5 complexes.
Wang Qianqian,Yu Min,Ma Yue,Zhang Xiaoming,Zhang Hui,Li Shuiming,Lan Rongfeng,Lu Fei
Laboratory investigation; a journal of technical methods and pathology
Transcriptional factor SOX2 regulates stem cell pluripotency, cell differentiation and tumorigenesis. As a key factor, the expression of SOX2 is tightly regulated at transcriptional and post-translational levels. However, the underlying mechanism of SOX2 protein stability remains to be elucidated. Here we show that the histone-lysine N-methyltransferase MLL1/WDR5 complexes physically interact with SOX2 and evoke SOX2 proteolysis, possibly through methylation on a potential site lysine 42 (K42). Small interfering RNA (siRNA)-mediated gene silencing of the components of the MLL1/WDR5 complexes WDR5, MLL1, RBBP5, and ASH2L lead to the accumulation of SOX2, while forced expression of WDR5 promotes SOX2 ubiquitination and proteolysis. Conversely, PHD finger protein 20-like protein 1 (PHF20L1) associates with SOX2, antagonizes SOX2 ubiquitination and the sequential degradation induced by the MLL1/WDR5 complexes. RNA interferences of PHF20L1 promote the degradation of SOX2, while forced expression of PHF20L1 stabilizes SOX2. Co-silencing of MLL1/WDR5 components and PHF20L1 preclude degradation of SOX2 induced by knockdown of PHF20L1. Moreover, co-expression of PHF20L1 and WDR5 prevent ubiquitination of SOX2 triggered by WDR5 over-expression. However, SOX2 mutant K42R is non-sensitive to the MLL1/WDR5 complexes or PHF20L1. In addition, PHF20L1 may regulate the stability of SOX2 through its malignant brain tumor (MBT) domain, since the degradation of SOX2 is accelerated by UNC1215 and UNC669, inhibitors that bind to the MBT domain. Furthermore, abundant expression of SOX2 is highly correlated to immature ovarian teratoma. Loss of PHF20L1 weakened the tumor initiation ability of PA-1 cells while ablation of MLL1 promoted the growth of tumors. Thus, our studies reveal an antagonistic mechanism by which the protein stability of SOX2 is regulated by the MLL1/WDR5 complexes and PHF20L1, possibly through methylation of SOX2 protein, and provide a novel perspective on SOX2-positive cancer treatment.
Down-regulated in OA cartilage, SFMBT2 contributes to NF-κB-mediated ECM degradation.
Hussain Safdar,Sun Mengyao,Min Zixin,Guo Yuanxu,Xu Jing,Mushtaq Nosheen,Heng Lisong,Huang Huang,Zhao Yitong,Yuan Ying,Hussain Nazim,Zhang Fujun,Han Yan,Xu Peng,Sun Jian,Lu Shemin
Journal of cellular and molecular medicine
The interplay between anabolic and catabolic factors regulates cartilage matrix homoeostasis. In OA, this balance is disrupted which results in cartilage degradation involving a plethora of inflammatory factors. Here, we identify a novel gene "Scm-like with four MBT domains protein 2" (SFMBT2) negatively regulated in OA cartilage. Articular cartilage from human OA patients undergoing knee arthroplasty surgery exhibited significantly decreased levels of SFMBT2 compared to the normal controls. Down-regulation of SFMBT2 by specific siRNA disturbed the metabolic homoeostasis and led to decreased expression of anabolic genes (SOX9, COL2A1) while increasing the expression of catabolic genes (MMP13 and ADAMTS4), in human chondrocytes. Finally, we revealed that SFMBT2 intervention by siRNA contributed to the catabolic phenotype of human chondrocytes mediated by NF-kB pathway.
Rearrangement of chromatin domains during development in Xenopus.
Vassetzky Y,Hair A,Méchali M
Genes & development
A dynamic change in the organization of different gene domains transcribed by RNA polymerase I, II, or III occurs during the progression from quiescent [pre-midblastula transition (pre-MBT)] to active (post-MBT) embryos during Xenopus development. In the rDNA, c-myc, and somatic 5S gene domains, a transition from random to specific anchorage to the nuclear matrix occurs when chromatin domains become active. The keratin gene domain was also randomly associated to the nuclear matrix before MBT, whereas a defined attachment site was found in keratinocytes. In agreement with this specification, ligation-mediated (LM)-PCR genomic footprinting carried out on the subpopulation of 5S domains specifically attached to the matrix reveals the hallmarks of determined chromatin after the midblastula transition. In contrast, the same analysis performed on the total 5S gene population does not reveal specific chromatin organization, validating the use of nuclear matrix fractionation to unveil active chromatin domains. These data provide a means for the determination of active chromosomal territories in the embryo and emphasize the role of nuclear architecture in regulated gene expression during development.
Solution structure of the PWWP domain of the hepatoma-derived growth factor family.
Nameki Nobukazu,Tochio Naoya,Koshiba Seizo,Inoue Makoto,Yabuki Takashi,Aoki Masaaki,Seki Eiko,Matsuda Takayoshi,Fujikura Yukiko,Saito Miyuki,Ikari Masaomi,Watanabe Megumi,Terada Takaho,Shirouzu Mikako,Yoshida Mayumi,Hirota Hiroshi,Tanaka Akiko,Hayashizaki Yoshihide,Güntert Peter,Kigawa Takanori,Yokoyama Shigeyuki
Protein science : a publication of the Protein Society
Among the many PWWP-containing proteins, the largest group of homologous proteins is related to hepatoma-derived growth factor (HDGF). Within a well-conserved region at the extreme N-terminus, HDGF and five HDGF-related proteins (HRPs) always have a PWWP domain, which is a module found in many chromatin-associated proteins. In this study, we determined the solution structure of the PWWP domain of HDGF-related protein-3 (HRP-3) by NMR spectroscopy. The structure consists of a five-stranded beta-barrel with a PWWP-specific long loop connecting beta2 and beta3 (PR-loop), followed by a helical region including two alpha-helices. Its structure was found to have a characteristic solvent-exposed hydrophobic cavity, which is composed of an abundance of aromatic residues in the beta1/beta2 loop (beta-beta arch) and the beta3/beta4 loop. A similar ligand binding cavity occurs at the corresponding position in the Tudor, chromo, and MBT domains, which have structural and probable evolutionary relationships with PWWP domains. These findings suggest that the PWWP domains of the HDGF family bind to some component of chromatin via the cavity.
Structure of the chromo barrel domain from the MOF acetyltransferase.
Nielsen Peter R,Nietlispach Daniel,Buscaino Alessia,Warner Rosemary J,Akhtar Asifa,Murzin Alexey G,Murzina Natalia V,Laue Ernest D
The Journal of biological chemistry
We report here the structure of the putative chromo domain from MOF, a member of the MYST family of histone acetyltransferases that acetylates histone H4 at Lys-16 and is part of the dosage compensation complex in Drosophila. We found that the structure of this domain is a beta-barrel that is distinct from the alpha + beta fold of the canonical chromo domain. Despite the differences, there are similarities that support an evolutionary relationship between the two domains, and we propose the name "chromo barrel." The chromo barrel domains may be divided into two groups, MSL3-like and MOF-like, on the basis of whether a group of conserved aromatic residues is present or not. The structure suggests that, although the MOF-like domains may have a role in RNA binding, the MSL3-like domains could instead bind methylated residues. The MOF chromo barrel shares a common fold with other chromatin-associated modules, including the MBT-like repeat, Tudor, and PWWP domains. This structural similarity suggests a probable evolutionary pathway from these other modules to the canonical chromo domains (or vice versa) with the chromo barrel domain representing an intermediate structure.
LIN-61, one of two Caenorhabditis elegans malignant-brain-tumor-repeat-containing proteins, acts with the DRM and NuRD-like protein complexes in vulval development but not in certain other biological processes.
Harrison Melissa M,Lu Xiaowei,Horvitz H Robert
Vulval development in Caenorhabiditis elegans is inhibited by the redundant functions of the synthetic multivulva (synMuv) genes. At least 26 synMuv genes have been identified, many of which appear to act via transcriptional repression. Here we report the molecular identification of the class B synMuv gene lin-61, which encodes a protein composed of four malignant brain tumor (MBT) repeats. MBT repeats, domains of approximately 100 amino acids, have been found in multiple copies in a number of transcriptional repressors, including Polycomb-group proteins. MBT repeats are important for the transcriptional repression mediated by these proteins and in some cases have been shown to bind modified histones. C. elegans contains one other MBT-repeat-containing protein, MBTR-1. We demonstrate that a deletion allele of mbtr-1 does not cause a synMuv phenotype nor does mbtr-1 appear to act redundantly with or in opposition to lin-61. We further show that lin-61 is phenotypically and biochemically distinct from other class B synMuv genes. Our data indicate that while the class B synMuv genes act together to regulate vulval development, lin-61 functions separately from some class B synMuv proteins in other biological processes.
L3MBTL1, a histone-methylation-dependent chromatin lock.
Trojer Patrick,Li Guohong,Sims Robert J,Vaquero Alejandro,Kalakonda Nagesh,Boccuni Piernicola,Lee Donghoon,Erdjument-Bromage Hediye,Tempst Paul,Nimer Stephen D,Wang Yuh-Hwa,Reinberg Danny
Distinct histone lysine methylation marks are involved in transcriptional repression linked to the formation and maintenance of facultative heterochromatin, although the underlying mechanisms remain unclear. We demonstrate that the malignant-brain-tumor (MBT) protein L3MBTL1 is in a complex with core histones, histone H1b, HP1gamma, and Rb. The MBT domain is structurally related to protein domains that directly bind methylated histone residues. Consistent with this, we found that the L3MBTL1 MBT domains compact nucleosomal arrays dependent on mono- and dimethylation of histone H4 lysine 20 and of histone H1b lysine 26. The MBT domains bind at least two nucleosomes simultaneously, linking repression of transcription to recognition of different histone marks by L3MBTL1. Consistently, L3MBTL1 was found to negatively regulate the expression of a subset of genes regulated by E2F, a factor that interacts with Rb.
Identification of a novel conserved mixed-isoform B56 regulatory subunit and spatiotemporal regulation of protein phosphatase 2A during Xenopus laevis development.
Baek Sungmin,Seeling Joni M
BMC developmental biology
BACKGROUND:Wnt signaling is a key regulator of development and tumorigenesis. Protein phosphatase 2A (PP2A), which consists of a catalytic C, a structural A, and a regulatory B subunit, plays diverse roles in Wnt signaling through its B56 subunits. B56 is a multigene family encoding for proteins with a conserved core domain and divergent amino- and carboxy-termini. Ectopic B56alpha and B56gamma reduce beta-catenin abundance and B56alpha reduces Wnt-dependent transcription, suggesting that B56alpha and B56gamma inhibit Wnt signaling. In contrast, B56epsilon is required for Wnt signaling. Knowledge of where and when B56 subunits are expressed during Xenopus development will aid in our understanding of their roles in Wnt signaling. RESULTS:We have undertaken expression analyses of B56alpha and B56gamma in Xenopus laevis. We cloned Xenopus B56alpha; it is 88% identical to human B56alpha. Xenopus B56gamma is 94% identical with human B56gamma, however, a novel evolutionarily conserved mixed-isoform transcript was identified that contains a B56delta-like amino-terminal domain and a B56gamma core domain. The B56delta-like variable domain exon is located upstream of the B56gamma variable domain exon at the human B56gamma locus, suggesting that the mixed-isoform transcript is due to alternative splicing. B56gamma transcripts with different 3' ends were identified that lack or possess a 35 base pair sequence, resulting in either a transcript similar to human B56gamma1, or an uncharacterized evolutionarily conserved sequence. Real time RT-PCR analyses revealed that B56alpha is expressed at moderate levels before the midblastula transition (MBT), at reduced levels during gastrulation and neurulation, and at high levels during organogenesis, while B56gamma is expressed at low levels until organogenesis. B56alpha is enriched in the ventral hemisphere pre-MBT, while B56gamma is ventrally enriched post-MBT. Aalpha, Abeta, Calpha and Cbeta are expressed in early Xenopus development, suggesting the presence of a functional heterotrimer. CONCLUSION:Our data suggest that B56 functional diversity is achieved in part through the synthesis of a novel mixed-isoform B56delta/gamma transcript. Our data also suggest that B56alpha functions pre-MBT, inhibiting Wnt signaling on the ventral side of the embryo, and again during organogenesis, while B56gamma functions primarily post-MBT.
Molecular recognition of histone lysine methylation by the Polycomb group repressor dSfmbt.
Grimm Clemens,Matos Raquel,Ly-Hartig Nga,Steuerwald Ulrich,Lindner Doris,Rybin Vladimir,Müller Jürg,Müller Christoph W
The EMBO journal
Polycomb group (PcG) proteins repress transcription by modifying chromatin structure in target genes. dSfmbt is a subunit of the Drosophila melanogaster PcG protein complex PhoRC and contains four malignant brain tumour (MBT) repeats involved in the recognition of various mono- and dimethylated histone peptides. Here, we present the crystal structure of the four-MBT-repeat domain of dSfmbt in complex with a mono-methylated histone H4 peptide. Only a single histone peptide binds to the four-MBT-repeat domain. Mutational analyses show high-affinity binding with low peptide sequence selectivity through combinatorial interaction of the methyl-lysine with an aromatic cage and positively charged flanking residues with the surrounding negatively charged surface of the fourth MBT repeat. dSfmbt directly interacts with the PcG protein Scm, a related MBT-repeat protein with similar methyl-lysine binding activity. dSfmbt and Scm co-occupy Polycomb response elements of target genes in Drosophila and they strongly synergize in the repression of these target genes, suggesting that the combined action of these two MBT proteins is crucial for Polycomb silencing.
Small-molecule ligands of methyl-lysine binding proteins.
Herold J Martin,Wigle Tim J,Norris Jacqueline L,Lam Robert,Korboukh Victoria K,Gao Cen,Ingerman Lindsey A,Kireev Dmitri B,Senisterra Guillermo,Vedadi Masoud,Tripathy Ashutosh,Brown Peter J,Arrowsmith Cheryl H,Jin Jian,Janzen William P,Frye Stephen V
Journal of medicinal chemistry
Proteins which bind methylated lysines ("readers" of the histone code) are important components in the epigenetic regulation of gene expression and can also modulate other proteins that contain methyl-lysine such as p53 and Rb. Recognition of methyl-lysine marks by MBT domains leads to compaction of chromatin and a repressed transcriptional state. Antagonists of MBT domains would serve as probes to interrogate the functional role of these proteins and initiate the chemical biology of methyl-lysine readers as a target class. Small-molecule MBT antagonists were designed based on the structure of histone peptide-MBT complexes and their interaction with MBT domains determined using a chemiluminescent assay and ITC. The ligands discovered antagonize native histone peptide binding, exhibiting 5-fold stronger binding affinity to L3MBTL1 than its preferred histone peptide. The first cocrystal structure of a small molecule bound to L3MBTL1 was determined and provides new insights into binding requirements for further ligand design.
Identification of IGF1, SLC4A4, WWOX, and SFMBT1 as hypertension susceptibility genes in Han Chinese with a genome-wide gene-based association study.
Yang Hsin-Chou,Liang Yu-Jen,Chen Jaw-Wen,Chiang Kuang-Mao,Chung Chia-Min,Ho Hung-Yun,Ting Chih-Tai,Lin Tsung-Hsien,Sheu Sheng-Hsiung,Tsai Wei-Chuan,Chen Jyh-Hong,Leu Hsin-Bang,Yin Wei-Hsian,Chiu Ting-Yu,Chern Ching-luan,Lin Shing-Jong,Tomlinson Brian,Guo Youling,Sham Pak C,Cherny Stacey S,Lam Tai Hing,Thomas G Neil,Pan Wen-Harn
Hypertension is a complex disorder with high prevalence rates all over the world. We conducted the first genome-wide gene-based association scan for hypertension in a Han Chinese population. By analyzing genome-wide single-nucleotide-polymorphism data of 400 matched pairs of young-onset hypertensive patients and normotensive controls genotyped with the Illumina HumanHap550-Duo BeadChip, 100 susceptibility genes for hypertension were identified and also validated with permutation tests. Seventeen of the 100 genes exhibited differential allelic and expression distributions between patient and control groups. These genes provided a good molecular signature for classifying hypertensive patients and normotensive controls. Among the 17 genes, IGF1, SLC4A4, WWOX, and SFMBT1 were not only identified by our gene-based association scan and gene expression analysis but were also replicated by a gene-based association analysis of the Hong Kong Hypertension Study. Moreover, cis-acting expression quantitative trait loci associated with the differentially expressed genes were found and linked to hypertension. IGF1, which encodes insulin-like growth factor 1, is associated with cardiovascular disorders, metabolic syndrome, decreased body weight/size, and changes of insulin levels in mice. SLC4A4, which encodes the electrogenic sodium bicarbonate cotransporter 1, is associated with decreased body weight/size and abnormal ion homeostasis in mice. WWOX, which encodes the WW domain-containing protein, is related to hypoglycemia and hyperphosphatemia. SFMBT1, which encodes the scm-like with four MBT domains protein 1, is a novel hypertension gene. GRB14, TMEM56 and KIAA1797 exhibited highly significant differential allelic and expressed distributions between hypertensive patients and normotensive controls. GRB14 was also found relevant to blood pressure in a previous genetic association study in East Asian populations. TMEM56 and KIAA1797 may be specific to Taiwanese populations, because they were not validated by the two replication studies. Identification of these genes enriches the collection of hypertension susceptibility genes, thereby shedding light on the etiology of hypertension in Han Chinese populations.
SFMBT1 functions with LSD1 to regulate expression of canonical histone genes and chromatin-related factors.
Zhang Jin,Bonasio Roberto,Strino Francesco,Kluger Yuval,Holloway J Kim,Modzelewski Andrew J,Cohen Paula E,Reinberg Danny
Genes & development
SFMBT1 (Scm [Sex comb on midleg] with four MBT [malignant brain tumor] domains 1) is a poorly characterized mammalian MBT domain-containing protein homologous to Drosophila SFMBT, a Polycomb group protein involved in epigenetic regulation of gene expression. Here, we show that SFMBT1 regulates transcription in somatic cells and during spermatogenesis through the formation of a stable complex with LSD1 and CoREST. When bound to its gene targets, SFMBT1 recruits its associated proteins and causes chromatin compaction and transcriptional repression. SFMBT1, LSD1, and CoREST share a large fraction of target genes, including those encoding replication-dependent histones. Simultaneous occupancy of histone genes by SFMBT1, LSD1, and CoREST is regulated during the cell cycle and correlates with the loss of RNA polymerase II at these promoters during G2, M, and G1. The interplay between the repressive SFMBT1-LSD1-CoREST complex and RNA polymerase II contributes to the timely transcriptional regulation of histone genes in human cells. SFMBT1, LSD1, and CoREST also form a stable complex in germ cells, and their chromatin binding activity is regulated during spermatogenesis.
Methyllysine reader plant homeodomain (PHD) finger protein 20-like 1 (PHF20L1) antagonizes DNA (cytosine-5) methyltransferase 1 (DNMT1) proteasomal degradation.
Estève Pierre-Olivier,Terragni Jolyon,Deepti Kanneganti,Chin Hang Gyeong,Dai Nan,Espejo Alexsandra,Corrêa Ivan R,Bedford Mark T,Pradhan Sriharsa
The Journal of biological chemistry
Inheritance of DNA cytosine methylation pattern during successive cell division is mediated by maintenance DNA (cytosine-5) methyltransferase 1 (DNMT1). Lysine 142 of DNMT1 is methylated by the SET domain containing lysine methyltransferase 7 (SET7), leading to its degradation by proteasome. Here we show that PHD finger protein 20-like 1 (PHF20L1) regulates DNMT1 turnover in mammalian cells. Malignant brain tumor (MBT) domain of PHF20L1 binds to monomethylated lysine 142 on DNMT1 (DNMT1K142me1) and colocalizes at the perinucleolar space in a SET7-dependent manner. PHF20L1 knockdown by siRNA resulted in decreased amounts of DNMT1 on chromatin. Ubiquitination of DNMT1K142me1 was abolished by overexpression of PHF20L1, suggesting that its binding may block proteasomal degradation of DNMT1K142me1. Conversely, siRNA-mediated knockdown of PHF20L1 or incubation of a small molecule MBT domain binding inhibitor in cultured cells accelerated the proteasomal degradation of DNMT1. These results demonstrate that the MBT domain of PHF20L1 reads and controls enzyme levels of methylated DNMT1 in cells, thus representing a novel antagonist of DNMT1 degradation.
MicroRNA-218 inhibits EMT, migration and invasion by targeting SFMBT1 and DCUN1D1 in cervical cancer.
Jiang Zhaojing,Song Qiancheng,Zeng Rong,Li Jing,Li Jingyu,Lin Xiaochun,Chen Xing,Zhang Jiren,Zheng Yanfang
Repeated infection with high-risk HPV is a major cause for the development and metastasis of human cervical cancer, even though the mechanism of the metastasis is still not completely understood. Here, we reported that miR-218 (microRNA-218) was downregulated in cervical cancer tissues, especially in metastatic cancer tissues. We found that miR-218 expression was associated with clinicopathological characteristics of patients with cervical cancer. MiR-218 overexpression inhibited Epithelial-Mesenchymal Transition (EMT), migration and invasiveness of cervical cancer cells in vitro. Moreover, miR-218 repressed the expression of SFMFBT1 (Scm-like with four MBT domains 1) and DCUN1D1 (defective in cullin neddylation 1, domain containing 1) by direct binding to the 3'UTRs of the mRNAs. The overexpression of SFMBT1 induced EMT and increased the migration and invasiveness of cervical cancer cells, while the overexpression of DCUN1D1 increased the migration and invasiveness of these cells, but did not induce EMT. An inverse correlation was observed between the expression of miR-218 and DCUN1D1 protein in cervical cancer tissues. Importantly, HPV16 E6 downregulated the expression of miR-218 in cervical cancer, while miR-218 rescued the promotion effect of HPV16 E6 on the expression of SFMBT1 and DCUN1D1. Taken together, our results revealed that HPV16 E6 promoted EMT and invasion in cervical cancer via the repression of miR-218, while miR-218 inhibited EMT and invasion in cervical cancer by targeting SFMBT1 and DCUN1D1.
Coordinated methyl readers: Functional communications in cancer.
Seminars in cancer biology
Methylation is a major post-translational modification (PTM) generated by methyltransferase on target proteins; it is recognized by the epigenetic reader to expand the functional diversity of proteins. Methylation can occur on specific lysine or arginine residues localized within regulatory domains in both histone and nonhistone proteins, thereby allowing distinguished properties of the targeted protein. Methylated residues are recognized by chromodomain, malignant brain tumor (MBT), Tudor, plant homeodomain (PHD), PWWP, WD-40, ADD, and ankyrin repeats by an induced-fit mechanism. Methylation-dependent activities regulate distinct aspects of target protein function and are largely reliant on methyl readers of histone and nonhistone proteins in various diseases. Methylation of nonhistone proteins that are recognized by methyl readers facilitates the degradation of unwanted proteins, as well as the stabilization of necessary proteins. Unlike nonhistone substrates, which are mainly monomethylated by methyltransferase, histones are di- or trimethylated by the same methyltransferases and then connected to other critical regulators by methyl readers. These fine-tuned controls by methyl readers are significant for the progression or inhibition of diseases, including cancers. Here, current knowledge and our perspectives about regulating protein function by methyl readers are summarized. We also propose that expanded research on the strong crosstalk mechanisms between methylation and other PTMs via methyl readers would augment therapeutic research in cancer.
Circ-SFMBT2 facilitates the malignant growth of acute myeloid leukemia cells by modulating miR-582-3p/ZBTB20 pathway.
Histology and histopathology
BACKGROUND:Acute myeloid leukemia (AML) is a highly heterogeneous hematological malignancy. Circular RNAs (circRNAs) play crucial roles in AML progression. This study aimed to explore the function and potential mechanism of circRNA Scm like with four mbt domains 2 (circ-SFMBT2; hsa_circ_0017639) in AML. METHODS:The levels of circ-SFMBT2, microRNA-582-3p (miR-582-3p) and zinc finger and BTB domain containing 20 (ZBTB20) were measured by quantitative real-time PCR and Western blot. Cell Counting Kit-8 (CCK-8), colony formation, flow cytometry and transwell assays were used to evaluate cell proliferation, apoptosis, migration and invasion. Glycolysis was assessed by detecting glucose consumption, lactate production and ATP/ADP ratios. The related protein expression was examined by Western blot. The binding relationship between miR-582-3p and circ-SFMBT2/ZBTB20 was verified by dual-luciferase reporter assay. RESULTS:Circ-SFMBT2 and ZBTB20 levels were elevated, while miR-582-3p level was reduced in AML patients and cells. Depletion of circ-SFMBT2/ZBTB20 impeded proliferation, migration, invasion and glycolysis and induced apoptosis in AML cells. Moreover, circ-SFMBT2 facilitated AML progression by sponging miR-582-3p, and miR-582-3p targeted ZBTB20 to hinder AML development. CONCLUSION:Knockdown of circ-SFMBT2 suppressed AML progression by regulating the miR-582-3p/ZBTB20 axis, which might provide a potential therapeutic strategy for AML.
promotes adipogenic differentiation and induces browning in preadipocytes.
Obesity is a major global health issue that contributes to the occurrence of metabolic disorders. Based on this fact, understanding the underlying mechanisms and to uncover promising therapeutic approaches for obesity have attracted intense investigation. Brown adipose tissue (BAT) can help burns excess calories. Therefore, promoting White adipose tissue (WAT) browning and BAT activation is an attractive strategy for obesity treatment. MicroRNAs (miRNAs) are small, non-coding RNAs, which are involved in regulation of adipogenic processes and metabolic functions. Evidence is accumulating that miRNAs are important regulators for both brown adipocyte differentiation and white adipocyte browning. Here we report that the expression of increases during the adipogenic differentiation of 3T3-L1 and C3H10T1/2 adipocytes. supplementation promotes adipogenic differentiation and causes browning of 3T3-L1 and C3H10T1/2 cells. Moreover, the expression of is upregulated in iWAT of mice exposed to cold. These data demonstrate that plays a role in regulating adipocyte differentiation and fat browning.: : long-chain acyl-Coenzyme A dehydrogenase; : medium-chain acyl-Coenzyme A dehydrogenase; : very long-chain acyl-Coenzyme A dehydrogenase, very long chain; : mitochondrial aconitase 2; BAT: brown adipose tissue; : BMP-binding endothelial regulator; :carnitine palmitoyltransferase 1b; : carnitine palmitoyltransferase 2; : carnitine acetyltransferase; : citrate synthase; C2MC: Chromosome 2 miRNA cluster; DMEM: Dulbecco's modified Eagle medium; eWAT: epididymal white adipose tissue; ETC: electron transport chain; FAO: fatty acid oxidation; :fatty acid binding protein 4; FBS: fetal bovine serum; : hydroxyacyl-CoA dehydrogenase trifunctional multienzyme complex subunit alpha; : hydroxyacyl-CoA dehydrogenase trifunctional multienzyme complex subunit beta; HFD: high fat diet; : isocitrate dehydrogenase 3 alpha; iWAT: inguinal subcutaneous white adipose tissue; : lipoprotein lipase; : malate dehydrogenase 2; NBCS: NewBorn Calf Serum; : mitochondrial NADH dehydrogenase 1; :ubiquinone oxidoreductase subunit B8; : nuclear respiratory factor 1; : peroxisome proliferative activated receptor gamma coactivator 1 alpha; : peroxisome proliferative activated receptor, gamma, coactivator 1 beta; : peroxisome proliferator activated receptor gamma; : PR domain containing 16; : regulator of G-protein signaling 4; : succinate dehydrogenase complex, subunit B; : succinate dehydrogenase complex, subunit C; : succinate dehydrogenase complex, subunit D; : SH3 domain containing 21; : Scm-like with four mbt domains 2; TG: triglyceride; TCA: tricarboxylic acid cycle; : transcription factor A, mitochondrial; TMRE: tetramethylrhodamine, methyl ester; : uncoupling protein 1; : ubiquinol cytochrome c reductase core protein 2; WAT: White adipose tissue.
Cloning of a novel murine gene Sfmbt, Scm-related gene containing four mbt domains, structurally belonging to the Polycomb group of genes.
Usui H,Ichikawa T,Kobayashi K,Kumanishi T
A novel cDNA clone encoding a protein structurally related to the transcriptional repressor Polycomb group (PcG) proteins, which regulate homeotic genes and others, was isolated from mouse and rat brain. The coding protein contained the SPM domain and mbt repeats, both of which are characteristic of the PcG proteins, and showed significant similarity in amino acid sequence to the Drosophila Sex comb on midleg (Scm) protein. Since this novel protein contains the mbt repeats in four tandem copies, we designated this murine gene as Sfmbt for Scm-related gene containing four mbt domains. Cloning and characterization of the mouse Sfmbt gene revealed that the coding sequence comprised 20 exons, dispersed along approximately 40kb, and mapped to the proximal part of Chromosome 14. Northern blot analysis showed that the Sfmbt mRNAs were expressed most abundantly in the adult testis, and less intensively in all other tissues examined.
The Tudor domain 'Royal Family': Tudor, plant Agenet, Chromo, PWWP and MBT domains.
Maurer-Stroh Sebastian,Dickens Nicholas J,Hughes-Davies Luke,Kouzarides Tony,Eisenhaber Frank,Ponting Chris P
Trends in biochemical sciences
We have identified a family of 'Agenet' domains that are plant-specific homologs of Tudor domains. This finding has been extended, using a combination of sequence- and structure-dependent approaches, to show that the three beta-stranded core regions of Tudor, PWWP, chromatin-binding (Chromo) and MBT domains are homologous because they originate from a common ancestor. In addition, we have revealed pairs of tandem repeats in the fragile X mental retardation protein (FMRP) family that are also members of this Tudor domain 'Royal Family'.
The human L(3)MBT polycomb group protein is a transcriptional repressor and interacts physically and functionally with TEL (ETV6).
Boccuni Piernicola,MacGrogan Donal,Scandura Joseph M,Nimer Stephen D
The Journal of biological chemistry
H-L(3)MBT, the human homolog of the Drosophila lethal(3)malignant brain tumor protein, is a member of the polycomb group (PcG) of proteins, which function as transcriptional regulators in large protein complexes. Homozygous mutations in the l(3)mbt gene cause brain tumors in Drosophila, identifying l(3)mbt as a tumor suppressor gene. The h-l(3)mbt gene maps to chromosome 20q12, within a common deleted region associated with myeloid hematopoietic malignancies. H-L(3)MBT contains three repeats of 100 residues called MBT repeats, whose function is unknown, and a C-terminal alpha-helical structure, the SPM (SCM, PH, MBT domain, which is structurally similar to the SAM (sterile alpha motif) protein-protein interaction domain, found in several ETS transcription factors, including TEL (translocation Ets leukemia). We report that H-L(3)MBT is a transcriptional repressor and that its activity is largely dependent on the presence of a region containing the three MBT repeats. H-L(3)MBT acts as a histone deacetylase-independent transcriptional repressor, based on its lack of sensitivity to trichostatin A. We found that H-L(3)MBT binds in vivo to TEL, and we have mapped the region of interaction to their respective SPM/SAM domains. We show that the ability of TEL to repress TEL-responsive promoters is enhanced by the presence of H-L(3)MBT, an effect dependent on the H-L(3)MBT and the TEL interacting domains. These experiments suggest that histone deacetylase-independent transcriptional repression by TEL depends on the recruitment of PcG proteins. We speculate that the interaction of TEL with H-L(3)MBT can direct a PcG complex to genes repressed by TEL, stabilizing their repressed state.
Crystal structure of the malignant brain tumor (MBT) repeats in Sex Comb on Midleg-like 2 (SCML2).
Sathyamurthy Aruna,Allen Mark D,Murzin Alexey G,Bycroft Mark
The Journal of biological chemistry
Sex Comb on Midleg (SCM) belongs to the Polycomb group of proteins, which are involved in transcriptional regulation in Drosophila. It is one of the components of Polycomb repressive complex 1, a multiprotein complex of Polycomb group proteins involved in the maintenance of repression and the blocking of chromatin remodeling. SCM contains two approximately 100-residue malignant brain tumor (MBT) repeats at the N terminus. These repeats are also found in other proteins involved in transcriptional repression. Here, we report the 1.78-A crystal structure of the two MBT repeats of SCM-like 2 (SCML2), a human homologue of SCM. Each repeat consists of an extended arm and a beta-barrel core. There are significant structural similarities to the Tudor, PWWP, and chromo domains, suggesting probable evolutionary relationships and functional similarities between the MBT repeats and these domains.
Proliferation-linked expression of the novel murine gene m4mbt encoding a nuclear zinc finger protein with four mbt domains.
Markus Jan,Feiková Sona,Sramko Marek,Wolff Linda,Bies Juraj
Here we describe the cloning and characterization of a novel murine gene named m4mbt that encodes a homolog of the lethal (3) malignant brain tumor (l(3)mbt) and Scm proteins. It is localized on mouse chromosome 15E2 and is organized into 17 exons. As demonstrated by Northern blot analysis, m4mbt mRNA is expressed in virtually all tested tissues and cell lines with the exception of stomach and muscle. The m4mbt transcript was most abundant in the testes. m4mbt expression was shown to initiate early during mouse embryonal development (before day 7) and continue until adulthood. The expression of m4mbt mRNA also appears to correlate with cellular proliferation, since we observed down-regulation of m4mbt expression during terminal monocytic differentiation and in contact-inhibited fibroblasts. Computer analysis of the amino acid (aa) sequence revealed that the M4mbt protein comprises an amino-terminally located atypical C2C2 zinc finger and four centrally located mbt repeats. Mbt repeats are also found in proteins of the Polycomb group (PcG) that associate with heterochromatin and function as long-term repressors of transcription. Using Western blot analysis and confocal fluorescent microscopy, we demonstrated that the M4mbt protein is localized in the nucleus. Since M4mbt has structural domains similar to chromatin-associated proteins, its expression is associated with proliferation, and it has a nuclear localization, it may have a regulatory role related to proliferation and/or differentiation.
Methylation-state-specific recognition of histones by the MBT repeat protein L3MBTL2.
Guo Yahong,Nady Nataliya,Qi Chao,Allali-Hassani Abdellah,Zhu Haizhong,Pan Patricia,Adams-Cioaba Melanie A,Amaya Maria F,Dong Aiping,Vedadi Masoud,Schapira Matthieu,Read Randy J,Arrowsmith Cheryl H,Min Jinrong
Nucleic acids research
The MBT repeat has been recently identified as a key domain capable of methyl-lysine histone recognition. Functional work has pointed to a role for MBT domain-containing proteins in transcriptional repression of developmental control genes such as Hox genes. In this study, L3MBTL2, a human homolog of Drosophila Sfmbt critical for Hox gene silencing, is demonstrated to preferentially recognize lower methylation states of several histone-derived peptides through its fourth MBT repeat. High-resolution crystallographic analysis of the four MBT repeats of this protein reveals its unique asymmetric rhomboid architecture, as well as binding mechanism, which preclude the interaction of the first three MBT repeats with methylated peptides. Structural elucidation of an L3MBTL2-H4K20me1 complex and comparison with other MBT-histone peptide complexes also suggests that an absence of distinct surface contours surrounding the methyl-lysine-binding pocket may underlie the lack of sequence specificity observed for members of this protein family.
Solution structure of the FCS zinc finger domain of the human polycomb group protein L(3)mbt-like 2.
Lechtenberg Bernhard C,Allen Mark D,Rutherford Trevor J,Freund Stefan M V,Bycroft Mark
Protein science : a publication of the Protein Society
Polycomb group proteins are epigenetic regulators that maintain patterns of gene expression over multiple rounds of cell division. Many of these proteins, including polyhomeotic and the MBT repeat containing proteins SCM and dSfmbt, contain an atypical C2C2 zinc finger with a characteristic phenylalanine-cysteine-serine sequence motif. The reoccurrence of this so-called FCS zinc finger in a variety of polycomb group proteins suggests that it has an important regulatory function. We have determined the solution structure of the FCS zinc finger of the human dSfmbt homologue L(3)mbt-like 2 (L3MBTL2). The structure consists of a beta-hairpin followed by an alpha-helix. The zinc ligands are situated in the beta-hairpin and at the N-terminus of the alpha-helix an arrangement typical of the treble clef class of zinc fingers. The structure is consistent with the proposal that FCS zinc fingers bind to regulatory RNAs.
Identification of non-peptide malignant brain tumor (MBT) repeat antagonists by virtual screening of commercially available compounds.
Kireev Dmitri,Wigle Tim J,Norris-Drouin Jacqueline,Herold J Martin,Janzen William P,Frye Stephen V
Journal of medicinal chemistry
The malignant brain tumor (MBT) repeat is an important epigenetic-code "reader" and is functionally associated with differentiation, gene silencing, and tumor suppression. (1-3) Small molecule probes of MBT domains should enable a systematic study of MBT-containing proteins and potentially reveal novel druggable targets. We designed and applied a virtual screening strategy that identified potential MBT antagonists in a large database of commercially available compounds. A small set of virtual hits was purchased and submitted to experimental testing. Nineteen of the purchased compounds showed a specific dose-dependent protein binding and will provide critical structure-activity information for subsequent lead generation and optimization.
Structural and histone binding ability characterizations of human PWWP domains.
Wu Hong,Zeng Hong,Lam Robert,Tempel Wolfram,Amaya Maria F,Xu Chao,Dombrovski Ludmila,Qiu Wei,Wang Yanming,Min Jinrong
BACKGROUND:The PWWP domain was first identified as a structural motif of 100-130 amino acids in the WHSC1 protein and predicted to be a protein-protein interaction domain. It belongs to the Tudor domain 'Royal Family', which consists of Tudor, chromodomain, MBT and PWWP domains. While Tudor, chromodomain and MBT domains have long been known to bind methylated histones, PWWP was shown to exhibit histone binding ability only until recently. METHODOLOGY/PRINCIPAL FINDINGS:The PWWP domain has been shown to be a DNA binding domain, but sequence analysis and previous structural studies show that the PWWP domain exhibits significant similarity to other 'Royal Family' members, implying that the PWWP domain has the potential to bind histones. In order to further explore the function of the PWWP domain, we used the protein family approach to determine the crystal structures of the PWWP domains from seven different human proteins. Our fluorescence polarization binding studies show that PWWP domains have weak histone binding ability, which is also confirmed by our NMR titration experiments. Furthermore, we determined the crystal structures of the BRPF1 PWWP domain in complex with H3K36me3, and HDGF2 PWWP domain in complex with H3K79me3 and H4K20me3. CONCLUSIONS:PWWP proteins constitute a new family of methyl lysine histone binders. The PWWP domain consists of three motifs: a canonical β-barrel core, an insertion motif between the second and third β-strands and a C-terminal α-helix bundle. Both the canonical β-barrel core and the insertion motif are directly involved in histone binding. The PWWP domain has been previously shown to be a DNA binding domain. Therefore, the PWWP domain exhibits dual functions: binding both DNA and methyllysine histones. ENHANCED VERSION:This article can also be viewed as an enhanced version in which the text of the article is integrated with interactive 3D representations and animated transitions. Please note that a web plugin is required to access this enhanced functionality. Instructions for the installation and use of the web plugin are available in Text S1.
ChEpiMod: a knowledgebase for chemical modulators of epigenome reader domains.
Meslamani Jamel,Smith Steven G,Sanchez Roberto,Zhou Ming-Ming
Bioinformatics (Oxford, England)
CONTEXT:Epigenome reader domains are rapidly emerging as a new class of drug targets for a wide array of human diseases. To facilitate study of structure-activity relationship and small-molecule ligand design for these domains, we have created ChEpiMod. ChEpiMod is a free knowledgebase of chemical modulators with documented modulatory activity for epigenome reader domains. METHODS:ChEpiMod organizes information about chemical modulators and their associated binding-affinity data, as well as available structures of epigenome readers from the Protein Data Bank. The data are gathered from the literature and patents. Entries are supplemented by annotation. The current version of ChEpiMod covers six epigenome reader domain families (Bromodomain, PHD finger, Chromodomain, MBT, PWWP and Tudor). The database can be used to browse existing chemical modulators and bioactivity data, as well as, all available structures of readers and their molecular interactions. The database is updated weekly. AVAILABILITY:ChEpiMod is freely available at http://chepimod.org CONTACT:email@example.com SUPPLEMENTARY INFORMATION:Supplementary data is available at Bioinformatics online.
Solution NMR structure of the DNA-binding domain from Scml2 (sex comb on midleg-like 2).
The Journal of biological chemistry
Scml2 is a member of the Polycomb group of proteins involved in epigenetic gene silencing. Human Scml2 is a part of a multisubunit protein complex, PRC1 (Polycomb repressive complex 1), which is responsible for maintenance of gene repression, prevention of chromatin remodeling, preservation of the "stemness" of the cell, and cell differentiation. Although the majority of PRC1 subunits have been recently characterized, the structure of Scml2 and its role in PRC1-mediated gene silencing remain unknown. In this work a conserved protein domain within human Scml2 has been identified, and its structure was determined by solution NMR spectroscopy. This module was named Scm-like embedded domain, or SLED. Evolutionarily, the SLED domain emerges in the first multicellular organisms, consistent with the role of Scml2 in cell differentiation. Furthermore, it is exclusively found within the Scm-like family of proteins, often accompanied by malignant brain tumor domain (MBT) and sterile α motif (SAM) domains. The domain adopts a novel α/β fold with no structural analogues found in the Protein Data Bank (PDB). The ability of the SLED to bind double-stranded DNA was also examined, and the isolated domain was shown to interact with DNA in a sequence-specific manner. Because PRC1 complexes localize to the promoters of a specific subset of developmental genes in vivo, the SLED domain of Scml2 may provide an important link connecting the PRC1 complexes to their target genes.
Interactions with RNA direct the Polycomb group protein SCML2 to chromatin where it represses target genes.
Bonasio Roberto,Lecona Emilio,Narendra Varun,Voigt Philipp,Parisi Fabio,Kluger Yuval,Reinberg Danny
Polycomb repressive complex-1 (PRC1) is essential for the epigenetic regulation of gene expression. SCML2 is a mammalian homolog of Drosophila SCM, a Polycomb-group protein that associates with PRC1. In this study, we show that SCML2A, an SCML2 isoform tightly associated to chromatin, contributes to PRC1 localization and also directly enforces repression of certain Polycomb target genes. SCML2A binds to PRC1 via its SPM domain and interacts with ncRNAs through a novel RNA-binding region (RBR). Targeting of SCML2A to chromatin involves the coordinated action of the MBT domains, RNA binding, and interaction with PRC1 through the SPM domain. Deletion of the RBR reduces the occupancy of SCML2A at target genes and overexpression of a mutant SCML2A lacking the RBR causes defects in PRC1 recruitment. These observations point to a role for ncRNAs in regulating SCML2 function and suggest that SCML2 participates in the epigenetic control of transcription directly and in cooperation with PRC1.DOI: http://dx.doi.org/10.7554/eLife.02637.001.
Tudor, MBT and chromo domains gauge the degree of lysine methylation.
Kim Jeesun,Daniel Jeremy,Espejo Alexsandra,Lake Aimee,Krishna Murli,Xia Li,Zhang Yi,Bedford Mark T
The post-translational modification of histones regulates many cellular processes, including transcription, replication and DNA repair. A large number of combinations of post-translational modifications are possible. This cipher is referred to as the histone code. Many of the enzymes that lay down this code have been identified. However, so far, few code-reading proteins have been identified. Here, we describe a protein-array approach for identifying methyl-specific interacting proteins. We found that not only chromo domains but also tudor and MBT domains bind to methylated peptides from the amino-terminal tails of histones H3 and H4. Binding specificity observed on the protein-domain microarray was corroborated using peptide pull-downs, surface plasma resonance and far western blotting. Thus, our studies expose tudor and MBT domains as new classes of methyl-lysine-binding protein modules, and also demonstrates that protein-domain microarrays are powerful tools for the identification of new domain types that recognize histone modifications.
Structural studies of a four-MBT repeat protein MBTD1.
Eryilmaz Jitka,Pan Patricia,Amaya Maria F,Allali-Hassani Abdellah,Dong Aiping,Adams-Cioaba Melanie A,Mackenzie Farrell,Vedadi Masoud,Min Jinrong
BACKGROUND:The Polycomb group (PcG) of proteins is a family of important developmental regulators. The respective members function as large protein complexes involved in establishment and maintenance of transcriptional repression of developmental control genes. MBTD1, Malignant Brain Tumor domain-containing protein 1, is one such PcG protein. MBTD1 contains four MBT repeats. METHODOLOGY/PRINCIPAL FINDINGS:We have determined the crystal structure of MBTD1 (residues 130-566aa covering the 4 MBT repeats) at 2.5 A resolution by X-ray crystallography. The crystal structure of MBTD1 reveals its similarity to another four-MBT-repeat protein L3MBTL2, which binds lower methylated lysine histones. Fluorescence polarization experiments confirmed that MBTD1 preferentially binds mono- and di-methyllysine histone peptides, like L3MBTL1 and L3MBTL2. All known MBT-peptide complex structures characterized to date do not exhibit strong histone peptide sequence selectivity, and use a "cavity insertion recognition mode" to recognize the methylated lysine with the deeply buried methyl-lysine forming extensive interactions with the protein while the peptide residues flanking methyl-lysine forming very few contacts . Nevertheless, our mutagenesis data based on L3MBTL1 suggested that the histone peptides could not bind to MBT repeats in any orientation. CONCLUSIONS:The four MBT repeats in MBTD1 exhibits an asymmetric rhomboid architecture. Like other MBT repeat proteins characterized so far, MBTD1 binds mono- or dimethylated lysine histones through one of its four MBT repeats utilizing a semi-aromatic cage. ENHANCED VERSION:This article can also be viewed as an enhanced version in which the text of the article is integrated with interactive 3D representations and animated transitions. Please note that a web plugin is required to access this enhanced functionality. Instructions for the installation and use of the web plugin are available in Text S1.
Hemp, an mbt domain-containing protein, plays essential roles in hematopoietic stem cell function and skeletal formation.
Honda Hiroaki,Takubo Keiyo,Oda Hideaki,Kosaki Kenjiro,Tazaki Tatsuya,Yamasaki Norimasa,Miyazaki Kazuko,Moore Kateri A,Honda Zen-ichiro,Suda Toshio,Lemischka Ihor R
Proceedings of the National Academy of Sciences of the United States of America
To clarify the molecular pathways governing hematopoietic stem cell (HSC) development, we screened a fetal liver (FL) HSC cDNA library and identified a unique gene, hematopoietic expressed mammalian polycomb (hemp), encoding a protein with a zinc-finger domain and four malignant brain tumor (mbt) repeats. To investigate its biological role, we generated mice lacking Hemp (hemp(-/-)). Hemp(-/-) mice exhibited a variety of skeletal malformations and died soon after birth. In the FL, hemp was preferentially expressed in the HSC and early progenitor cell fractions, and analyses of fetal hematopoiesis revealed that the number of FL mononuclear cells, including HSCs, was reduced markedly in hemp(-/-) embryos, especially during early development. In addition, colony-forming and competitive repopulation assays demonstrated that the proliferative and reconstitution abilities of hemp(-/-) FL HSCs were significantly impaired. Microarray analysis revealed alterations in the expression levels of several genes implicated in hematopoietic development and differentiation in hemp(-/-) FL HSCs. These results demonstrate that Hemp, an mbt-containing protein, plays essential roles in HSC function and skeletal formation. It is also hypothesized that Hemp might be involved in certain congenital diseases, such as Klippel-Feil anomaly.
H3K9me2/3 binding of the MBT domain protein LIN-61 is essential for Caenorhabditis elegans vulva development.
Koester-Eiserfunke Nora,Fischle Wolfgang
MBT domain proteins are involved in developmental processes and tumorigenesis. In vitro binding and mutagenesis studies have shown that individual MBT domains within clustered MBT repeat regions bind mono- and dimethylated histone lysine residues with little to no sequence specificity but discriminate against the tri- and unmethylated states. However, the exact function of promiscuous histone methyl-lysine binding in the biology of MBT domain proteins has not been elucidated. Here, we show that the Caenorhabditis elegans four MBT domain protein LIN-61, in contrast to other MBT repeat factors, specifically interacts with histone H3 when methylated on lysine 9, displaying a strong preference for di- and trimethylated states (H3K9me2/3). Although the fourth MBT repeat is implicated in this interaction, H3K9me2/3 binding minimally requires MBT repeats two to four. Further, mutagenesis of residues conserved with other methyl-lysine binding MBT regions in the fourth MBT repeat does not abolish interaction, implicating a distinct binding mode. In vivo, H3K9me2/3 interaction of LIN-61 is required for C. elegans vulva development within the synMuvB pathway. Mutant LIN-61 proteins deficient in H3K9me2/3 binding fail to rescue lin-61 synMuvB function. Also, previously identified point mutant synMuvB alleles are deficient in H3K9me2/3 interaction although these target residues that are outside of the fourth MBT repeat. Interestingly, lin-61 genetically interacts with two other synMuvB genes, hpl-2, an HP1 homologous H3K9me2/3 binding factor, and met-2, a SETDB1 homologous H3K9 methyl transferase (H3K9MT), in determining C. elegans vulva development and fertility. Besides identifying the first sequence specific and di-/trimethylation binding MBT domain protein, our studies imply complex multi-domain regulation of ligand interaction of MBT domains. Our results also introduce a mechanistic link between LIN-61 function and biology, and they establish interplay of the H3K9me2/3 binding proteins, LIN-61 and HPL-2, as well as the H3K9MT MET-2 in distinct developmental pathways.
Histone recognition by human malignant brain tumor domains.
Nady Nataliya,Krichevsky Liubov,Zhong Nan,Duan Shili,Tempel Wolfram,Amaya Maria F,Ravichandran Mani,Arrowsmith Cheryl H
Journal of molecular biology
Histone methylation has emerged as an important covalent modification involved in a variety of biological processes, especially regulation of transcription and chromatin dynamics. Lysine methylation is found in three distinct states (monomethylation, dimethylation and trimethylation), which are recognized by specific protein domains. The malignant brain tumor (MBT) domain is one such module found in several chromatin regulatory complexes including Polycomb repressive complex 1. Here, we present a comprehensive characterization of the human MBT family with emphasis on histone binding specificity. SPOT-blot peptide arrays were used to screen for the methyllysine-containing histone peptides that bind to MBT domains found in nine human proteins. Selected interactions were quantified using fluorescence polarization assays. We show that all MBT proteins recognize only monomethyllysine and/or dimethyllysine marks and provide evidence that some MBT domains recognize a defined consensus sequence while others bind in a promiscuous, non-sequence-specific manner. Furthermore, using structure-based mutants, we identify a triad of residues in the methyllysine binding pocket that imparts discrimination between monomethyllysine and dimethyllysine. This study represents a comprehensive analysis of MBT substrate specificity, establishing a foundation for the rational design of selective MBT domain inhibitors that may enable elucidation of their role in human biology and disease.
The histone H4 lysine 20 monomethyl mark, set by PR-Set7 and stabilized by L(3)mbt, is necessary for proper interphase chromatin organization.
Sakaguchi Ayako,Joyce Eric,Aoki Tsutomu,Schedl Paul,Steward Ruth
Drosophila PR-Set7 or SET8 is a histone methyltransferase that specifically monomethylates histone H4 lysine 20 (H4K20). L(3)MBT has been identified as a reader of methylated H4K20. It contains several conserved domains including three MBT repeats binding mono- and dimethylated H4K20 peptides. We find that the depletion of PR-Set7 blocks de novo H4K20me1 resulting in the immediate activation of the DNA damage checkpoint, an increase in the size of interphase nuclei, and drastic reduction of cell viability. L(3)mbt on the other hand stabilizes the monomethyl mark, as L(3)mbt-depleted S2 cells show a reduction of more than 60% of bulk monomethylated H4K20 (H4K20me1) while viability is barely affected. Ploidy and basic chromatin structure show only small changes in PR-Set7-depleted cells, but higher order interphase chromatin organization is significantly affected presumably resulting in the activation of the DNA damage checkpoint. In the absence of any other known functions of PR-Set7, the setting of the de novo monomethyl mark appears essential for cell viability in the presence or absence of the DNA damage checkpoint, but once newly assembled chromatin is established the monomethyl mark, protected by L(3)mbt, is dispensable.
Regulation of DU145 prostate cancer cell growth by Scm-like with four mbt domains 2.
Lee Kwanghyun,Na Wonho,Maeng Je-Heon,Wu Hongjin,Ju Bong-Gun
Journal of biosciences
Mammalian SFMBTs have been considered to be polycomb group repressors. However, molecular mechanisms underlying mammalian SFMBTs-mediated gene regulation and their biological function have not been characterized. In the present study, we identified YY1 and methylated histones as interacting proteins of human SFMBT2. We also found that human SFMBT2 binds preferentially to methylated histone H3 and H4 that are associated with transcriptional repression. Using DU145 prostate cancer cells as a model, we showed that SFMBT2 has a transcriptional repression activity on HOXB13 gene expression. In addition, occupancy of SFMBT2 coincided with enrichment of diand tri-methylated H3K9 and H4K20 as well as tri-methylated H3K27 at the HOXB13 gene promoter. When SFMBT2 was depleted by siRNA in DU145 prostate cancer cells, significant up-regulation of HOXB13 gene expression and decreased cell growth were observed. Collectively, our findings indicate that human SFMBT2 may regulate cell growth via epigenetic regulation of HOXB13 gene expression in DU145 prostate cancer cells.
A role for the malignant brain tumour (MBT) domain protein LIN-61 in DNA double-strand break repair by homologous recombination.
Johnson Nicholas M,Lemmens Bennie B L G,Tijsterman Marcel
Malignant brain tumour (MBT) domain proteins are transcriptional repressors that function within Polycomb complexes. Some MBT genes are tumour suppressors, but how they prevent tumourigenesis is unknown. The Caenorhabditis elegans MBT protein LIN-61 is a member of the synMuvB chromatin-remodelling proteins that control vulval development. Here we report a new role for LIN-61: it protects the genome by promoting homologous recombination (HR) for the repair of DNA double-strand breaks (DSBs). lin-61 mutants manifest numerous problems associated with defective HR in germ and somatic cells but remain proficient in meiotic recombination. They are hypersensitive to ionizing radiation and interstrand crosslinks but not UV light. Using a novel reporter system that monitors repair of a defined DSB in C. elegans somatic cells, we show that LIN-61 contributes to HR. The involvement of this MBT protein in HR raises the possibility that MBT-deficient tumours may also have defective DSB repair.
The malignant brain tumor (MBT) domain protein SFMBT1 is an integral histone reader subunit of the LSD1 demethylase complex for chromatin association and epithelial-to-mesenchymal transition.
Tang Ming,Shen Huangxuan,Jin Yue,Lin Tong,Cai Qingsong,Pinard Melissa A,Biswas Shyamasri,Tran Quyen,Li Guangyao,Shenoy Anitha K,Tongdee Emily,Lin Shuibin,Gu Yumei,Law Brian K,Zhou Lei,Mckenna Robert,Wu Lizi,Lu Jianrong
The Journal of biological chemistry
Chromatin readers decipher the functional readouts of histone modifications by recruiting specific effector complexes for subsequent epigenetic reprogramming. The LSD1 (also known as KDM1A) histone demethylase complex modifies chromatin and represses transcription in part by catalyzing demethylation of dimethylated histone H3 lysine 4 (H3K4me2), a mark for active transcription. However, none of its currently known subunits recognizes methylated histones. The Snai1 family transcription factors are central drivers of epithelial-to-mesenchymal transition (EMT) by which epithelial cells acquire enhanced invasiveness. Snai1-mediated transcriptional repression of epithelial genes depends on its recruitment of the LSD1 complex and ensuing demethylation of H3K4me2 at its target genes. Through biochemical purification, we identified the MBT domain-containing protein SFMBT1 as a novel component of the LSD1 complex associated with Snai1. Unlike other mammalian MBT domain proteins characterized to date that selectively recognize mono- and dimethylated lysines, SFMBT1 binds di- and trimethyl H3K4, both of which are enriched at active promoters. We show that SFMBT1 is essential for Snai1-dependent recruitment of LSD1 to chromatin, demethylation of H3K4me2, transcriptional repression of epithelial markers, and induction of EMT by TGFβ. Carcinogenic metal nickel is a widespread environmental and occupational pollutant. Nickel alters gene expression and induces EMT. We demonstrate the nickel-initiated effects are dependent on LSD1-SFMBT1-mediated chromatin modification. Furthermore, in human cancer, expression of SFMBT1 is associated with mesenchymal markers and unfavorable prognosis. These results highlight a critical role of SFMBT1 in epigenetic regulation, EMT, and cancer.
Chromatin regulation: how complex does it get?
Meier Karin,Brehm Alexander
Gene transcription is tightly regulated at different levels to ensure that the transcriptome of the cell is appropriate for developmental stage and cell type. The chromatin state in which a gene is embedded determines its expression level to a large extent. Activation or repression of transcription is typically accomplished by the recruitment of chromatin-associated multisubunit protein complexes that combine several molecular tools, such as histone-binding and chromatin-modifying activities. Recent biochemical purifications of such complexes have revealed a substantial diversity. On the one hand, complexes that were thought to be unique have been revealed to be part of large complex families. On the other hand, protein subunits that were thought to only exist in separate complexes have been shown to coexist in novel assemblies. In this review we discuss our current knowledge of repressor complexes that contain MBT domain proteins and/or the CoREST co-repressor and use them as a paradigm to illustrate the unexpected heterogeneity and tool sharing of chromatin regulating protein complexes. These recent insights also challenge the ways we define and think about protein complexes in general.
The L3MBTL3 Methyl-Lysine Reader Domain Functions As a Dimer.
Baughman Brandi M,Pattenden Samantha G,Norris Jacqueline L,James Lindsey I,Frye Stephen V
ACS chemical biology
L3MBTL3 recognizes mono- and dimethylated lysine residues on histone tails. The recently reported X-ray cocrystal structures of the chemical probe UNC1215 and inhibitor UNC2533 bound to the methyl-lysine reading MBT domains of L3MBTL3 demonstrate a unique and flexible 2:2 dimer mode of recognition. In this study, we describe our in vitro analysis of L3MBTL3 dimerization via its MBT domains and additionally show that this dimerization occurs within a cellular context in the absence of small molecule ligands. Furthermore, mutations to the first and second MBT domains abrogated L3MBTL3 dimerization both in vitro and in cells. These observations are consistent with the hypothesis that L3MBTL3 engages methylated histone tails as a dimer while carrying out its normal function and provides an explanation for the presence of repeated MBT domains within L3MBTL3.
Plant homologs of mammalian MBT-domain protein-regulated KDM1 histone lysine demethylases do not interact with plant Tudor/PWWP/MBT-domain proteins.
Sadiq Irfan,Keren Ido,Citovsky Vitaly
Biochemical and biophysical research communications
Histone lysine demethylases of the LSD1/KDM1 family play important roles in epigenetic regulation of eukaryotic chromatin, and they are conserved between plants and animals. Mammalian LSD1 is thought to be targeted to its substrates, i.e., methylated histones, by an MBT-domain protein SFMBT1 that represents a component of the LSD1-based repressor complex and binds methylated histones. Because MBT-domain proteins are conserved between different organisms, from animals to plants, we examined whether the KDM1-type histone lysine demethylases KDM1C and FLD of Arabidopsis interact with the Arabidopsis Tudor/PWWP/MBT-domain SFMBT1-like proteins SL1, SL2, SL3, and SL4. No such interaction was detected using the bimolecular fluorescence complementation assay in living plant cells. Thus, plants most likely direct their KDM1 chromatin-modifying enzymes to methylated histones of the target chromatin by a mechanism different from that employed by the mammalian cells.
SFMBT2 (Scm-like with four mbt domains 2) negatively regulates cell migration and invasion in prostate cancer cells.
Gwak Jungsug,Shin Jee Yoon,Lee Kwanghyun,Hong Soon Ki,Oh Sangtaek,Goh Sung-Ho,Kim Won Sun,Ju Bong Gun
Metastatic prostate cancer is the leading cause of morbidity and mortality in men. In this study, we found that expression level of SFMBT2 is altered during prostate cancer progression and has been associated with the migration and invasion of prostate cancer cells. The expression level of SFMBT2 is high in poorly metastatic prostate cancer cells compared to highly metastatic prostate cancer cells. We also found that SFMBT2 knockdown elevates MMP-2, MMP-3, MMP-9, and MMP-26 expression, leading to increased cell migration and invasion in LNCaP and VCaP cells. SFMBT2 interacts with YY1, RNF2, N-CoR and HDAC1/3, as well as repressive histone marks such as H3K9me2, H4K20me2, and H2AK119Ub which are associated with transcriptional repression. In addition, SFMBT2 knockdown decreased KAI1 gene expression through up-regulation of N-CoR gene expression. Expression of SFMBT2 in prostate cancer was strongly associated with clinicopathological features. Patients having higher Gleason score (≥ 8) had substantially lower SFMBT2 expression than patients with lower Gleason score. Moreover, tail vein or intraprostatic injection of SFMBT2 knockdown LNCaP cells induced metastasis. Taken together, our findings suggest that regulation of SFMBT2 may provide a new therapeutic strategy to control prostate cancer metastasis as well as being a potential biomarker of metastatic prostate cancer.
Circular RNA SFMBT2 Inhibits the Proliferation and Metastasis of Glioma Cells Through Mir-182-5p/Mtss1 Pathway.
Zhang Shoudan,Qin Wanxiang,Yang Shuo,Guan Ning,Sui Xin,Guo Wenshi
Technology in cancer research & treatment
Glioma is a common type of tumor in human central nervous system, and it is characterized with high mobility and mortality. The prognosis of patients with advanced glioma remains poor. Thus, it is necessary to develop novel therapeutic approaches for the treatment of this disease. Circular RNAs are a group of noncoding RNAs which have been detected in eukaryotic cells. They are tissue-specific and characterized with a more stable structure compared with linear RNAs. Recently, studies have revealed that certain circular RNAs are involved in biological processes such as gene regulation; however, the functions of most circular RNAs remain unknown and require further investigation. Furthermore, circular RNAs can act as "sponges" of its target microRNA, consequently suppressing their activity. Additionally, impaired expression of circular RNAs is reported in different diseases including cancer. In our study, low expression of circular RNA Scm like with 4 Mbt domains 2 was detected in glioma samples. Furthermore, reduced circRNA Scm like with 4 Mbt domains 2 expression was observed in human glioma cell lines compared to normal astrocyte cells. Additionally, overexpression of circRNA Scm like with 4 Mbt domains 2 suppressed the growth and metastasis of glioma cells . Moreover, microRNA-182-5p could be a downstream molecule of circRNA Scm like with 4 Mbt domains 2. The influenced of microRNA-182-5p-induced proliferation, migration, and invasion of glioma cells could be abrogated by overexpressed circRNA Scm like with 4 Mbt domains 2. In addition, metastasis suppressor 1 was predicted as a novel target of microRNA-182-5p, and its expression was restored by circRNA Scm like with 4 Mbt domains 2. In summary, our findings provided novel insight into the roles of circRNA Scm like with 4 Mbt domains 2 in glioma. More importantly, circRNA Scm like with 4 Mbt domains 2/microRNA-182-5p/metastasis suppressor 1 axis could be a putative therapeutic target for the treatment of patients with glioma.
Small Molecules Targeting the Specific Domains of Histone-Mark Readers in Cancer Therapy.
Zhu Huihui,Wei Tao,Cai Yong,Jin Jingji
Molecules (Basel, Switzerland)
Epigenetic modifications (or epigenetic tags) on DNA and histones not only alter the chromatin structure, but also provide a recognition platform for subsequent protein recruitment and enable them to acquire executive instructions to carry out specific intracellular biological processes. In cells, different epigenetic-tags on DNA and histones are often recognized by the specific domains in proteins (readers), such as bromodomain (BRD), chromodomain (CHD), plant homeodomain (PHD), Tudor domain, Pro-Trp-Trp-Pro (PWWP) domain and malignant brain tumor (MBT) domain. Recent accumulating data reveal that abnormal intracellular histone modifications (histone marks) caused by tumors can be modulated by small molecule-mediated changes in the activity of the above domains, suggesting that small molecules targeting histone-mark reader domains may be the trend of new anticancer drug development. Here, we summarize the protein domains involved in histone-mark recognition, and introduce recent research findings about small molecules targeting histone-mark readers in cancer therapy.
SSRP1-mediated histone H1 eviction promotes replication origin assembly and accelerated development.
Falbo Lucia,Raspelli Erica,Romeo Francesco,Fiorani Simona,Pezzimenti Federica,Casagrande Francesca,Costa Ilaria,Parazzoli Dario,Costanzo Vincenzo
In several metazoans, the number of active replication origins in embryonic nuclei is higher than in somatic ones, ensuring rapid genome duplication during synchronous embryonic cell divisions. High replication origin density can be restored by somatic nuclear reprogramming. However, mechanisms underlying high replication origin density formation coupled to rapid cell cycles are poorly understood. Here, using Xenopus laevis, we show that SSRP1 stimulates replication origin assembly on somatic chromatin by promoting eviction of histone H1 through its N-terminal domain. Histone H1 removal derepresses ORC and MCM chromatin binding, allowing efficient replication origin assembly. SSRP1 protein decays at mid-blastula transition (MBT) when asynchronous somatic cell cycles start. Increasing levels of SSRP1 delay MBT and, surprisingly, accelerate post-MBT cell cycle speed and embryo development. These findings identify a major epigenetic mechanism regulating DNA replication and directly linking replication origin assembly, cell cycle duration and embryo development in vertebrates.
Argonaute NRDE-3 and MBT domain protein LIN-61 redundantly recruit an H3K9me3 HMT to prevent embryonic lethality and transposon expression.
Padeken Jan,Methot Stephen,Zeller Peter,Delaney Colin E,Kalck Veronique,Gasser Susan M
Genes & development
The establishment and maintenance of chromatin domains shape the epigenetic memory of a cell, with the methylation of histone H3 lysine 9 (H3K9me) defining transcriptionally silent heterochromatin. We show here that the SET-25 (SUV39/G9a) histone methyltransferase (HMT), which catalyzes H3K9me1, me2 and me3, can establish repressed chromatin domains de novo unlike the SETDB1 homolog MET-2. Thus, SET-25 is needed to silence novel insertions of RNA or DNA transposons, and repress tissue-specific genes de novo during development. We identify two partially redundant pathways that recruit SET-25 to its targets. One pathway requires LIN-61 (L3MBTL2), which uses its four MBT domains to bind the H3K9me2 deposited by MET-2. The second pathway functions independently of MET-2 and involves the somatic Argonaute NRDE-3 and small RNAs. This pathway targets primarily highly conserved RNA and DNA transposons. These redundant SET-25 targeting pathways (MET-2-LIN-61-SET-25 and NRDE-3-SET-25) ensure repression of intact transposons and de novo insertions, while MET-2 can act alone to repress simple and satellite repeats. Removal of both pathways in the double mutant leads to the loss of somatic H3K9me2 and me3 and the synergistic derepression of transposons in embryos, strongly elevating embryonic lethality.