CG hypomethylation in Lsh ^−/− mouse embryonic fibroblasts is associated with de novo H3K4me1 formation and altered cellular plasticity

Abstract

DNA methylation patterns are established in early embryogenesis and are critical for cellular differentiation. To investigate the role of CG methylation in potential enhancer formation, we assessed H3K4me1 modification in murine embryonic fibroblasts (MEFs) derived from the DNA methylation mutant Lsh −/− mice. We report here de novo formation of putative enhancer elements at CG hypomethylated sites that can be dynamically altered. We found a subset of differentially enriched H3K4me1 regions clustered at neuronal lineage genes and overlapping with known cis-regulatory elements present in brain tissue. C ytosine methylation is an epigenetic mechanism conserved among diverse eukaryotes whereby a family of conserved DNA methyltransferases (DNMTs) is responsible for cytosine methylation at cytosine-phosphate-guanosine (CpG) sites. Deletion of DNMTs in mammals alters DNA methylation patterns and results in early embryonic lethality, indicating a critical role of DNMTs in development (1). Genome-wide methylation changes have been reported after deletion of Uhfr1, a hemimethylation binding protein supporting maintenance of DNA methylation, and after deletion of Lsh (2-4). Lsh and the Arabidopsis thaliana homolog DDM1 belong to the SNF2 family of chromatin remodeling proteins, and DDM1 can cause nucleosomal sliding in vitro (5).Nucleosome templates provide limited access to DNMTs, and the addition of SNF2 proteins can improve de novo methylation in vitro (6). DDM1 mutants show reduced cytosine methylation at genomic regions rich in the linker histone H1, suggesting that the chromatin remodeling activity of DDM1 promotes access to densely packaged chromatin (7).Deletion of Lsh in mice is perinatally lethal, indicating that Lsh is not a redundant SNF2 family member and plays a critical role in murine development (8, 9). Lsh promotes de novo methylation of exogenous retroviral sequences and endogenous genomic loci, including stem cell-associated genes (10, 11). Lsh can interact with DNMT3b in vitro and supports the association of DNMT3b to some genomic targets. In addition, deletion of Lsh alters genomewide cytosine methylation pattern, suggesting a regulatory role for Lsh in the establishment of DNA methylation (3,4,12,13).DNA methylation patterns are highly dynamic and undergo large transitions during germ cell development, early embryogenesis, and reprogramming into induced pluripotent stem (iPS) cells. Dynamic alterations in DNA methylation and H3K4me1 modifications occur at putative enhancers upon cellular differentiation (14, 15). Despite their close association, the causal relationship of CG methylation changes and the establishment of cis-regulatory regions are only partially understood. Engagement of CCCTCbinding factor or repressor element-1 silencing transcription factor to specific loci causes local CG hypomethylation, indicating that DNA-binding factors, such as transcription factors, can shape the methylome (16). Whether CG hypomethylation in turn can influence the formation of enhancer sites and affect cellul...

Chromatin Remodeling Factor LSH Drives Cancer Progression by Suppressing the Activity of Fumarate Hydratase

“…LSH is critical for chromatin function and establishment of DNA methylation (6,7,32,39). Reports show that LSH contributes to the malignant progression of prostate cancer, melanoma, head and neck cancer, etc.…”

Genome-wide DNA methylation patterns in LSH mutant reveals de-repression of repeat elements and redundant epigenetic silencing pathways

“…Our observation that relative transcript abundance is stable in KO MEFs (despite CG methylation loss) suggests redundancies in epigenetic silencing pathways. Interestingly, several genes that are embedded in repressed chromatin (and still silenced upon CG hypomethylation) are in part marked by de novo H3K4me1 modification in KO MEFs and belong to the neuronal lineage (Yu et al 2014). H3K4me1 can mark potential enhancers, and the lack of histone acetylation indicated that these sites are not active yet in KO MEFs but represent putative enhancers.…”

“…Multiple pathways regulate DNA methylation, but it remains largely unknown how specific factors control methylation at a given site. Previous studies using Hells mutants examined specific genomic loci, such as immediate promoter regions or potential enhancers, and applied techniques that are biased for regions of low CG density and that have limited resolution (Xi et al 2007(Xi et al , 2009Myant et al 2011;Tao et al 2011;Yu et al 2014). In addition, fibroblast cell lines were analyzed that may have developed aberrant methylation patterns due to prolonged culture in vitro (Myant et al 2011;Tao et al 2011).…”

HELLS (Helicase, Lymphoid-Specific)

“…Knockout mice die perinatally with defects in lymphocyte proliferation, embryonic growth, and kidney development (Geiman et al, 2001;Geiman and Muegge, 2000). Loss leads to global DNA methylation changes including global hypomethylation at repetive sequences, as well as both hypo and hypermethylation of single copy genes (Myant et al, 2011;Tao et al, 2011b;Dunican et al, 2013;Yu et al, 2014). HELLS has been identified as a marker of mammalian stem cells with expression decreasing upon differentiation.…”

“…In addition, activation of lymphocytes leads to apoptosis intead of cell proliferation (Geiman et al, 2000). Molecular studies on this mouse led to identifying HELLS as a gene necessary for DNA methylation and important for correct chromatin packaging and histone methylation (Dennis et al, 2001;Yan et al, 2003b ;Huang et al, 2004;Myant et al, 2011;Tao et al, 2011b;Yu et al, 2014).…”