Figure 7: Usp16 but not the enzymatically inactive mutant rescues the undifferentiated phenotype of Usp16−/− ESCs. | Nature Communications

Figure 7: Usp16 but not the enzymatically inactive mutant rescues the undifferentiated phenotype of Usp16−/− ESCs.

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

Figure 7

(a) 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 were arbitrarily set as 1. GAPDH and histone H3 were used as loading controls. Original scans for blots are shown in Supplementary Fig. 7. (b) Haematoxylin 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 bar, 100 μm. (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+s.d. 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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