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Embryonic stem cells (ESCs) possess a distinct chromatin conformation maintained by specialized chromatin proteins. To identify chromatin regulators in ESCs, we developed a simple biochemical assay named D-CAP (differential chromatin-associated proteins), using brief micrococcal nuclease digestion of chromatin, followed by liquid chromatography tandem mass spectrometry (LC-MS/MS). Using D-CAP, we identified several differentially chromatin-associated proteins between undifferentiated and differentiated ESCs, including the chromatin remodeling protein SMARCD1. SMARCD1 depletion in ESCs led to altered chromatin and enhanced endodermal differentiation. Gene expression and chromatin immunoprecipitation sequencing (ChIP-seq) analyses suggested that SMARCD1 is both an activator and a repressor and is enriched at developmental regulators and that its chromatin binding coincides with H3K27me3. SMARCD1 knockdown caused H3K27me3 redistribution and increased H3K4me3 around the transcription start site (TSS). One of the identified SMARCD1 targets was Klf4. In SMARCD1-knockdown clones, KLF4, as well as H3K4me3 at the Klf4 locus, remained high and H3K27me3 was abolished. These results propose a role for SMARCD1 in restricting pluripotency and activating lineage pathways by regulating H3K27 methylation., (Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.)

The global impact of DNA methylation on alternative splicing is largely unknown. Using a genome-wide approach in wild-type and methylation-deficient embryonic stem cells, we found that DNA methylation can either enhance or silence exon recognition and affects the splicing of more than 20% of alternative exons. These exons are characterized by distinct genetic and epigenetic signatures. Alternative splicing regulation of a subset of these exons can be explained by heterochromatin protein 1 (HP1), which silences or enhances exon recognition in a position-dependent manner. We constructed an experimental system using site-specific targeting of a methylated/unmethylated gene and demonstrate a direct causal relationship between DNA methylation and alternative splicing. HP1 regulates this gene's alternative splicing in a methylation-dependent manner by recruiting splicing factors to its methylated form. Our results demonstrate DNA methylation's significant global influence on mRNA splicing and identify a specific mechanism of splicing regulation mediated by HP1., (Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.)