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The histone H2A deubiquitinase Usp16 regulates embryonic stem cell gene expression and lineage commitment.

Yang W, Lee YH, Jones AE, Woolnough JL, Zhou D, Dai Q, Wu Q, Giles KE, Townes TM, Wang H - Nat Commun (2014)

Bottom Line: Polycomb Repressive Complex 1 and histone H2A ubiquitination (ubH2A) contribute to embryonic stem cell (ESC) pluripotency by repressing lineage-specific gene expression.Usp16 binds to the promoter regions of a large number of genes in ESCs, and Usp16 binding is inversely correlated with ubH2A levels, and positively correlates with gene expression levels.Therefore, this study identifies Usp16 and H2A deubiquitination as critical regulators of ESC gene expression and differentiation.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Genetics, Stem Cell Institute, University of Alabama at Birmingham, Kaul Human Genetics Building 430, 720 South 20th Street, Birmingham, Alabama 35294, USA.

ABSTRACT
Polycomb Repressive Complex 1 and histone H2A ubiquitination (ubH2A) contribute to embryonic stem cell (ESC) pluripotency by repressing lineage-specific gene expression. However, whether active deubiquitination co-regulates ubH2A levels in ESCs and during differentiation is not known. Here we report that Usp16, a histone H2A deubiquitinase, regulates H2A deubiquitination and gene expression in ESCs, and importantly, is required for ESC differentiation. Usp16 knockout is embryonic lethal in mice, but does not affect ESC viability or identity. Usp16 binds to the promoter regions of a large number of genes in ESCs, and Usp16 binding is inversely correlated with ubH2A levels, and positively correlates with gene expression levels. Intriguingly, Usp16(-/-) ESCs fail to differentiate due to ubH2A-mediated repression of lineage-specific genes. Finally, Usp16, but not a catalytically inactive mutant, rescues the differentiation defects of Usp16(-/-) ESCs. Therefore, this study identifies Usp16 and H2A deubiquitination as critical regulators of ESC gene expression and differentiation.

No MeSH data available.


Related in: MedlinePlus

The failure to activate lineage specific gene expression is the cause of undifferentiated phenotype of Usp16−/− ESCsa. Knockdown of Oct4 has no effect on Usp16−/− ESC differentiation. RT-qPCR analysis of genes in differentiated Usp16+/+ and Usp16−/− ESCs with or without infection of shRNA against Oct4. Bars shown represent means + SD. Number of biological replicates n=2.b. Expression of HoxB4 triggers Usp16−/− ESC differentiation. RT-qPCR analysis of genes in differentiated control and Usp16−/− ESCs with or without infection of HoxB4 lentivirus. Bars shown represent means + SD. Number of biological replicates n=2.
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Figure 6: The failure to activate lineage specific gene expression is the cause of undifferentiated phenotype of Usp16−/− ESCsa. Knockdown of Oct4 has no effect on Usp16−/− ESC differentiation. RT-qPCR analysis of genes in differentiated Usp16+/+ and Usp16−/− ESCs with or without infection of shRNA against Oct4. Bars shown represent means + SD. Number of biological replicates n=2.b. Expression of HoxB4 triggers Usp16−/− ESC differentiation. RT-qPCR analysis of genes in differentiated control and Usp16−/− ESCs with or without infection of HoxB4 lentivirus. Bars shown represent means + SD. Number of biological replicates n=2.

Mentions: Our studies reveal that Usp16−/− ESCs do not activate lineage-specific genes and repress pluripotency genes when induced to differentiate (Figs. 4 and 5). To determine the primary cause for the undifferentiated phenotype of Usp16−/− EBs, we tested whether knockdown of pluripotent genes or overexpression of lineage-specific genes could rescue this phenotype. Since EB formation requires prolonged culture and selection of infected cells caused substantial cell death during EB formation , we tested whether the undifferentiated phenotype of Usp16−/− ESCs could be recapitulated in other culturing systems. For this purpose, we cultured control and Usp16−/− ESCs in gelatin-coated plates without LIF to induce ESC differentiation. As shown in Fig. 6a and 6b, when cultured for 6 to 8 days, control Usp16+/+ ESCs successfully differentiated, as evidenced by the repression of pluripotent genes Oct4 and Nanog, and activation of lineage-specific genes including Gata4, Gata6, Hoxc6 (control Usp16+/+). In this culture system, Usp16−/− ESCs also failed to differentiate, as evidenced by the high expression of Oct4 and Nanog, and low levels of expression of differentiation-associated genes including Gata4, Gata6, Hoxc6, etc. (Fig. 6a and 6b). We next tested whether knockdown of Oct4 could rescue the undifferentiated phenotypes. As shown in Fig. 6a, when Oct4 was significantly knocked down in Usp16−/− ESCs, Nanog remains highly expressed and lineage-associated genes Gata4, Gata6, Igfbf5 and Hoxc6 remains silenced in these cells (compare Usp16+/+ and Usp16−/− with Oct4 KD). These data indicate that the failure to repress pluripotent gene expression is unlikely the primary cause for the undifferentiated phenotype of Usp16−/− ESCs.


The histone H2A deubiquitinase Usp16 regulates embryonic stem cell gene expression and lineage commitment.

Yang W, Lee YH, Jones AE, Woolnough JL, Zhou D, Dai Q, Wu Q, Giles KE, Townes TM, Wang H - Nat Commun (2014)

The failure to activate lineage specific gene expression is the cause of undifferentiated phenotype of Usp16−/− ESCsa. Knockdown of Oct4 has no effect on Usp16−/− ESC differentiation. RT-qPCR analysis of genes in differentiated Usp16+/+ and Usp16−/− ESCs with or without infection of shRNA against Oct4. Bars shown represent means + SD. Number of biological replicates n=2.b. Expression of HoxB4 triggers Usp16−/− ESC differentiation. RT-qPCR analysis of genes in differentiated control and Usp16−/− ESCs with or without infection of HoxB4 lentivirus. Bars shown represent means + SD. Number of biological replicates n=2.
© Copyright Policy
Related In: Results  -  Collection

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Figure 6: The failure to activate lineage specific gene expression is the cause of undifferentiated phenotype of Usp16−/− ESCsa. Knockdown of Oct4 has no effect on Usp16−/− ESC differentiation. RT-qPCR analysis of genes in differentiated Usp16+/+ and Usp16−/− ESCs with or without infection of shRNA against Oct4. Bars shown represent means + SD. Number of biological replicates n=2.b. Expression of HoxB4 triggers Usp16−/− ESC differentiation. RT-qPCR analysis of genes in differentiated control and Usp16−/− ESCs with or without infection of HoxB4 lentivirus. Bars shown represent means + SD. Number of biological replicates n=2.
Mentions: Our studies reveal that Usp16−/− ESCs do not activate lineage-specific genes and repress pluripotency genes when induced to differentiate (Figs. 4 and 5). To determine the primary cause for the undifferentiated phenotype of Usp16−/− EBs, we tested whether knockdown of pluripotent genes or overexpression of lineage-specific genes could rescue this phenotype. Since EB formation requires prolonged culture and selection of infected cells caused substantial cell death during EB formation , we tested whether the undifferentiated phenotype of Usp16−/− ESCs could be recapitulated in other culturing systems. For this purpose, we cultured control and Usp16−/− ESCs in gelatin-coated plates without LIF to induce ESC differentiation. As shown in Fig. 6a and 6b, when cultured for 6 to 8 days, control Usp16+/+ ESCs successfully differentiated, as evidenced by the repression of pluripotent genes Oct4 and Nanog, and activation of lineage-specific genes including Gata4, Gata6, Hoxc6 (control Usp16+/+). In this culture system, Usp16−/− ESCs also failed to differentiate, as evidenced by the high expression of Oct4 and Nanog, and low levels of expression of differentiation-associated genes including Gata4, Gata6, Hoxc6, etc. (Fig. 6a and 6b). We next tested whether knockdown of Oct4 could rescue the undifferentiated phenotypes. As shown in Fig. 6a, when Oct4 was significantly knocked down in Usp16−/− ESCs, Nanog remains highly expressed and lineage-associated genes Gata4, Gata6, Igfbf5 and Hoxc6 remains silenced in these cells (compare Usp16+/+ and Usp16−/− with Oct4 KD). These data indicate that the failure to repress pluripotent gene expression is unlikely the primary cause for the undifferentiated phenotype of Usp16−/− ESCs.

Bottom Line: Polycomb Repressive Complex 1 and histone H2A ubiquitination (ubH2A) contribute to embryonic stem cell (ESC) pluripotency by repressing lineage-specific gene expression.Usp16 binds to the promoter regions of a large number of genes in ESCs, and Usp16 binding is inversely correlated with ubH2A levels, and positively correlates with gene expression levels.Therefore, this study identifies Usp16 and H2A deubiquitination as critical regulators of ESC gene expression and differentiation.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Genetics, Stem Cell Institute, University of Alabama at Birmingham, Kaul Human Genetics Building 430, 720 South 20th Street, Birmingham, Alabama 35294, USA.

ABSTRACT
Polycomb Repressive Complex 1 and histone H2A ubiquitination (ubH2A) contribute to embryonic stem cell (ESC) pluripotency by repressing lineage-specific gene expression. However, whether active deubiquitination co-regulates ubH2A levels in ESCs and during differentiation is not known. Here we report that Usp16, a histone H2A deubiquitinase, regulates H2A deubiquitination and gene expression in ESCs, and importantly, is required for ESC differentiation. Usp16 knockout is embryonic lethal in mice, but does not affect ESC viability or identity. Usp16 binds to the promoter regions of a large number of genes in ESCs, and Usp16 binding is inversely correlated with ubH2A levels, and positively correlates with gene expression levels. Intriguingly, Usp16(-/-) ESCs fail to differentiate due to ubH2A-mediated repression of lineage-specific genes. Finally, Usp16, but not a catalytically inactive mutant, rescues the differentiation defects of Usp16(-/-) ESCs. Therefore, this study identifies Usp16 and H2A deubiquitination as critical regulators of ESC gene expression and differentiation.

No MeSH data available.


Related in: MedlinePlus