Histone Methyltransferase SETDB1 Research Articles

High‐frequency p16INK4A promoter methylation is associated with histone methyltransferase SETDB1 expression in sporadic cutaneous melanoma

Epigenetic mechanisms participate in melanoma development and progression. The effect of histone modifications and their catalysing enzymes over euchromatic promoter DNA methylation in melanoma remains unclear. This study investigated the potential association of p16(INK) (4A) promoter methylation with histone methyltransferase SETDB1 expression in Greek patients with sporadic melanoma and their correlation with clinicopathological characteristics. Promoter methylation was detected by methylation-specific PCR in 100 peripheral blood samples and 58 melanoma tissues from the same patients. Cell proliferation (Ki-67 index), p16(INK) (4A) and SETDB1 expression were evaluated by immunohistochemistry. High-frequency promoter methylation (25.86%) was observed in tissue samples and correlated with increased cell proliferation (P=0.0514). p16(INK) (4A) promoter methylation was higher in vertical growth-phase (60%) melanomas than in radial (40%, P=0.063) and those displaying epidermal involvement (P=0.046). Importantly, p16(INK) (4A) methylation correlated with increased melanoma thickness according to Breslow index (P=0.0495) and marginally with increased Clark level (I/II vs III/IV/V, P=0.070). Low (1-30%) p16(INK) (4A) expression was detected at the majority (19 of 54) of melanoma cases (35.19%), being marginally correlated with tumor lymphocytic infiltration (P=0.078). SETDB1 nuclear immunoreactivity was observed in 47 of 57 (82.46%) cases, whereas 27 of 57 (47.37%) showed cytoplasmic immunoexpression. Cytoplasmic SETDB1 expression correlated with higher frequency of p16(INK) (4A) methylation and p16(INK) (4A) expression (P=0.033, P=0.011, respectively). Increased nuclear SETDB1 levels were associated with higher mitotic count (0-5/mm(2) vs >5/mm(2) , P=0.0869), advanced Clark level (III-V, P=0.0380), epidermal involvement (P=0.0331) and the non-chronic sun exposure-associated melanoma type (P=0.0664). Our data demonstrate for the first time the association of histone methyltransferase SETDB1 with frequent methylation of the euchromatic p16(INK) (4A) promoter and several prognostic parameters in melanomas.

Regulation of DNA methylation turnover at LTR retrotransposons and imprinted loci by the histone methyltransferase Setdb1.

During mammalian development, DNA methylation patterns need to be reset in primordial germ cells (PGCs) and preimplantation embryos. However, many LTR retrotransposons and imprinted genes are impervious to such global epigenetic reprogramming via hitherto undefined mechanisms. Here, we report that a subset of such genomic regions are resistant to widespread erasure of DNA methylation in mouse embryonic stem cells (mESCs) lacking the de novo DNA methyltransferases (Dnmts) Dnmt3a and Dnmt3b. Intriguingly, these loci are enriched for H3K9me3 in mESCs, implicating this mark in DNA methylation homeostasis. Indeed, deletion of the H3K9 methyltransferase SET domain bifurcated 1 (Setdb1) results in reduced H3K9me3 and DNA methylation levels at specific loci, concomitant with increased 5-hydroxymethylation (5hmC) and ten-eleven translocation 1 binding. Taken together, these data reveal that Setdb1 promotes the persistence of DNA methylation in mESCs, likely reflecting one mechanism by which DNA methylation is maintained at LTR retrotransposons and imprinted genes during developmental stages when DNA methylation is reprogrammed.

SETDB1 in Early Embryos and Embryonic Stem Cells

The histone methyltransferase SETDB1 contributes to the silencing of local chromatin and the target specificity appears to be determined through various proteins that SETDB1 interacts with. This fundamental function endows SETDB1 with specialized roles in embryonic cells. Keeping the genomic and transcriptomic integrity via proviral silencing and maintaining the pluripotency by repressing the differentiation-associated genes have been demonstrated as the roles of SETDB1 in embryonic stem cells. In early developing embryos, SETDB1 exhibits characteristic nuclear mobilizations that might account for its pleiotropic roles in these rapidly changing cells as well. Early lethality of SETDB1-null embryos, along with other immunolocalization findings, suggests that SETDB1 is necessary for reprogramming and preparing the genomes of zygotes and pluripotent cells for the post-implantation developmental program.

Transcriptional Derepression of the ERVWE1 Locus following Influenza A Virus Infection

Syncytin-1, a fusogenic protein encoded by a human endogenous retrovirus of the W family (HERV-W) element (ERVWE1), is expressed in the syncytiotrophoblast layer of the placenta. This locus is transcriptionally repressed in adult tissues through promoter CpG methylation and suppressive histone modifications. Whereas syncytin-1 appears to be crucial for the development and functioning of the human placenta, its ectopic expression has been associated with pathological conditions, such as multiple sclerosis and schizophrenia. We previously reported on the transactivation of HERV-W elements, including ERVWE1, during influenza A/WSN/33 virus infection in a range of human cell lines. Here we report the results of quantitative PCR analyses of transcripts encoding syncytin-1 in both cell lines and primary fibroblast cells. We observed that spliced ERVWE1 transcripts and those encoding the transcription factor glial cells missing 1 (GCM1), acting as an enhancer element upstream of ERVWE1, are prominently upregulated in response to influenza A/WSN/33 virus infection in nonplacental cells. Knockdown of GCM1 by small interfering RNA followed by infection suppressed the transactivation of ERVWE1. While the infection had no influence on CpG methylation in the ERVWE1 promoter, chromatin immunoprecipitation assays detected decreased H3K9 trimethylation (H3K9me3) and histone methyltransferase SETDB1 levels along with influenza virus proteins associated with ERVWE1 and other HERV-W loci in infected CCF-STTG1 cells. The present findings suggest that an exogenous influenza virus infection can transactivate ERVWE1 by increasing transcription of GCM1 and reducing H3K9me3 in this region and in other regions harboring HERV-W elements. Syncytin-1, a protein encoded by the env gene in the HERV-W locus ERVWE1, appears to be crucial for the development and functioning of the human placenta and is transcriptionally repressed in nonplacental tissues. Nevertheless, its ectopic expression has been associated with pathological conditions, such as multiple sclerosis and schizophrenia. In the present paper, we report findings suggesting that an exogenous influenza A virus infection can transactivate ERVWE1 by increasing the transcription of GCM1 and reducing the repressive histone mark H3K9me3 in this region and in other regions harboring HERV-W elements. These observations have implications of potential relevance for viral pathogenesis and for conditions associated with the aberrant transcription of HERV-W loci.

Histone methyltransferase SETDB1 is required for prostate cancer cell proliferation, migration and invasion

SETDB1 has been established as an oncogene in a number of human carcinomas. The present study was to evaluate the expression of SETDB1 in prostate cancer (PCa) tissues and cells and to preliminarily investigate the role of SETDB1 in prostate tumorigenesis in vitro. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) and immunohistochemistry (IHC) were used to detect the expression of SETDB1 in PCa tissues, adjacent normal tissues, benign prostatic hyperplasia (BPH) tissues, PCa cell lines and normal prostate epithelial cells. The results suggested that SETDB1 was upregulated in human PCa tissues compared with normal tissues at the mRNA and protein levels. The role of SETDB1 in proliferation was analyzed with cell counting kit-8, colony-forming efficiency and flow cytometry assays. The results indicated that downregulation of SETDB1 by siRNA inhibited PCa cell growth, and induced G0/G1 cell cycle arrest. The PCa cell migration and invasion decreased by silcencing SETDB1 which were assessed by using in vitro scratch and transwell invasion assay respectively. Our data suggested that SETDB1 is overexpressed in human PCa. Silencing SETDB1 inhibited PCa cell proliferation, migration and invasion.

A Conditional Zebrafish MITF Mutation Reveals MITF Levels Are Critical for Melanoma Promotion vs. Regression In Vivo

The microphthalmia-associated transcription factor (MITF) is the “master melanocyte transcription factor” with a complex role in melanoma. MITF protein levels vary between and within clinical specimens, and amplifications and gain- and loss-of-function mutations have been identified in melanoma. How MITF functions in melanoma development and the effects of targeting MITF in vivo are unknown because MITF levels have not been directly tested in a genetic animal model. Here, we use a temperature-sensitive mitf zebrafish mutant to conditionally control endogenous MITF activity. We show that low levels of endogenous MITF activity are oncogenic with BRAFV600E to promote melanoma that reflects the pathology of the human disease. Remarkably, abrogating MITF activity in BRAFV600Emitf melanoma leads to dramatic tumor regression marked by melanophage infiltration and increased apoptosis. These studies are significant because they show that targeting MITF activity is a potent antitumor mechanism, but also show that caution is required because low levels of wild-type MITF activity are oncogenic.

Deletion of the histone methyltransferase Setdb1 during hematopoiesis results in hematopoietic stem cell failure and abrogates B cell development

Hematopoietic stem cells (HSCs) give rise to all blood lineages. The fact that transcription factors (TFs) determine gene expression profiles during hematopoiesis has been broadly demonstrated. However, we still lack a detailed understanding about how the chromatin context influences the ability of TFs in activating or repressing genes. Because chromatin modifications might play significant roles for TF binding and function, we investigate the importance of H3K9me3, a repressive chromatin mark established by the histone methyltransferase Setdb1.

Gene amplification of the histone methyltransferase SETDB1 contributes to human lung tumorigenesis

Disruption of the histone modification patterns is one of the most common features of human tumors. However, few genetic alterations in the histone modifier genes have been described in tumorigenesis. Herein we show that the histone methyltransferase SETDB1 undergoes gene amplification in non-small and small lung cancer cell lines and primary tumors. The existence of additional copies of the SETDB1 gene in these transformed cells is associated with higher levels of the corresponding mRNA and protein. From a functional standpoint, the depletion of SETDB1 expression in amplified cells reduces cancer growth in cell culture and nude mice models, whereas its overexpression increases the tumor invasiveness. The increased gene dosage of SETDB1 is also associated with enhanced sensitivity to the growth inhibitory effect mediated by the SETDB1-interfering drug mithramycin. Overall, the findings identify SETDB1 as a bona fide oncogene undergoing gene amplification-associated activation in lung cancer and suggest its potential for new therapeutic strategies.

Methionine Adenosyltransferase II-dependent Histone H3K9 Methylation at the COX-2 Gene Locus

MATII biosynthesizes AdoMet, which supplies methyl group for methylation of molecules, including histone. MATII interacts with histone methyltransferase SETDB1 and inhibits COX-2 gene expression. AdoMet synthesis and histone methylation are coupled on chromatin by a physical interaction of MATII and SETDB1 at the MafK target genes. MATII may be important for both gene-specific and epigenome-wide regulation of histone methylation. Methionine adenosyltransferase (MAT) synthesizes S-adenosylmethionine (AdoMet), which is utilized as a methyl donor in transmethylation reactions involving histones. MATIIα, a MAT isozyme, serves as a transcriptional corepressor in the oxidative stress response and forms the AdoMet-integrating transcription regulation module, affecting histone methyltransferase activities. However, the identities of genes regulated by MATIIα or its associated methyltransferases are unclear. We show that MATIIα represses the expression of cyclooxygenase 2 (COX-2), encoded by Ptgs2, by specifically interacting with histone H3K9 methyltransferase SETDB1, thereby promoting the trimethylation of H3K9 at the COX-2 locus. We discuss both gene-specific and epigenome-wide functions of MATIIα.

HP1a, Su(var)3-9, SETDB1 and POF stimulate or repress gene expression depending on genomic position, gene length and expression pattern in Drosophila melanogaster

Heterochromatin protein 1a (HP1a) is a chromatin-associated protein important for the formation and maintenance of heterochromatin. In Drosophila, the two histone methyltransferases SETDB1 and Su(var)3-9 mediate H3K9 methylation marks that initiates the establishment and spreading of HP1a-enriched chromatin. Although HP1a is generally regarded as a factor that represses gene transcription, several reports have linked HP1a binding to active genes, and in some cases, it has been shown to stimulate transcriptional activity. To clarify the function of HP1a in transcription regulation and its association with Su(var)3-9, SETDB1 and the chromosome 4-specific protein POF, we conducted genome-wide expression studies and combined the results with available binding data in Drosophila melanogaster. The results suggest that HP1a, SETDB1 and Su(var)3-9 repress genes on chromosome 4, where non-ubiquitously expressed genes are preferentially targeted, and stimulate genes in pericentromeric regions. Further, we showed that on chromosome 4, Su(var)3-9, SETDB1 and HP1a target the same genes. In addition, we found that transposons are repressed by HP1a and Su(var)3-9 and that the binding level and expression effects of HP1a are affected by gene length. Our results indicate that genes have adapted to be properly expressed in their local chromatin environment.

The histone methyltransferase SETDB1 is recurrently amplified in melanoma and accelerates its onset

The most common mutation in melanoma, BRAF(V600E), activates the BRAF serine/threonine kinase and causes excessive MAPK pathway activity1,2. BRAF(V600E)mutations are also present in benign melanocytic nevi3, highlighting the importance of additional genetic alterations in the genesis of malignant tumors. Such changes include recurrent copy number variations that result in the amplification of oncogenes4,5. For certain amplifications, the large number of genes in the interval has precluded an understanding of cooperating oncogenic events. Here, we have used a zebrafish melanoma model to test genes in a recurrently amplified region on chromosome 1 for the ability to cooperate with BRAF(V600E) and accelerate melanoma. SETDB1, an enzyme that methylates histone H3 on lysine 9 (H3K9), was found to significantly accelerate melanoma formation in the zebrafish. Chromatin immunoprecipitation coupled with massively parallel DNA sequencing (ChIP-Seq) and gene expression analyses revealed target genes, including Hox genes, that are transcriptionally dysregulated in response to elevated SETDB1. Our studies establish SETDB1 as an oncogene in melanoma and underscore the role of chromatin factors in regulating tumorigenesis.

ZNF274 Recruits the Histone Methyltransferase SETDB1 to the 3′ Ends of ZNF Genes

Only a small percentage of human transcription factors (e.g. those associated with a specific differentiation program) are expressed in a given cell type. Thus, cell fate is mainly determined by cell type-specific silencing of transcription factors that drive different cellular lineages. Several histone modifications have been associated with gene silencing, including H3K27me3 and H3K9me3. We have previously shown that genes for the two largest classes of mammalian transcription factors are marked by distinct histone modifications; homeobox genes are marked by H3K27me3 and zinc finger genes are marked by H3K9me3. Several histone methyltransferases (e.g. G9a and SETDB1) may be involved in mediating the H3K9me3 silencing mark. We have used ChIP-chip and ChIP-seq to demonstrate that SETDB1, but not G9a, is associated with regions of the genome enriched for H3K9me3. One current model is that SETDB1 is recruited to specific genomic locations via interaction with the corepressor TRIM28 (KAP1), which is in turn recruited to the genome via interaction with zinc finger transcription factors that contain a Kruppel-associated box (KRAB) domain. However, specific KRAB-ZNFs that recruit TRIM28 (KAP1) and SETDB1 to the genome have not been identified. We now show that ZNF274 (a KRAB-ZNF that contains 5 C2H2 zinc finger domains), can interact with KAP1 both in vivo and in vitro and, using ChIP-seq, we show that ZNF274 binding sites co-localize with SETDB1, KAP1, and H3K9me3 at the 3′ ends of zinc finger genes. Knockdown of ZNF274 with siRNAs reduced the levels of KAP1 and SETDB1 recruitment to the binding sites. These studies provide the first identification of a KRAB domain-containing ZNF that is involved in recruitment of the KAP1 and SETDB1 to specific regions of the human genome.

KAP1 controls endogenous retroviruses in embryonic stem cells

More than forty per cent of the mammalian genome is derived from retroelements, of which about one-quarter are endogenous retroviruses (ERVs). Some are still active, notably in mice the highly polymorphic early transposon (ETn)/MusD and intracisternal A-type particles (IAP). ERVs are transcriptionally silenced during early embryogenesis by histone and DNA methylation (and reviewed in ref. 7), although the initiators of this process, which is essential to protect genome integrity, remain largely unknown. KAP1 (KRAB-associated protein 1, also known as tripartite motif-containing protein 28, TRIM28) represses genes by recruiting the histone methyltransferase SETDB1, heterochromatin protein 1 (HP1) and the NuRD histone deacetylase complex, but few of its physiological targets are known. Two lines of evidence suggest that KAP1-mediated repression could contribute to the control of ERVs: first, KAP1 can trigger permanent gene silencing during early embryogenesis, and second, a KAP1 complex silences the retrovirus murine leukaemia virus in embryonic cells. Consistent with this hypothesis, here we show that KAP1 deletion leads to a marked upregulation of a range of ERVs, in particular IAP elements, in mouse embryonic stem (ES) cells and in early embryos. We further demonstrate that KAP1 acts synergistically with DNA methylation to silence IAP elements, and that it is enriched at the 5' untranslated region (5'UTR) of IAP genomes, where KAP1 deletion leads to the loss of histone 3 lysine 9 trimethylation (H3K9me3), a hallmark of KAP1-mediated repression. Correspondingly, IAP 5'UTR sequences can impose in cis KAP1-dependent repression on a heterologous promoter in ES cells. Our results establish that KAP1 controls endogenous retroelements during early embryonic development.

Wnt and PPARγ signaling in osteoblastogenesis and adipogenesis

Osteoblasts and adipocytes differentiate from a common pluripotent precursor, the mesenchymal stem cell (MSC). Studies have identified numerous transcription factors, and multiple extracellular and intracellular signaling pathways that regulate the closely linked processes of adipogenesis and osteoblastogenesis. Interestingly, inducers of differentiation along one lineage often inhibit differentiation along the other; for example, the transcription factor peroxisome proliferator-activated receptor gamma (PPARgamma) is a prime inducer of adipogenesis that inhibits osteoblastogenesis. The latest research has shown that inducers of osteoblastogenesis (such as bone morphogenetic protein 2 and Wnt ligands) use different mechanisms to suppress the transactivation function of PPARgamma during osteoblastogenesis from MSCs. Signaling via the canonical Wnt-beta-catenin pathway inhibits PPARgamma mRNA expression, whereas signaling via the noncanonical Wnt pathway results in activation of a histone methyltransferase SETDB1 that represses PPARgamma transactivation through histone H3K9 methylation of target genes. This article summarizes Wnt and PPARgamma signaling in MSCs and the crosstalk between these pathways, and speculates on future clinical application of this knowledge as the basis of novel approaches for regeneration therapy.

TIF1β/KAP-1 Is a Coactivator of the Orphan Nuclear Receptor NGFI-B/Nur77

In efforts to define mechanisms of transcriptional activation by the orphan nuclear receptor NGFI-B (Nur77), we identified TIF1beta by mass spectrometry within a nuclear protein complex containing NGFI-B. TIF1beta, also known as KAP-1 (KRAB domain-associated protein) or KRIP-1, acts as a transcriptional corepressor for many transcription factors, in particular for the Krüppel-associated box domain-containing zinc finger transcription factors. TIF1beta is also an intrinsic component of two chromatin remodeling and histone deacetylase complexes, the N-CoR1 and nucleosome remodeling and deacetylation complexes. In contrast to these activities, we report that TIF1beta is a coactivator of NGFI-B and that it is as potent as the SRC coactivators in this context. Using pull-down assays and immunoprecipitation, we showed that TIF1beta interacts directly with NGFI-B and with other Nur family members. NGFI-B is an important mediator of hypothalamic corticotropin-releasing hormone (CRH) activation of proopiomelanocortin (POMC) transcription, and TIF1beta enhances transcription mediated through the NGFI-B target, the Nur response element (NurRE). The NurRE binds Nur factor dimers and is responsive to signaling pathways. In keeping with the role of NGFI-B as mediator of CRH signaling, we found that TIF1beta is recruited to the POMC promoter following CRH stimulation and that TIF1beta potentiates CRH and protein kinase A signaling through the NurRE; it acts synergistically with the SRC2 coactivator. However, the actions of TIF1beta and SRC2 were mapped to different NGFI-B AF-1 subdomains. Taken together, these results indicate that TIF1beta is an important coactivator of NGFI-B-dependent transcription.

SUMO‐modified Sp3 represses transcription by provoking local heterochromatic gene silencing

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.

Esco2 is a novel corepressor that associates with various chromatin modifying enzymes

Accurate chromosome segregation during cell division requires that sister chromatids are kept together by cohesin complex until anaphase, when the chromatids separate and distribute to the two daughter cells. Esco2 is an acetyltransferase that is required for the establishment of sister chromatid cohesion during S phase. Here, we report that Esco2 interacts with several component proteins of the CoREST complex, including a transcription corepressor CoREST, histone demethlyase LSD1, HDAC1, HDAC2, BRAF35, and PHF21A. Esco2 also interacts with various histone methyltransferases Suv39h1, SETDB1 and G9a. Esco2 complex purified from HeLa nuclear extract possesses histone H3 K9 methylation activity and functions as a transcription repressor. Esco2 fused to Gal4 DNA binding domain represses transcription by increasing methylation of histone H3 K9 in the promoter region. These results suggest a novel function of Esco2 in transcription repression through modulation of the chromatin structure.

Inhibition of Transcription and DNA Replication by the Papillomavirus E8⁁E2C Protein Is Mediated by Interaction with Corepressor Molecules

Papillomavirus genomes replicate as nuclear plasmids at a low copy number in undifferentiated keratinocytes. Papillomaviruses encode the E1 and E2 proteins that bind to the origin of replication and are required for the activation of replication. In addition to E2, several papillomaviruses express an E8-E2C protein, which is generated by alternative splicing and functions as a transcriptional repressor and inhibitor of the E1/E2-dependent replication of the viral origin. Previous analyses suggested that the E8 domain functions as a transferable repression domain. In this report we present evidence that the E8 domain is responsible for the interaction with cellular corepressor molecules such as histone deacetylases, the histone methyltransferase SETDB1, and the TRIM28/KAP-1/TIF1beta/KRIP-1 protein. Whereas the interaction with histone deacetylases is involved only in transcriptional repression, the interaction with TRIM28/KAP-1/TIF1beta/KRIP-1 contributes to the inhibition of E1/E2-dependent replication. The corepressor TRIM28/KAP-1/TIF1beta/KRIP-1 has been described to be part of multicomponent complexes involved in transcriptional regulation and functions as a scaffold protein. Since neither histone deacetylases nor the histone methyltransferase SETDB1 appears to be involved in the inhibition of E1/E2-dependent replication, most likely the modification of non-histone proteins contributes to the replication repression activity of E8-E2C.

Fat or bone? A non-canonical decision

The transcription factor PPAR-γ determines whether mesenchymal stem cells differentiate into adipocytes or osteoblasts. The finding that non-canonical Wnt signalling represses PPAR-γ through assembly of a nuclear corepressor complex containing the histone methyltransferase SETDB1 provides the first insight into how extracellular signals can regulate chromatin modification.

Regulation of E2F1 Function by the Nuclear Corepressor KAP1

KAP1 is a nuclear corepressor with conserved domains for RING finger, B boxes, leucine zipper alpha helical coiled-coil region, plant homeo domain finger, and bromo domain. The plant homeo domain finger and bromo domain of KAP1 cooperatively function as a transcription repression domain by recruiting the histone deacetylase complex NuRD and histone H3 lysine 9-specific methyltransferase SETDB1. Here we report that KAP1 binds the E2F1 transcription factor in a retinoblastoma protein (pRb)-independent fashion and inhibits E2F1 activity. KAP1 stimulates formation of E2F1-HDAC1 complex and inhibits E2F1 acetylation. Ectopic expression of KAP1 represses E2F1 transcription and apoptosis functions independent of pRb. Depletion of endogenous KAP1 in pRb-deficient Saos2 cells by RNA interference increases E2F1 acetylation level, stimulates E2F1 transcriptional activity, and sensitizes apoptosis response to DNA damage. Therefore, KAP1 contributes to the negative regulation of E2F1 and may serve as a partial backup to prevent E2F1-mediated apoptosis in the absence of pRb.