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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

Usp16 but not the enzymatically inactive mutant rescues the undifferentiated phenotype of Usp16−/− ESCsa. Western blot analysis of Usp16 (top panel) and H2A ubiquitination levels (third panel) in Usp16+/− ESCs, and Usp16−/− ESCs rescued with wild type or enzymatically inactive C205Smutant Usp16. ubH2A signals were quantified as in Fig. 2c. Signals in Usp16+/+ ESCs werearbitrarily set as 1. GAPDH and histone H3 were used as loading controls.b. Hematoxylin and eosin staining of day12 EBs formed by Usp16−/− ESCs rescued with wild type or enzymatically inactive C205S mutant Usp16. A bar chart summary of EBs exhibiting differentiated phenotype is shown. Scale car, 100μm.c. RT-qPCR analysis of genes in EBs formed by control, Usp16+/− ESCs, and Usp16−/− ESCs rescued with wild type or enzymatic inactive C205S mutant Usp16. Bars shown represent means + SD. Number of biological replicates n=2.d. A proposed model for Usp16 and H2A deubiquitination in ESC gene expression and lineage commitment. In ESCs, Usp16 binds to a large number of genes and Usp16 binding inversely correlates with ubH2A levels and positively correlates with gene expression. During ESC differentiation, Usp16 is responsible for reversing H2A ubiquitination at developmental genes, enabling ESC differentiation.
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Figure 7: Usp16 but not the enzymatically inactive mutant rescues the undifferentiated phenotype of Usp16−/− ESCsa. Western blot analysis of Usp16 (top panel) and H2A ubiquitination levels (third panel) in Usp16+/− ESCs, and Usp16−/− ESCs rescued with wild type or enzymatically inactive C205Smutant Usp16. ubH2A signals were quantified as in Fig. 2c. Signals in Usp16+/+ ESCs werearbitrarily set as 1. GAPDH and histone H3 were used as loading controls.b. Hematoxylin and eosin staining of day12 EBs formed by Usp16−/− ESCs rescued with wild type or enzymatically inactive C205S mutant Usp16. A bar chart summary of EBs exhibiting differentiated phenotype is shown. Scale car, 100μm.c. RT-qPCR analysis of genes in EBs formed by control, Usp16+/− ESCs, and Usp16−/− ESCs rescued with wild type or enzymatic inactive C205S mutant Usp16. Bars shown represent means + SD. Number of biological replicates n=2.d. A proposed model for Usp16 and H2A deubiquitination in ESC gene expression and lineage commitment. In ESCs, Usp16 binds to a large number of genes and Usp16 binding inversely correlates with ubH2A levels and positively correlates with gene expression. During ESC differentiation, Usp16 is responsible for reversing H2A ubiquitination at developmental genes, enabling ESC differentiation.

Mentions: To determine whether defects in Usp16−/− EB differentiation are due to Usp16 deubiquitinase activity, we electroporated wild type and catalytically inactive Usp16 targeting vectors into Usp16−/− ESCs. As shown in Fig. 7a, wild-type and mutant Usp16 were expressed at levels similar to that in Usp16+/− ESCs. Expression of wild-type but not enzymatically inactive mutant Usp16 decreased the ubH2A level in Usp16−/− ESCs (Fig. 7a). When these ESCs were used for EB formation, we found that a significant number of EBs formed from Usp16−/− ESCs rescued with wild-type Usp16 displayed a differentiated phenotype (Fig. 7b, see insertion for relative ratio of differentiated EBs) while virtually all EBs formed from ESCs rescued by catalytically inactive Usp16 exhibited an undifferentiated phenotype (Fig. 7b). The differentiated phenotype was further confirmed by RT-qPCR analysis of lineage-specific genes in these EBs. As shown in Fig. 7c, expression of the Igf2, Vimentin, Gata4, Gata6, and Foxa2 genes was significantly increased in EBs formed from Usp16−/− ESCs rescued by wild-type Usp16. Surprisingly, although there was a modest reduction in the expression of pluripotent genes Oct4 and Nanog, we did not observe a difference between EBs rescued by wild-type or catalytically inactive mutant Usp16 (Fig. 7c). This is likely due to the presence of both undifferentiated and differentiated EB populations derived from Usp16−/− ESCs rescued by wild-type Usp16 (Fig. 7b, see insertion for relative ratio of differentiated EB).


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)

Usp16 but not the enzymatically inactive mutant rescues the undifferentiated phenotype of Usp16−/− ESCsa. Western blot analysis of Usp16 (top panel) and H2A ubiquitination levels (third panel) in Usp16+/− ESCs, and Usp16−/− ESCs rescued with wild type or enzymatically inactive C205Smutant Usp16. ubH2A signals were quantified as in Fig. 2c. Signals in Usp16+/+ ESCs werearbitrarily set as 1. GAPDH and histone H3 were used as loading controls.b. Hematoxylin and eosin staining of day12 EBs formed by Usp16−/− ESCs rescued with wild type or enzymatically inactive C205S mutant Usp16. A bar chart summary of EBs exhibiting differentiated phenotype is shown. Scale car, 100μm.c. RT-qPCR analysis of genes in EBs formed by control, Usp16+/− ESCs, and Usp16−/− ESCs rescued with wild type or enzymatic inactive C205S mutant Usp16. Bars shown represent means + SD. Number of biological replicates n=2.d. A proposed model for Usp16 and H2A deubiquitination in ESC gene expression and lineage commitment. In ESCs, Usp16 binds to a large number of genes and Usp16 binding inversely correlates with ubH2A levels and positively correlates with gene expression. During ESC differentiation, Usp16 is responsible for reversing H2A ubiquitination at developmental genes, enabling ESC differentiation.
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Figure 7: Usp16 but not the enzymatically inactive mutant rescues the undifferentiated phenotype of Usp16−/− ESCsa. Western blot analysis of Usp16 (top panel) and H2A ubiquitination levels (third panel) in Usp16+/− ESCs, and Usp16−/− ESCs rescued with wild type or enzymatically inactive C205Smutant Usp16. ubH2A signals were quantified as in Fig. 2c. Signals in Usp16+/+ ESCs werearbitrarily set as 1. GAPDH and histone H3 were used as loading controls.b. Hematoxylin and eosin staining of day12 EBs formed by Usp16−/− ESCs rescued with wild type or enzymatically inactive C205S mutant Usp16. A bar chart summary of EBs exhibiting differentiated phenotype is shown. Scale car, 100μm.c. RT-qPCR analysis of genes in EBs formed by control, Usp16+/− ESCs, and Usp16−/− ESCs rescued with wild type or enzymatic inactive C205S mutant Usp16. Bars shown represent means + SD. Number of biological replicates n=2.d. A proposed model for Usp16 and H2A deubiquitination in ESC gene expression and lineage commitment. In ESCs, Usp16 binds to a large number of genes and Usp16 binding inversely correlates with ubH2A levels and positively correlates with gene expression. During ESC differentiation, Usp16 is responsible for reversing H2A ubiquitination at developmental genes, enabling ESC differentiation.
Mentions: To determine whether defects in Usp16−/− EB differentiation are due to Usp16 deubiquitinase activity, we electroporated wild type and catalytically inactive Usp16 targeting vectors into Usp16−/− ESCs. As shown in Fig. 7a, wild-type and mutant Usp16 were expressed at levels similar to that in Usp16+/− ESCs. Expression of wild-type but not enzymatically inactive mutant Usp16 decreased the ubH2A level in Usp16−/− ESCs (Fig. 7a). When these ESCs were used for EB formation, we found that a significant number of EBs formed from Usp16−/− ESCs rescued with wild-type Usp16 displayed a differentiated phenotype (Fig. 7b, see insertion for relative ratio of differentiated EBs) while virtually all EBs formed from ESCs rescued by catalytically inactive Usp16 exhibited an undifferentiated phenotype (Fig. 7b). The differentiated phenotype was further confirmed by RT-qPCR analysis of lineage-specific genes in these EBs. As shown in Fig. 7c, expression of the Igf2, Vimentin, Gata4, Gata6, and Foxa2 genes was significantly increased in EBs formed from Usp16−/− ESCs rescued by wild-type Usp16. Surprisingly, although there was a modest reduction in the expression of pluripotent genes Oct4 and Nanog, we did not observe a difference between EBs rescued by wild-type or catalytically inactive mutant Usp16 (Fig. 7c). This is likely due to the presence of both undifferentiated and differentiated EB populations derived from Usp16−/− ESCs rescued by wild-type Usp16 (Fig. 7b, see insertion for relative ratio of differentiated EB).

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