Abstract
In mouse embryonic stem (mES) cells, ubiquitylation of histone H2A lysine 119 represses a large number of developmental genes and maintains mES cell pluripotency. It has been suggested that a number of H2A ubiquitin ligases as well as deubiquitylases and related peptide fragments contribute to a delicate balance between self-renewal and multi-lineage differentiation in mES cells. Here, we tested whether known H2A ubiquitin ligases and deubiquitylases are involved in mES cell regulation and discovered that Dzip3, the E3 ligase of H2AK119, represses differentiation-inducible genes, as does Ring1B. The two sets of target genes partially overlapped but had different spectra. We found that Dzip3 represses gene expression by orchestrating changes in 3D organization, in addition to regulating ubiquitylation of H2A. Our results shed light on the epigenetic mechanism of transcriptional regulation, which depends on 3D chromatin reorganization to regulate mES cell differentiation.
Highlights
Which, like Ring1B, is an E3 ligase targeting H2AK119
KD of Dzip[3] and Ring1B was confirmed by RT-qPCR analysis and western blotting (Fig. 1B and Supplementary Fig. 2a), and the amount of H2A ubiquitylation in the cell was determined by western blotting
Lineage-affiliated gene expression is strongly repressed when mouse ES (mES) cells are cultured in the presence of the two inhibitors PD0325901 and CHIR99021 plus LIF compared with culturing in the presence of fetal bovine serum (FBS) plus LIF. These results indicate that, in the presence of FBS plus LIF, the mES cell is in a metastable state rather than in a state exhibiting the inherent properties of pluripotent cells[20,21]
Summary
Even double knockout of Ring1A and Ring1B does not lead to the complete removal of ubH2A around the transcription start site (TSS)[6] This result suggests the existence of additional site-specific factors that are involved in mediating ubH2A modifications and repressing a specific set of genes. In C2C12 cells, Dzip[3] shows nuclear localization and modulates specific histone modifications, rather than exerting global effects through interactions with Nco-R, HDAC1, and HDAC38. It is of great interest whether Dzip[3] contributes to gene regulation in ES cells. We tested whether H2A ubiquitin ligases and deubiquitylases are involved in the regulation of pluripotency and the differentiation process in mES cells and demonstrated that Dzip[3] regulates developmental genes in mES cells by reorganizing 3D chromatin conformation
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