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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.


Knockdown of Dzip3 results in a decrease in the percentage of tightly packed cell colonies and upregulates differentiation-inducible gene expression.(A) The morphology of mES cells 72 h after siRNA transfection. Knockdown (KD) of Ring1B and Dzip3 resulted in a decrease in the percentage of tightly packed cell colonies. There were three categories of colony morphology: “tightly packed” (observed in pluripotency); “flattened” (observed in differentiation); and “packed” (intermediate between tightly packed and flattened). The n value represents the number of colonies classified. Statistical significance was assessed using the HYPGEOMDIST function (P = 2.90 × 10−18, negative control [NC] versus Dzip3 KD samples). (B) The efficiency of knockdown was evaluated in mES cells (serum + LIF) transfected with the indicated specific and control siRNAs. RT-qPCR analysis was performed to document the efficiency of Dzip3 siRNA and Ring1B siRNA knockdown to diminish endogenous Dzip3 and Ring1B. Values (normalized to the corresponding values of the internal control gene GAPDH) are the mean ± SEM of three independent experiments. Protein levels were determined by western blotting using the indicated antibodies. Equal loading was confirmed by Amido Black staining. The two major bands correspond to the alternatively spliced versions of Dzip3. Full-length blots are presented in Supplementary Fig. 2a,b. (C) The ubH2A level was determined by western blotting. Full-length blots are presented in Supplementary Fig. 2c. (D) The relative expression levels of pluripotent marker genes in NC, Ring1B KD, and Dzip3 KD mES cells. (E) The relative expression levels of differentiation-inducible genes adjusted with GAPDH in NC, Ring1B KD, and Dzip3 KD mES cells. Statistical significance was assessed by two-tailed Student’s t-test. NTC, no-treatment control. Error bars, standard deviation.
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f1: Knockdown of Dzip3 results in a decrease in the percentage of tightly packed cell colonies and upregulates differentiation-inducible gene expression.(A) The morphology of mES cells 72 h after siRNA transfection. Knockdown (KD) of Ring1B and Dzip3 resulted in a decrease in the percentage of tightly packed cell colonies. There were three categories of colony morphology: “tightly packed” (observed in pluripotency); “flattened” (observed in differentiation); and “packed” (intermediate between tightly packed and flattened). The n value represents the number of colonies classified. Statistical significance was assessed using the HYPGEOMDIST function (P = 2.90 × 10−18, negative control [NC] versus Dzip3 KD samples). (B) The efficiency of knockdown was evaluated in mES cells (serum + LIF) transfected with the indicated specific and control siRNAs. RT-qPCR analysis was performed to document the efficiency of Dzip3 siRNA and Ring1B siRNA knockdown to diminish endogenous Dzip3 and Ring1B. Values (normalized to the corresponding values of the internal control gene GAPDH) are the mean ± SEM of three independent experiments. Protein levels were determined by western blotting using the indicated antibodies. Equal loading was confirmed by Amido Black staining. The two major bands correspond to the alternatively spliced versions of Dzip3. Full-length blots are presented in Supplementary Fig. 2a,b. (C) The ubH2A level was determined by western blotting. Full-length blots are presented in Supplementary Fig. 2c. (D) The relative expression levels of pluripotent marker genes in NC, Ring1B KD, and Dzip3 KD mES cells. (E) The relative expression levels of differentiation-inducible genes adjusted with GAPDH in NC, Ring1B KD, and Dzip3 KD mES cells. Statistical significance was assessed by two-tailed Student’s t-test. NTC, no-treatment control. Error bars, standard deviation.

Mentions: First, we focused on morphological changes under pluripotent conditions (serum + LIF)19. KD of Ring1B and Dzip3 resulted in a decrease in tight packing of the cells and led to an expansion of the colony (Fig. 1A and Supplementary Fig. 1b). KD of Dzip3 and Ring1B was confirmed by RT-qPCR analysis and western blotting (Fig. 1B and Supplementary Fig. 2a), and the amount of H2A ubiquitylation (ubH2A) in the cell was determined by western blotting. The total amount of ubH2A in the cell decreased after Ring1B KD but not after Dzip3 KD (Fig. 1C), which suggests that Dzip3 functions by modulating a specific histone modification in the promoter region, rather than by global effects.


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)

Knockdown of Dzip3 results in a decrease in the percentage of tightly packed cell colonies and upregulates differentiation-inducible gene expression.(A) The morphology of mES cells 72 h after siRNA transfection. Knockdown (KD) of Ring1B and Dzip3 resulted in a decrease in the percentage of tightly packed cell colonies. There were three categories of colony morphology: “tightly packed” (observed in pluripotency); “flattened” (observed in differentiation); and “packed” (intermediate between tightly packed and flattened). The n value represents the number of colonies classified. Statistical significance was assessed using the HYPGEOMDIST function (P = 2.90 × 10−18, negative control [NC] versus Dzip3 KD samples). (B) The efficiency of knockdown was evaluated in mES cells (serum + LIF) transfected with the indicated specific and control siRNAs. RT-qPCR analysis was performed to document the efficiency of Dzip3 siRNA and Ring1B siRNA knockdown to diminish endogenous Dzip3 and Ring1B. Values (normalized to the corresponding values of the internal control gene GAPDH) are the mean ± SEM of three independent experiments. Protein levels were determined by western blotting using the indicated antibodies. Equal loading was confirmed by Amido Black staining. The two major bands correspond to the alternatively spliced versions of Dzip3. Full-length blots are presented in Supplementary Fig. 2a,b. (C) The ubH2A level was determined by western blotting. Full-length blots are presented in Supplementary Fig. 2c. (D) The relative expression levels of pluripotent marker genes in NC, Ring1B KD, and Dzip3 KD mES cells. (E) The relative expression levels of differentiation-inducible genes adjusted with GAPDH in NC, Ring1B KD, and Dzip3 KD mES cells. Statistical significance was assessed by two-tailed Student’s t-test. NTC, no-treatment control. Error bars, standard deviation.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f1: Knockdown of Dzip3 results in a decrease in the percentage of tightly packed cell colonies and upregulates differentiation-inducible gene expression.(A) The morphology of mES cells 72 h after siRNA transfection. Knockdown (KD) of Ring1B and Dzip3 resulted in a decrease in the percentage of tightly packed cell colonies. There were three categories of colony morphology: “tightly packed” (observed in pluripotency); “flattened” (observed in differentiation); and “packed” (intermediate between tightly packed and flattened). The n value represents the number of colonies classified. Statistical significance was assessed using the HYPGEOMDIST function (P = 2.90 × 10−18, negative control [NC] versus Dzip3 KD samples). (B) The efficiency of knockdown was evaluated in mES cells (serum + LIF) transfected with the indicated specific and control siRNAs. RT-qPCR analysis was performed to document the efficiency of Dzip3 siRNA and Ring1B siRNA knockdown to diminish endogenous Dzip3 and Ring1B. Values (normalized to the corresponding values of the internal control gene GAPDH) are the mean ± SEM of three independent experiments. Protein levels were determined by western blotting using the indicated antibodies. Equal loading was confirmed by Amido Black staining. The two major bands correspond to the alternatively spliced versions of Dzip3. Full-length blots are presented in Supplementary Fig. 2a,b. (C) The ubH2A level was determined by western blotting. Full-length blots are presented in Supplementary Fig. 2c. (D) The relative expression levels of pluripotent marker genes in NC, Ring1B KD, and Dzip3 KD mES cells. (E) The relative expression levels of differentiation-inducible genes adjusted with GAPDH in NC, Ring1B KD, and Dzip3 KD mES cells. Statistical significance was assessed by two-tailed Student’s t-test. NTC, no-treatment control. Error bars, standard deviation.
Mentions: First, we focused on morphological changes under pluripotent conditions (serum + LIF)19. KD of Ring1B and Dzip3 resulted in a decrease in tight packing of the cells and led to an expansion of the colony (Fig. 1A and Supplementary Fig. 1b). KD of Dzip3 and Ring1B was confirmed by RT-qPCR analysis and western blotting (Fig. 1B and Supplementary Fig. 2a), and the amount of H2A ubiquitylation (ubH2A) in the cell was determined by western blotting. The total amount of ubH2A in the cell decreased after Ring1B KD but not after Dzip3 KD (Fig. 1C), which suggests that Dzip3 functions by modulating a specific histone modification in the promoter region, rather than by global effects.

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.