Limits...
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 knockout does not affect ESC viability and identitya. Schematic representation of the strategy used to delete Usp16 (top panels) and an image of genotyping result of Usp16 deleted ESCs (bottom panel). PCR primers used for genotyping are indicated in the top panel.b. Phase contrast images (top panels) and alkaline phosphatase staining images (bottom panels) of Usp16+/+ and Usp16−/− ESCs. Scale bar, 50μm.c. Western blot analysis of Usp16 (top panel), H2A ubiquitination (third panel), and H2B ubiquitination (fifth panel) levels in two independent Usp16+/+ and Usp16−/− ESC lines. The quantitation of ubH2A signals was labeled. Signals in control Usp16+/+ ESCs were arbitrarily set as 1. GAPDH, histone H2A, H2B, and H3 were used as loading controls.d. Western blot analysis of the levels of pluripotent genes and PRC subunits in two independent control Usp16+/+ and Usp16−/− ESC lines. β-tubulin was used as a loading control.e. Growth curve of two independent control Usp16+/+ and Usp16−/− ESC lines. 1 × 104 ESCs were seeded in 12 well plates and cell numbers were counted every other day. The same seeding procedure was repeated every other day.f. Fluorescence activated cell sorting (FACS) analysis of control Usp16+/+ and Usp16−/− ESC lines. The percentages of cell populations at each cell cycle phase were labeled.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4060806&req=5

Figure 2: Usp16 knockout does not affect ESC viability and identitya. Schematic representation of the strategy used to delete Usp16 (top panels) and an image of genotyping result of Usp16 deleted ESCs (bottom panel). PCR primers used for genotyping are indicated in the top panel.b. Phase contrast images (top panels) and alkaline phosphatase staining images (bottom panels) of Usp16+/+ and Usp16−/− ESCs. Scale bar, 50μm.c. Western blot analysis of Usp16 (top panel), H2A ubiquitination (third panel), and H2B ubiquitination (fifth panel) levels in two independent Usp16+/+ and Usp16−/− ESC lines. The quantitation of ubH2A signals was labeled. Signals in control Usp16+/+ ESCs were arbitrarily set as 1. GAPDH, histone H2A, H2B, and H3 were used as loading controls.d. Western blot analysis of the levels of pluripotent genes and PRC subunits in two independent control Usp16+/+ and Usp16−/− ESC lines. β-tubulin was used as a loading control.e. Growth curve of two independent control Usp16+/+ and Usp16−/− ESC lines. 1 × 104 ESCs were seeded in 12 well plates and cell numbers were counted every other day. The same seeding procedure was repeated every other day.f. Fluorescence activated cell sorting (FACS) analysis of control Usp16+/+ and Usp16−/− ESC lines. The percentages of cell populations at each cell cycle phase were labeled.

Mentions: Since Usp16−/− ESCs generated by the above described approach have been subjected to multiple rounds of electroporation, which might alter cellular functions, we also derived Usp16−/− ESCs by an alternative approach. We took advantage of Usp16 conditional knockout mice, which were generated by injecting Usp162Lox ESCs (Supplementary Fig. 1a) into blastocysts (data not published). These conditional Usp16 knockout (Usp162lox/2lox) mice were crossed with the inducible Cre expression mouse line, CAGG-Cre, to obtain Usp162lox/+: CAGG-Cre mice. These mice were then interbred or bred with Usp162lox/2lox mice. ESCs were derived from the offspring of these crossings and Usp162lox/2lox: CAGG-Cre ESCs were identified by PCR mediated genotyping and confirmed by sequencing (Fig. 2a). Usp16 was deleted by culturing Usp162lox/2lox:CAGG-Cre ESCs with 1μM 4-hydroxytamoxifen (4-OHT), which induces nuclear localization of the Cre recombinase, for 3–5 days (Fig. 2a) before switching to 4-OHT free medium. The efficiency of Usp16 deletion was confirmed by western blot assay (Fig. 2c). Consistent with our previous studies, Usp16 knockout caused a specific increase of ubH2A levels, but had no effect on ubH2B levels (Fig. 2c, the intensity of ubH2A signal were quantified and labeled). Usp16−/− ESCs exhibited normal morphology (Fig. 2b) and have growth rates (Fig. 2e) and cell cycle profiles (Fig. 2f) similar to Usp16+/+ ESCs. This contrasts with studies in HeLa cells, where USP16 deletion causes a slow growth phenotype, partially due to the defects of H2A deubiquitination during cell cycle G2/M phase progression26,41. Usp16−/− ESCs stained positively for alkaline phosphatase (Fig. 2b). Pluripotent genes Oct4, Sox2, and Nanog and PRC subunits Ring1B, Suz12, and Ezh2) were not affected by Usp16 deletion (Fig. 2d). Together, these studies demonstrate that Usp16 regulates ubH2A levels in ESCs, and is not required for ESC viability and identity.


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 knockout does not affect ESC viability and identitya. Schematic representation of the strategy used to delete Usp16 (top panels) and an image of genotyping result of Usp16 deleted ESCs (bottom panel). PCR primers used for genotyping are indicated in the top panel.b. Phase contrast images (top panels) and alkaline phosphatase staining images (bottom panels) of Usp16+/+ and Usp16−/− ESCs. Scale bar, 50μm.c. Western blot analysis of Usp16 (top panel), H2A ubiquitination (third panel), and H2B ubiquitination (fifth panel) levels in two independent Usp16+/+ and Usp16−/− ESC lines. The quantitation of ubH2A signals was labeled. Signals in control Usp16+/+ ESCs were arbitrarily set as 1. GAPDH, histone H2A, H2B, and H3 were used as loading controls.d. Western blot analysis of the levels of pluripotent genes and PRC subunits in two independent control Usp16+/+ and Usp16−/− ESC lines. β-tubulin was used as a loading control.e. Growth curve of two independent control Usp16+/+ and Usp16−/− ESC lines. 1 × 104 ESCs were seeded in 12 well plates and cell numbers were counted every other day. The same seeding procedure was repeated every other day.f. Fluorescence activated cell sorting (FACS) analysis of control Usp16+/+ and Usp16−/− ESC lines. The percentages of cell populations at each cell cycle phase were labeled.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4060806&req=5

Figure 2: Usp16 knockout does not affect ESC viability and identitya. Schematic representation of the strategy used to delete Usp16 (top panels) and an image of genotyping result of Usp16 deleted ESCs (bottom panel). PCR primers used for genotyping are indicated in the top panel.b. Phase contrast images (top panels) and alkaline phosphatase staining images (bottom panels) of Usp16+/+ and Usp16−/− ESCs. Scale bar, 50μm.c. Western blot analysis of Usp16 (top panel), H2A ubiquitination (third panel), and H2B ubiquitination (fifth panel) levels in two independent Usp16+/+ and Usp16−/− ESC lines. The quantitation of ubH2A signals was labeled. Signals in control Usp16+/+ ESCs were arbitrarily set as 1. GAPDH, histone H2A, H2B, and H3 were used as loading controls.d. Western blot analysis of the levels of pluripotent genes and PRC subunits in two independent control Usp16+/+ and Usp16−/− ESC lines. β-tubulin was used as a loading control.e. Growth curve of two independent control Usp16+/+ and Usp16−/− ESC lines. 1 × 104 ESCs were seeded in 12 well plates and cell numbers were counted every other day. The same seeding procedure was repeated every other day.f. Fluorescence activated cell sorting (FACS) analysis of control Usp16+/+ and Usp16−/− ESC lines. The percentages of cell populations at each cell cycle phase were labeled.
Mentions: Since Usp16−/− ESCs generated by the above described approach have been subjected to multiple rounds of electroporation, which might alter cellular functions, we also derived Usp16−/− ESCs by an alternative approach. We took advantage of Usp16 conditional knockout mice, which were generated by injecting Usp162Lox ESCs (Supplementary Fig. 1a) into blastocysts (data not published). These conditional Usp16 knockout (Usp162lox/2lox) mice were crossed with the inducible Cre expression mouse line, CAGG-Cre, to obtain Usp162lox/+: CAGG-Cre mice. These mice were then interbred or bred with Usp162lox/2lox mice. ESCs were derived from the offspring of these crossings and Usp162lox/2lox: CAGG-Cre ESCs were identified by PCR mediated genotyping and confirmed by sequencing (Fig. 2a). Usp16 was deleted by culturing Usp162lox/2lox:CAGG-Cre ESCs with 1μM 4-hydroxytamoxifen (4-OHT), which induces nuclear localization of the Cre recombinase, for 3–5 days (Fig. 2a) before switching to 4-OHT free medium. The efficiency of Usp16 deletion was confirmed by western blot assay (Fig. 2c). Consistent with our previous studies, Usp16 knockout caused a specific increase of ubH2A levels, but had no effect on ubH2B levels (Fig. 2c, the intensity of ubH2A signal were quantified and labeled). Usp16−/− ESCs exhibited normal morphology (Fig. 2b) and have growth rates (Fig. 2e) and cell cycle profiles (Fig. 2f) similar to Usp16+/+ ESCs. This contrasts with studies in HeLa cells, where USP16 deletion causes a slow growth phenotype, partially due to the defects of H2A deubiquitination during cell cycle G2/M phase progression26,41. Usp16−/− ESCs stained positively for alkaline phosphatase (Fig. 2b). Pluripotent genes Oct4, Sox2, and Nanog and PRC subunits Ring1B, Suz12, and Ezh2) were not affected by Usp16 deletion (Fig. 2d). Together, these studies demonstrate that Usp16 regulates ubH2A levels in ESCs, and is not required for ESC viability and identity.

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