Non-germ Line Restoration of Genomic Imprinting for a Small Subset of Imprinted Genes in Ubiquitin-like PHD and RING Finger Domain-Containing 1 (Uhrf1) Null Mouse Embryonic Stem Cells

The underlying mechanism for the establishment and maintenance of differential DNA methylation in imprinted genes is largely unknown. Previous studies using Dnmt1 knock-out embryonic stem (ES) cells demonstrated that, although re-expression of DNMT1 restored DNA methylation in the non-imprinted regions, the methylation patterns of imprinted genes could be restored only through germ line passage. Knock-out of Uhrf1, an accessory factor essential for DNMT1-mediated DNA methylation, in mouse ES cells also led to impaired global DNA methylation and loss of genomic imprinting. Here, we demonstrate that, although re-expression of UHRF1 in Uhrf1^−/− ES cells restored DNA methylation for the bulk genome but not for most of the imprinted genes, it did rescue DNA methylation for the imprinted H19, Nnat, and Dlk1 genes. Analysis of histone modifications at the differential methylated regions of the imprinted genes by ChIP assays revealed that for the imprinted genes whose DNA methylation could be restored upon re-expression of UHRF1, the active histone markers (especially H3K4me3) were maintained at considerably low levels, and low levels were maintained even in Uhrf1^−/− ES cells. In contrast, for the imprinted genes whose DNA methylation could not be restored upon UHRF1 re-expression, the active histone markers (especially H3K4me3) were relatively high and became even higher in Uhrf1^−/− ES cells. Our study thus supports a role for histone modifications in determining the establishment of imprinting-related DNA methylation and demonstrates that mouse ES cells can be a valuable model for mechanistic study of the establishment and maintenance of differential DNA methylation in imprinted genes.     

UHRF1 recruits the histone acetyltransferase Tip60 and controls its expression and activity

Tat-interactive protein, 60 kDa (Tip60) is a histone acetyltransferase with specificity toward lysine 5 of histone H2A (H2AK5) and plays multiple roles in chromatin remodeling processes. Co-immunoprecipitation experiments performed on Jurkat cells, showed that Tip60 is present in the same macro-molecular complex as UHRF1 (Ubiquitin-like containing PHD and RING domain 1), DNMT1 (DNA methyltransferase 1), and HDAC1 (histone deacetylase 1). Furthermore, immunocytochemistry experiments confirmed that Tip60 co-localizes with the UHRF1/DNMT1 complex. Although down-regulation of UHRF1 by RNA interference enhanced Tip60 expression, a significant decrease of the level of acetylated H2AK5 was observed. Consistently, we have observed that down-regulation of Tip60 and DNMT1 by RNA interference, dramatically reduced the levels of acetylated H2AK5. Altogether, these results suggest that Tip60 is a novel partner of the epigenetic integration platform interplayed by UHRF1, DNMT1 and HDAC1 involved in the epigenetic code replication.     

S phase-dependent interaction with DNMT1 dictates the role of UHRF1 but not UHRF2 in DNA methylation maintenance

Recent studies demonstrate that UHRF1 is required for DNA methylation maintenance by targeting DNMT1 to DNA replication foci, presumably through its unique hemi-methylated DNA-binding activity and interaction with DNMT1. UHRF2, another member of the UHRF family proteins, is highly similar to UHRF1 in both sequence and structure, raising questions about its role in DNA methylation. In this study, we demonstrate that, like UHRF1, UHRF2 also binds preferentially to methylated histone H3 lysine 9 (H3K9) through its conserved tudor domain and hemi-methylated DNA through the SET and Ring associated domain. Like UHRF1, UHRF2 is enriched in pericentric heterochromatin. The heterochromatin localization depends to large extent on its methylated H3K9-binding activity and to less extent on its methylated DNA-binding activity. Coimmunoprecipitation experiments demonstrate that both UHRF1 and UHRF2 interact with DNMT1, DNMT3a, DNMT3b and G9a. Despite all these conserved functions, we find that UHRF2 is not able to rescue the DNA methylation defect in Uhrf1 null mouse embryonic stem cells. This can be attributed to the inability for UHRF2 to recruit DNMT1 to replication foci during S phase of the cell cycle. Indeed, we find that while UHRF1 interacts with DNMT1 in an S phase-dependent manner in cells, UHRF2 does not. Thus, our study demonstrates that UHRF2 and UHRF1 are not functionally redundant in DNA methylation maintenance and reveals the cell-cycle-dependent interaction between UHRF1 and DNMT1 as a key regulatory mechanism targeting DNMT1 for DNA methylation.     

DNA methylation requires a DNMT1 ubiquitin interacting motif (UIM) and histone ubiquitination

DNMT1 is recruited by PCNA and UHRF1 to maintain DNA methylation after replication. UHRF1 recognizes hemimethylated DNA substrates via the SRA domain, but also repressive H3K9me3 histone marks with its TTD. With systematic mutagenesis and functional assays, we could show that chromatin binding further involved UHRF1 PHD binding to unmodified H3R2. These complementation assays clearly demonstrated that the ubiquitin ligase activity of the UHRF1 RING domain is required for maintenance DNA methylation. Mass spectrometry of UHRF1-deficient cells revealed H3K18 as a novel ubiquitination target of UHRF1 in mammalian cells. With bioinformatics and mutational analyses, we identified a ubiquitin interacting motif (UIM) in the N-terminal regulatory domain of DNMT1 that binds to ubiquitinated H3 tails and is essential for DNA methylation in vivo. H3 ubiquitination and subsequent DNA methylation required UHRF1 PHD binding to H3R2. These results show the manifold regulatory mechanisms controlling DNMT1 activity that require the reading and writing of epigenetic marks by UHRF1 and illustrate the multifaceted interplay between DNA and histone modifications. The identification and functional characterization of the DNMT1 UIM suggests a novel regulatory principle and we speculate that histone H2AK119 ubiquitination might also lead to UIM-dependent recruitment of DNMT1 and DNA methylation beyond classic maintenance.     

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.     

Histone deacetylase inhibitor depsipeptide activates silenced genes through decreasing both CpG and H3K9 methylation on the promoter

Histone deacetylase inhibitor (HDACi) has been shown to demethylate the mammalian genome, which further strengthens the concept that DNA methylation and histone modifications interact in regulation of gene expression. Here, we report that an HDAC inhibitor, depsipeptide, exhibited significant demethylating activity on the promoters of several genes, including p16, SALL3, and GATA4 in human lung cancer cell lines H719 and H23, colon cancer cell line HT-29, and pancreatic cancer cell line PANC1. Although expression of DNA methyltransferase 1 (DNMT1) was not affected by depsipeptide, a decrease in binding of DNMT1 to the promoter of these genes played a dominant role in depsipeptide-induced demethylation and reactivation. Depsipeptide also suppressed expression of histone methyltransferases G9A and SUV39H1, which in turn resulted in a decrease of di- and trimethylated H3K9 around these genes' promoter. Furthermore, both loading of heterochromatin-associated protein 1 (HP1alpha and HP1beta) to methylated H3K9 and binding of DNMT1 to these genes' promoter were significantly reduced in depsipeptide-treated cells. Similar DNA demethylation was induced by another HDAC inhibitor, apicidin, but not by trichostatin A. Our data describe a novel mechanism of HDACi-mediated DNA demethylation via suppression of histone methyltransferases and reduced recruitment of HP1 and DNMT1 to the genes' promoter.     

Mutant p53 binds to estrogen receptor negative promoter via DNMT1 and HDAC1 in MDA-MB-468 breast cancer cells

DNA methylation and histone deacetylation are two epigenetic mechanisms involved in the lack of estrogen receptor (ER) expression. Our previous studies demonstrated that mutant p53 along with repression complex proteins including DNMT1, HDAC1 and MeCP2 is associated with ER-negative promoter in MDA-MB-468 cells. To elucidate the molecular mechanism of estrogen receptor 1 (ESR1) gene silencing in these cells, we down-regulated DNMT1 and HDAC1 expression using siRNAs and studied the ability of DNMT1, HDAC1, MeCP2 and p53 in binding to ESR1 promoter CpG island. Our results showed that DNMT1 or HDAC1 down-regulation disassembled the repression complex proteins and mutant p53 from ER-negative promoter. The partial demethylation of ESR1 promoter and ER re-expression in down-regulated cells supports these findings. In vivo binding studies demonstrated that mutation of p53 protein in this cell line did not affect its binding capacity to DNMT1, HDAC1 and MeCP2 proteins. Our observations suggest that not only histone deacetylase activity of HDAC1 contributes to inactivation of methylated ESR1 gene but also HDAC1 presence on ESR1 promoter is important for assembly of DNMT1 in repression complex. In addition, our data revealed that mutant p53 protein binds to the promoter of ESR1 through direct interaction with HDAC1 and indirect interaction with DNMT1, MeCP2 proteins in the ER-negative MDA-MB-468 cells.     

Epigenetic regulation of p62/SQSTM1 overcomes the radioresistance of head and neck cancer cells via autophagy-dependent senescence induction

Tumors are composed of subpopulations of cancer cells with functionally distinct features. Intratumoral heterogeneity limits the therapeutic effectiveness of cancer drugs. To address this issue, it is important to understand the regulatory mechanisms driving a subclonal variety within a therapy-resistant tumor. We identified tumor subclones of HN9 head and neck cancer cells showing distinct responses to radiation with different levels of p62 expression. Genetically identical grounds but epigenetic heterogeneity of the p62 promoter regions revealed that radioresistant HN9-R clones displayed low p62 expression via the creation of repressive chromatin architecture, in which cooperation between DNMT1 (DNA methyltransferases 1) and HDAC1 (histone deacetylases 1) resulted in DNA methylation and repressive H3K9me3 and H3K27me3 marks in the p62 promoter. Combined inhibition of DNMT1 and HDAC1 by genetic depletion or inhibitors enhanced the suppressive effects on proliferative capacity and in vivo tumorigenesis following irradiation. Importantly, ectopically p62-overexpressed HN9-R clones increased the induction of senescence along with p62-dependent autophagy activation. These results demonstrate the heterogeneous expression of p62 as the key component of clonal variation within a tumor against irradiation. Understanding the epigenetic diversity of p62 heterogeneity among subclones allows for improved identification of the functional state of subclones and provides a novel treatment option to resolve resistance to current therapies.Subject terms: Cancer, Cancer therapy     

Epigallocatechin-3-gallate up-regulates tumor suppressor gene expression via a reactive oxygen species-dependent down-regulation of UHRF1

Ubiquitin-like containing PHD and Ring finger 1 (UHRF1) contributes to silencing of tumor suppressor genes by recruiting DNA methyltransferase 1 (DNMT1) to their hemi-methylated promoters. Conversely, demethylation of these promoters has been ascribed to the natural anti-cancer drug, epigallocatechin-3-gallate (EGCG). The aim of the present study was to investigate whether the UHRF1/DNMT1 pair is an important target of EGCG action. Here, we show that EGCG down-regulates UHRF1 and DNMT1 expression in Jurkat cells, with subsequent up-regulation of p73 and p16^INK4A genes. The down-regulation of UHRF1 is dependent upon the generation of reactive oxygen species by EGCG. Up-regulation of p16^INK4A is strongly correlated with decreased promoter binding by UHRF1. UHRF1 over-expression counteracted EGCG-induced G1-arrested cells, apoptosis, and up-regulation of p16^INK4A and p73. Mutants of the Set and Ring Associated (SRA) domain of UHRF1 were unable to down-regulate p16^INK4A and p73, either in the presence or absence of EGCG. Our results show that down-regulation of UHRF1 is upstream to many cellular events, including G1 cell arrest, up-regulation of tumor suppressor genes and apoptosis.