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Dzip3 regulates developmental genes in mouse embryonic stem cells by reorganizing 3D chromatin conformation.

Inoue D, Aihara H, Sato T, Mizusaki H, Doiguchi M, Higashi M, Imamura Y, Yoneda M, Miyanishi T, Fujii S, Okuda A, Nakagawa T, Ito T - Sci Rep (2015)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Nagasaki University School of Medicine.

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.

No MeSH data available.


Dzip3 regulates developmental genes by reorganizing 3D chromatin conformation.(A) 3C-qPCR analysis of four Dzip3 target genes. Upper panel: ChIP-seq signal profile of target genes. The arrows indicate the primer regions for 3C-qPCR. Lower panel: 3C-qPCR results. Chromatin from Dzip3 knockdown (KD) and negative control (NC) cells was treated with or without digestion and ligation, and the resulting samples were analyzed by qPCR. ChIP-seq signal profile of target genes on a whole-gene scale are presented in Supplementary Fig. 4a. (B) Models for transcriptional repression by Dzip3, which promotes interactions between the promoter and regions distal to its binding site. These changes in 3D chromatin structure repress transcription redundantly with Ring1B. Txn, transcription. Error bars, standard deviation.
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f4: Dzip3 regulates developmental genes by reorganizing 3D chromatin conformation.(A) 3C-qPCR analysis of four Dzip3 target genes. Upper panel: ChIP-seq signal profile of target genes. The arrows indicate the primer regions for 3C-qPCR. Lower panel: 3C-qPCR results. Chromatin from Dzip3 knockdown (KD) and negative control (NC) cells was treated with or without digestion and ligation, and the resulting samples were analyzed by qPCR. ChIP-seq signal profile of target genes on a whole-gene scale are presented in Supplementary Fig. 4a. (B) Models for transcriptional repression by Dzip3, which promotes interactions between the promoter and regions distal to its binding site. These changes in 3D chromatin structure repress transcription redundantly with Ring1B. Txn, transcription. Error bars, standard deviation.

Mentions: To explore the molecular mechanisms by which Dzip3 represses gene expression, we focused on those of its target genes around which significant Dzip3 binding sites exist. A recent study revealed that about 25% of Ring1B-bound genes in ESCs possess prominent Ring1B binding sites (RBS) outside of their promoter regions (TSS ± 4 kb) and that Ring1B represses target genes by orchestrating 3D chromatin structural changes22. We hypothesized that Dzip3 also participates in regulating target genes by reorganizing 3D chromatin conformation. To assess the validity of this hypothesis, we performed a chromosome conformation capture (3C) assay. We first designed PCR primers for the 3C assay around the promoter region and the 3’ end of the gene, as indicated by arrows (Fig. 4A and Supplementary Fig. 4a). Undigested and unligated samples were prepared as negative controls. For Neurog1, Cdh2, Bspry, and Shank3, we detected signals implicating an interaction between the promoter region and the 3’ end of the gene in the presence of the restriction enzyme Mse I and T4 DNA ligase, but not in their absence. Furthermore, the signal indicating chromatin conformation was decreased by Dzip3 KD (Fig. 4A and Supplementary Fig. 5a). Therefore, we suggest that Dzip3 regulates gene expression by changing the local chromatin conformation.


Dzip3 regulates developmental genes in mouse embryonic stem cells by reorganizing 3D chromatin conformation.

Inoue D, Aihara H, Sato T, Mizusaki H, Doiguchi M, Higashi M, Imamura Y, Yoneda M, Miyanishi T, Fujii S, Okuda A, Nakagawa T, Ito T - Sci Rep (2015)

Dzip3 regulates developmental genes by reorganizing 3D chromatin conformation.(A) 3C-qPCR analysis of four Dzip3 target genes. Upper panel: ChIP-seq signal profile of target genes. The arrows indicate the primer regions for 3C-qPCR. Lower panel: 3C-qPCR results. Chromatin from Dzip3 knockdown (KD) and negative control (NC) cells was treated with or without digestion and ligation, and the resulting samples were analyzed by qPCR. ChIP-seq signal profile of target genes on a whole-gene scale are presented in Supplementary Fig. 4a. (B) Models for transcriptional repression by Dzip3, which promotes interactions between the promoter and regions distal to its binding site. These changes in 3D chromatin structure repress transcription redundantly with Ring1B. Txn, transcription. Error bars, standard deviation.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4645096&req=5

f4: Dzip3 regulates developmental genes by reorganizing 3D chromatin conformation.(A) 3C-qPCR analysis of four Dzip3 target genes. Upper panel: ChIP-seq signal profile of target genes. The arrows indicate the primer regions for 3C-qPCR. Lower panel: 3C-qPCR results. Chromatin from Dzip3 knockdown (KD) and negative control (NC) cells was treated with or without digestion and ligation, and the resulting samples were analyzed by qPCR. ChIP-seq signal profile of target genes on a whole-gene scale are presented in Supplementary Fig. 4a. (B) Models for transcriptional repression by Dzip3, which promotes interactions between the promoter and regions distal to its binding site. These changes in 3D chromatin structure repress transcription redundantly with Ring1B. Txn, transcription. Error bars, standard deviation.
Mentions: To explore the molecular mechanisms by which Dzip3 represses gene expression, we focused on those of its target genes around which significant Dzip3 binding sites exist. A recent study revealed that about 25% of Ring1B-bound genes in ESCs possess prominent Ring1B binding sites (RBS) outside of their promoter regions (TSS ± 4 kb) and that Ring1B represses target genes by orchestrating 3D chromatin structural changes22. We hypothesized that Dzip3 also participates in regulating target genes by reorganizing 3D chromatin conformation. To assess the validity of this hypothesis, we performed a chromosome conformation capture (3C) assay. We first designed PCR primers for the 3C assay around the promoter region and the 3’ end of the gene, as indicated by arrows (Fig. 4A and Supplementary Fig. 4a). Undigested and unligated samples were prepared as negative controls. For Neurog1, Cdh2, Bspry, and Shank3, we detected signals implicating an interaction between the promoter region and the 3’ end of the gene in the presence of the restriction enzyme Mse I and T4 DNA ligase, but not in their absence. Furthermore, the signal indicating chromatin conformation was decreased by Dzip3 KD (Fig. 4A and Supplementary Fig. 5a). Therefore, we suggest that Dzip3 regulates gene expression by changing the local chromatin conformation.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Nagasaki University School of Medicine.

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.

No MeSH data available.