Holocarboxylase synthetase synergizes with methyl CpG binding protein 2 and DNA methyltransferase 1 in the transcriptional repression of long-terminal repeats

Holocarboxylase synthetase (HLCS) is a chromatin protein that facilitates the creation of histone H3 lysine 9-methylation (H3K9me) gene repression marks through physical interactions with the histone methyltransferase EHMT-1. HLCS knockdown causes a depletion of H3K9me marks in mammalian cell cultures and severe phenotypes such as short lifespan and low stress resistance in Drosophila melanogaster. HLCS displays a punctuate distribution pattern in chromatin despite lacking a strong DNA-binding domain. Previous studies suggest that the binding of HLCS to chromatin depends on DNA methylation. We tested the hypothesis that HLCS interacts physically with the DNA methyltransferase DNMT1 and the methyl CpG binding protein MeCP2 to facilitate the binding of HLCS to chromatin, and that these interactions contribute toward the repression of long-terminal repeats (LTRs) by H3K9me marks. Co-immunoprecipitation and limited proteolysis assays provided evidence suggesting that HLCS interacts physically with both DNMT1 and MeCP2. The abundance of H3K9me marks was 207% greater in the LTR15 locus in HLCS overexpression human embryonic kidney HEK293 cells compared with controls. This gain in H3K9me was inversely linked with a 87% decrease in mRNA coding for LTRs. Effects of HLCS abundance on LTR expression were abolished when DNA methylation marks were erased by treating cells with 5-azacytidine. We conclude that interactions between DNA methylation and HLCS are crucial for mediating gene repression by H3K9me, thereby providing evidence for epigenetic synergies between the protein biotin ligase HLCS and dietary methyl donors.     

Histone deacetylation directs DNA methylation in survivin gene silencing

DNA methylation and histone acetylation are major epigenetic modifications in gene silencing. In our previous research, we found that the methylated oligonucleotide (SurKex) complementary to a region of promoter of survivin could induce DNA methylation in a site-specific manner leading to survivin silencing. Here, we further studied the role of histone acetylation in survivin silencing and the relationship between histone acetylation and DNA methylation.First we observed the levels of histone H4 and H4K16 acetylation that were decreased after SurKex treatment by using the chromatin immunoprecipitation (ChIP) assay. Next, we investigated the roles of histone acetylation and DNA methylation in survivin silencing after blockade of histone deacetylation with Trichostatin A (TSA). We assessed survivin mRNA expression by RT-PCR, measured survivin promoter methylation by bisulfite sequencing and examined the level of histone acetylation by the ChIP assay. The results showed that histone deacetylation blocked by TSA reversed the effects of SurKex on inhibiting the expression of survivin mRNA, inducing a site-specific methylation on survivin promoter and decreasing the level of histone acetylation. Finally, we examined the role of histone acetylation in the expression of DNA methyltransferase 1 (DNMT1) mRNA. The results showed that histone deacetylation blocked by TSA reversed the increasing effect of histone deacetylation on the expression of survivin mRNA. This study suggests that histone deacetylation guides SurKex-induced DNA methylation in survivin silencing possibly through increasing the expression of DNMT1 mRNA.     

Induced DNA demethylation can reshape chromatin topology at the IGF2-H19 locus

Choriocarcinomas are embryonal tumours with loss of imprinting and hypermethylation at the insulin-like growth factor 2 (IGF2)-H19 locus. The DNA methyltransferase inhibitor, 5-Aza-2′deoxycytidine (5-AzaCdR) is an approved epigenetic cancer therapy. However, it is not known to what extent 5-AzaCdR influences other epigenetic marks. In this study, we set out to determine whether 5-AzaCdR treatment can reprogram the epigenomic organization of the IGF2-H19 locus in a choriocarcinoma cancer cell line (JEG3). We found that localized DNA demethylation at the H19 imprinting control region (ICR) induced by 5-AzaCdR, reduced IGF2, increased H19 expression, increased CTCF and cohesin recruitment and changed histone modifications. Furthermore chromatin accessibility was increased locus-wide and chromatin looping topography was altered such that a CTCF site downstream of the H19 enhancers switched its association with the CTCF site upstream of the IGF2 promoters to associate with the ICR. We identified a stable chromatin looping domain, which forms independently of DNA methylation. This domain contains the IGF2 gene and is marked by a histone H3 lysine 27 trimethylation block between CTCF site upstream of the IGF2 promoters and the Centrally Conserved Domain upstream of the ICR. Together, these data provide new insights into the responsiveness of chromatin topography to DNA methylation changes.     

Enhanced targeted DNA methylation of the CMV and endogenous promoters with dCas9-DNMT3A3L entails distinct subsequent histone modification changes in CHO cells

With the emergence of new CRISPR/dCas9 tools that enable site specific modulation of DNA methylation and histone modifications, more detailed investigations of the contribution of epigenetic regulation to the precise phenotype of cells in culture, including recombinant production subclones, is now possible. These also allow a wide range of applications in metabolic engineering once the impact of such epigenetic modifications on the chromatin state is available.In this study, enhanced DNA methylation tools were targeted to a recombinant viral promoter (CMV), an endogenous promoter that is silenced in its native state in CHO cells, but had been reactivated previously (β-galactoside α-2,6-sialyltransferase 1) and an active endogenous promoter (α-1,6-fucosyltransferase), respectively. Comparative ChIP-analysis of histone modifications revealed a general loss of active promoter histone marks and the acquisition of distinct repressive heterochromatin marks after targeted methylation. On the other hand, targeted demethylation resulted in autologous acquisition of active promoter histone marks and loss of repressive heterochromatin marks. These data suggest that DNA methylation directs the removal or deposition of specific histone marks associated with either active, poised or silenced chromatin. Moreover, we show that de novo methylation of the CMV promoter results in reduced transgene expression in CHO cells. Although targeted DNA methylation is not efficient, the transgene is repressed, thus offering an explanation for seemingly conflicting reports about the source of CMV promoter instability in CHO cells.Importantly, modulation of epigenetic marks enables to nudge the cell into a specific gene expression pattern or phenotype, which is stabilized in the cell by autologous addition of further epigenetic marks. Such engineering strategies have the added advantage of being reversible and potentially tunable to not only turn on or off a targeted gene, but also to achieve the setting of a desirable expression level.     

Estrogen,through estrogen receptor 1, regulates histone modifications and chromatin remodeling during spermatogenesis in adult rats

Estrogen receptors (ESR1 and ESR2) play crucial roles in various processes during spermatogenesis. To elucidate individual roles of ESRs in male fertility, we developed in vivo selective ESR agonist administration models. Adult male rats treated with ESR1 and ESR2 agonist for 60 days show spermatogenic defects leading to reduced sperm counts and fertility. While studying epigenetic changes in the male germ line that could have affected fertility, we earlier observed a decrease in DNA methylation and its machinery upon ESR2 agonist treatment. Here, we explored the effects on histone modifications, which could contribute to decreased male fertility upon ESR agonist administration. ESR1 agonist treatment affected testicular levels of histone modifications associated with active and repressed chromatin states, along with heterochromatin marks. This was concomitant with deregulation of corresponding histone modifying enzymes in the testis. In addition, there was increased retention of histones along with protamine deficiency in the caudal spermatozoa after ESR1 agonist treatment. This could be due to the observed decrease in several chromatin remodeling proteins implicated in mediating histone-to-protamine exchange during spermiogenesis. The activating and repressing histone marks in spermatozoa, which play a critical role in early embryo development, were deregulated after both the ESR agonist treatments. Together, these epigenetic defects in the male germ line could affect the spermatozoa quality and lead to the observed decrease in fertility. Our results thus highlight the importance of ESRs in regulating different epigenetic processes during spermatogenesis, which are crucial for male fertility.     

UHRF1 Induces Methylation of the TXNIP Promoter and Down-Regulates Gene Expression in Cervical Cancer

DNA methylation, and consequent down-regulation, of tumour suppressor genes occurs in response to epigenetic stimuli during cancer development. Similarly, human oncoviruses, including human papillomavirus (HPV), up-regulate and augment DNA methyltransferase (DNMT) and histone deacetylase (HDAC) activities, thereby decreasing tumour suppressor genes (TSGs) expression. Ubiquitin-like containing PHD and RING finger domain 1 (UHRF1), an epigenetic regulator of DNA methylation, is overexpressed in HPV-induced cervical cancers. Here, we investigated the role of UHRF1 in cervical cancer by knocking down its expression in HeLa cells using lentiviral-encoded short hairpin (sh)RNA and performing cDNA microarrays. We detected significantly elevated expression of thioredoxin-interacting protein (TXNIP), a known TSG, in UHRF1-knockdown cells, and this gene is hypermethylated in cervical cancer tissue and cell lines, as indicated by whole-genome methylation analysis. Up-regulation of UHRF1 and decreased TXNIP were further detected in cervical cancer by western blot and immunohistochemistry and confirmed by Oncomine database analysis. Using chromatin immunoprecipitation, we identified the inverted CCAAT domain-containing UHRF1-binding site in the TXNIP promoter and demonstrated UHRF1 knockdown decreases UHRF1 promoter binding and enhances TXNIP expression through demethylation of this region. TXNIP promoter CpG methylation was further confirmed in cervical cancer tissue by pyrosequencing and methylation-specific polymerase chain reaction. Critically, down-regulation of UHRF1 by siRNA or UHRF1 antagonist (thymoquinone) induces cell cycle arrest and apoptosis, and ubiquitin-specific protease 7 (USP7), which stabilises and promotes UHRF1 function, is increased by HPV viral protein E6/E7 overexpression. These results indicate HPV might induce carcinogenesis through UHRF1-mediated TXNIP promoter methylation, thus suggesting a possible link between CpG methylation and cervical cancer.     

Epigenomic regulation of human T-cell leukemia virus by chromatin-insulator CTCF

Human T-cell leukemia virus type 1 (HTLV-1) is a retrovirus that causes an aggressive T-cell malignancy and a variety of inflammatory conditions. The integrated provirus includes a single binding site for the epigenomic insulator, CCCTC-binding protein (CTCF), but its function remains unclear. In the current study, a mutant virus was examined that eliminates the CTCF-binding site. The mutation did not disrupt the kinetics and levels of virus gene expression, or establishment of or reactivation from latency. However, the mutation disrupted the epigenetic barrier function, resulting in enhanced DNA CpG methylation downstream of the CTCF binding site on both strands of the integrated provirus and H3K4Me3, H3K36Me3, and H3K27Me3 chromatin modifications both up- and downstream of the site. A majority of clonal cell lines infected with wild type HTLV-1 exhibited increased plus strand gene expression with CTCF knockdown, while expression in mutant HTLV-1 clonal lines was unaffected. These findings indicate that CTCF binding regulates HTLV-1 gene expression, DNA and histone methylation in an integration site dependent fashion.     

DNA methylation in animal development

Nuclear transfer experiments have demonstrated that epigenetic mechanisms operate to limit gene expression during animal development. In somatic cells, silenced genes are associated with defined chromatin states which are characterised by hypermethylation of DNA, hypoacetylation of histones and specific patterns of methylation at distinct residues of the N-terminal tails of histone H3 and H4. This review describes the role of the DNA methylation-mediated repression system (Dnmt1's, MeCPs and MBDs and associated chromatin remodelling activities) in animal development. DNA methylation is essential for normal vertebrate development but has distinct regulatory roles in non-mammalian and mammalian vertebrates. In mammals, DNA methylation has an additional role in regulating imprinting. This suggests that epigenetic regulation is plastic in its application and should be considered in a developmental context that may be species specific.     

Position effect variegation and epigenetic modification of a transgene in a pig model

Sequences proximal to transgene integration sites are able to regulate transgene expression, resulting in complex position effect variegation. Position effect variegation can cause differences in epigenetic modifications, such as DNA methylation and histone acetylation. However, it is not known which factor, position effect or epigenetic modification, plays a more important role in the regulation of transgene expression. We analyzed transgene expression patterns and epigenetic modifications of transgenic pigs expressing green fluorescent protein, driven by the cytomegalovirus (CMV) promoter. DNA hypermethylation and loss of acetylation of specific histone H3 and H4 lysines, except H4K16 acetylation in the CMV promoter, were consistent with a low level of transgene expression. Moreover, the degree of DNA methylation and histone H3/H4 acetylation in the promoter region depended on the integration site; consequently, position effect variegation caused variations in epigenetic modifications. The transgenic pig fibroblast cell lines were treated with DNA methyltransferase inhibitor 5-Aza-2'-deoxycytidine and/or histone deacetylase inhibitor trichostatin A. Transgene expression was promoted by reversing the DNA hypermethylation and histone hypoacetylation status. The differences in DNA methylation and histone acetylation in the CMV promoter region in these cell lines were not significant; however, significant differences in transgene expression were detected, demonstrating that variegation of transgene expression is affected by the integration site. We conclude that in this pig model, position effect variegation affects transgene expression.     

Methyl-CpG binding protein MBD1 couples histone H3 methylation at lysine 9 by SETDB1 to DNA replication and chromatin assembly

In mammals, heterochromatin is characterized by DNA methylation at CpG dinucleotides and methylation at lysine 9 of histone H3. It is currently unclear whether there is a coordinated transmission of these two epigenetic modifications through DNA replication. Here we show that the methyl-CpG binding protein MBD1 forms a stable complex with histone H3-K9 methylase SETDB1. Moreover, during DNA replication, MBD1 recruits SETDB1 to the large subunit of chromatin assembly factor CAF-1 to form an S phase-specific CAF-1/MBD1/SETDB1 complex that facilitates methylation of H3-K9 during replication-coupled chromatin assembly. In the absence of MBD1, H3-K9 methylation is lost at multiple genomic loci and results in activation of p53BP2 gene, normally repressed by MBD1 in HeLa cells. Our data suggest a model in which H3-K9 methylation by SETDB1 is dependent on MBD1 and is heritably maintained through DNA replication to support the formation of stable heterochromatin at methylated DNA.     

A methylation rendezvous: reader meets writers

It is well established that two marks of silent chromatin, DNA methylation and histone H3 methylation at lysine 9, engage in an epigenetic conversation. In a recent issue of Genes & Development, Smallwood et al. (2007) report that the mammalian HP1 adaptor "translates" methylation information from histone to DNA, helping to cement epigenetic expression states.