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A Primary Role of TET Proteins in Establishment and Maintenance of De Novo Bivalency at CpG Islands of Developmental Genes

ABSTRACT: TET protein-catalyzed 5mC oxidation not only creates novel DNA modifications such as 5hmC, but also initiates active or passive DNA demethylation. However, the TETsâ function in crosstalk with specific histone modifications is largely elusive. Here, we show that TET2-mediated DNA demethylation plays a primary role in the de novo establishment and maintenance of H3K4me3/H3K27me3 bivalent domain underlying the methylated DNA CpG islands (CGIs). Overexpression of wild type (WT) but not catalytic inactive mutant (Mut) TET2 in TET-low-expressing cells results in increase of 5hmC level and accompanying DNA demethylation at a subset of CGIs. Importantly, this is sufficient to create de novo bivalent domains at these loci. Genome-wide analysis reveals that these de novo synthesized bivalent domains are largely associated with a subset of key developmental gene promoters, which are often located within CpG islands that are previously hyper-methylated and silenced. On the other hand, depletion of Tet1 and Tet2 in mouse ES cells results in an apparent loss of H3K27me3 at bivalent domains, which are located within CGIs and associated with a particular set of key developmental gene promoters. Collectively, these data suggest that TET proteins have a primary role in charge of regulating the crosstalk between two key epigenetic mechanisms, DNA methylation and histone methylation (H3K4me3 and H3K27me3), particularly at a subset of CpG islands associated with developmental genes. We examined H3K4me3,H3K27me3,5mC and 5hmC in 293T and mES cell types,using Illumina Hiseq2500.

ORGANISM(S): Mus musculus

SUBMITTER: Shi Yang

PROVIDER: E-GEOD-72481 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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A primary role of TET proteins in establishment and maintenance of De Novo bivalency at CpG islands.

Kong Lingchun L Tan Li L Lv Ruitu R Shi Zhennan Z Xiong Lijun L Wu Feizhen F Rabidou Kimberlie K Smith Michael M He Celestine C Zhang Lei L Qian Yanyan Y Ma Duan D Lan Fei F Shi Yang Y Shi Yujiang Geno YG

Nucleic acids research 20160610 18

Ten Eleven Translocation (TET) protein-catalyzed 5mC oxidation not only creates novel DNA modifications, such as 5hmC, but also initiates active or passive DNA demethylation. TETs' role in the crosstalk with specific histone modifications, however, is largely elusive. Here, we show that TET2-mediated DNA demethylation plays a primary role in the de novo establishment and maintenance of H3K4me3/H3K27me3 bivalent domains underlying methylated DNA CpG islands (CGIs). Overexpression of wild type (WT ...[more]

PMID: 27288448

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Project description:Ten-eleven translocation (TET) proteins oxidize 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), 5-formylcytsosine (5fC), and 5-carboxylcytosine (5caC). 5fC/5caC can be excised and repaired by the base excision repair (BER) pathway, implicating 5mC oxidation in active DNA demethylation. Genome-wide DNA methylation is erased in the transition from metastable states to ground state of embryonic stem cells (ESCs) and in migrating primordial germ cells (PGCs), while some resistant regions become demethylated only in gonadal PGCs. Understanding the mechanisms underlying global hypomethylation in naïve ESCs and developing PGCs will be useful for realizing cellular pluripotency and totipotency. In this study, we found that PRDM14, the PR-domain-containing transcriptional regulator, accelerates the TET-BER cycle, resulting in the promotion of active DNA demethylation in ESCs. Induction of PRDM14 expression rapidly removed the 5mC associated with transient elevation of 5hmC at pluripotency-associated genes, germline-specific genes, and imprinted loci but not across the entire genome, which resemble second wave of DNA demethylation in gonadal PGCs. PRDM14 physically interacts with TET1/TET2 and enhances the recruitment of TET1/TET2 at target loci. Knockdown of Tet1/Tet2 impaired transcriptional regulation and DNA demethylation by PRDM14. The repression of the BER pathway by administration of pharmacological inhibitors against APE1 and PARP1 and the knockdown of thymine DNA glycosylase (TDG) also impaired DNA demethylation by PRDM14. Furthermore, DNA demethylation induced by PRDM14 normally takes place in the presence of aphidicolin, which is an inhibitor of G1/S progression. Together, our analysis provides mechanistic insight into DNA demethylation in naive pluripotent stem cells and developing PGCs. To investigate the function of TET1/TET2 in transcriptional regulation by PRDM14 in ESCs, we exploited microarray analysis using total mRNA derived from Scramble, Scramble + PRDM14, Tet1/Tet2 KD, Tet1/Tet2 KD + PRDM14 mouse ESC.

Project description:BS69 (aka ZMYND11) was initially discovered as a direct target of adenoviral E1A oncoprotein1. Subsequent studies implicated BS69 as a tumor suppressor and a transcriptional regulator2. But exactly how BS69 regulates gene expression has remained elusive. BS69 contains tandemly arranged PHD, BROMO and PWWP domains, which are known to function as chromatin recognition modalities. Here we show that BS69 selectively recognizes histone variant H3.3 lysine 36 trimethylation (H3.3K36me3) via these chromatin recognition modules. Using a proteomics approach, we further identified association of BS69 with a host of RNA splicing regulators including EFTUD2 and other U5 snRNP components of the spliceosome. Remarkably, RNA-seq analysis shows that BS69 mainly regulates intron retention (IR), which is one of the least well-understood RNA alternative splicing events in mammalian cells. Specifically, loss of BS69 results in a decrease in IR, while in contrast, knockdown of EFTUD2 leads to an increase in IR, consistent with its role as a core member of the splicesome machinery. Importantly, wildtype BS69, but not a BS69 mutant defective in its interaction with EFTUD2, rescues the IR events. We also show that knockdown of SETD2, the main enzyme that mediates H3K36 trimethylation3, similarly results in a decreased retention of the same introns regulated by BS69. Significantly, a BS69 mutant unable to bind H3.3K36me3 in vitro fails to rescue the reduced IR phenotype associated with the loss of BS69. Taken together, our findings identify an antagonistic relationship between BS69 and the core splicing machinery and demonstrate that BS69-mediated RNA splicing regulation is dependent both on its ability to bind chromatin decorated by H3K36me3 and its ability to physically interact with the core spliceosome machinery. Our study reveals a novel and unexpected role of BS69 in connecting histone H3.3K36 trimethylation to regulated RNA splicing, providing significant new insights into chromatin regulation of RNA splicing. HeLaS cells stably expressing BS69 were used for HA-tagged BS69 ChIP-seq. HeLa cells were used for BS69 ChIP-seq. HeLa cells were used for H3K36me3 ChIP-seq.

Dataset Information

A Primary Role of TET Proteins in Establishment and Maintenance of De Novo Bivalency at CpG Islands of Developmental Genes

Publications

A primary role of TET proteins in establishment and maintenance of De Novo bivalency at CpG islands.

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