ABSTRACT
The CCCTC-binding factor (CTCF) protein and its modified forms regulate gene expression and genome organization. However, information on CTCF acetylation and its biological function is still lacking. Here, we show that CTCF can be acetylated at lysine 20 (CTCF-K20) by CREB-binding protein (CBP) and deacetylated by histone deacetylase 6 (HDAC6). CTCF-K20 is required for the CTCF interaction with CBP. A CTCF point mutation at lysine 20 had no effect on self-renewal but blocked the mesoderm differentiation of mouse embryonic stem cells (mESCs). The CTCF-K20 mutation reduced CTCF binding to the promoters and enhancers of genes associated with early cardiac mesoderm differentiation, resulting in diminished chromatin accessibility and decreased enhancer-promoter interactions, impairing gene expression. In summary, this study reveals the important roles of CTCF-K20 in regulating CTCF genomic functions and mESC differentiation into mesoderm.
ABSTRACT
CTCF plays a pivotal role in mediating chromatin interactions, but it does not do so alone. A number of factors have been reported to co-localize with CTCF and regulate CTCF loops, but no comprehensive analysis of binding partners has been performed. This prompted us to identify CTCF loop participants and regulators by co-localization analysis with CTCF. We screened all factors that had ChIP-seq data in humans by co-localization analysis with human super conserved CTCF (hscCTCF) binding sites, and identified many new factors that overlapped with hscCTCF binding sites. Combined with CTCF loop information, we observed that clustered factors could promote CTCF loops. After in-depth mining of each factor, we found that many factors might have the potential to promote CTCF loops. Our data further demonstrated that BHLHE40 affected CTCF loops by regulating CTCF binding. Together, this study revealed that many factors have the potential to participate in or regulate CTCF loops, and discovered a new role for BHLHE40 in modulating CTCF loop formation.
Subject(s)
Basic Helix-Loop-Helix Transcription Factors/metabolism , CCCTC-Binding Factor/genetics , CCCTC-Binding Factor/metabolism , Chromatin/metabolism , Homeodomain Proteins/metabolism , Basic Helix-Loop-Helix Transcription Factors/genetics , Binding Sites , Chromatin/genetics , Genome, Human , HEK293 Cells , HeLa Cells , Homeodomain Proteins/genetics , Humans , Protein Interaction Domains and Motifs , Protein Interaction MapsABSTRACT
Polycomb repressive complex 1 (PRC1) is an important regulator of gene expression and development. PRC1 contains the E3 ligases RING1A/B, which monoubiquitinate lysine 119 at histone H2A (H2AK119ub1), and has been sub-classified into six major complexes based on the presence of a PCGF subunit. Here, we report that PCGF5, one of six PCGF paralogs, is an important requirement in the differentiation of mouse embryonic stem cells (mESCs) towards a neural cell fate. Although PCGF5 is not required for mESC self-renewal, its loss blocks mESC neural differentiation by activating the SMAD2/TGF-ß signaling pathway. PCGF5 loss-of-function impairs the reduction of H2AK119ub1 and H3K27me3 around neural specific genes and keeps them repressed. Our results suggest that PCGF5 might function as both a repressor for SMAD2/TGF-ß signaling pathway and a facilitator for neural differentiation. Together, our findings reveal a critical context-specific function for PCGF5 in directing PRC1 to control cell fate.
Subject(s)
Mouse Embryonic Stem Cells/cytology , Mouse Embryonic Stem Cells/metabolism , Neural Stem Cells/cytology , Neural Stem Cells/metabolism , Polycomb-Group Proteins/metabolism , Animals , Cell Differentiation/genetics , Cell Differentiation/physiology , Cell Line , Gene Expression Regulation, Developmental , Gene Knockout Techniques , Histones/metabolism , Humans , Mice , Neurogenesis/genetics , Neurogenesis/physiology , Polycomb Repressive Complex 1/metabolism , Polycomb-Group Proteins/deficiency , Polycomb-Group Proteins/genetics , Signal Transduction , Smad2 Protein/metabolism , Transforming Growth Factor beta/metabolism , Ubiquitin-Protein Ligases/metabolismABSTRACT
Induced pluripotent stem cells can be derived from somatic cells through ectopic expression of transcription factors or chemical cocktails. Chemical iPSCs (C-iPSCs) and OSKM-iPSCs (4F-iPSCs) have been suggested to have similar characteristics to mouse embryonic stem cells (mESCs). However, their epigenetic equivalence remains incompletely understood throughout the genome. In this study, we have generated mouse C-iPSCs and 4F-iPSCs, and further compared the genome-wide DNA methylomes of C-iPSCs, 4F-iPSCs, and mESCs that were maintained in 2i and LIF. Three pluripotent stem cells tend to be low methylated overall, however, DNA methylations in some specific regions (such as retrotransposons) are cell type-specific. Importantly, C-iPSCs are more hypomethylated than 4F-iPSCs. Bisulfite sequencing indicated that DNA methylation status in several known imprinted clusters, such as: Dlk1-Dio3 and Peg12-Ube3a, in C-iPSCs are closer to those of mESCs than 4F-iPSCs. Overall, our data demonstrate the reprogramming methods-dependent epigenetic differences of C-iPSCs and 4F-iPSCs and reveal that C-iPSCs are more hypomethylated than OSKM-integrated iPSCs.
