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.

The metabolism of trans-18-'4Cjzeatin was examined in embryos of Phaseolus vulgaris cv Great Northern (GN) and P. lunatus cv Kingston (K) in an attempt to detect genetic variations in organized plant tissues. Metabolites were fractionated by HPLC, and identified by chemical and enzymic tests Metabolism of '4CiZeatin. Immature embryos at three developmental stages (measuring 3, 6, and 9 mm in length) were dissected from the pods under sterile condition. The embryos corresponded to late heart and early and mid cotyledonary stages at the respective length. ['4C]Zeatin (0.05 MCi, 0.002 umol) dissolved in 250 ,d of H20 was applied aseptically to 250 mg of immature embryos. The vials were sealed and maintained at 27°C in the dark for 2, 4, and 8 h. In addition, embryos were incubated with radioactively labeled zeatin dissolved in 0.05 M Tris-HCI (pH 6.0) buffer with all other conditions identical to those described above. To determine the amount of radioactivity recovered at time 0, ['4C]zeatin was applied and metabolites extracted immediately. This determination was made for the three sizes of embryos of both genotypes. Each experiment was repeated at least once and averages of two experiments are presented.To extract metabolites, the embryos were homogenized with a Tissuemizer equipped with a Microprobe Shaft (Tekmar) in 2.5 parts (v/w) of cold 95% ethanol. Cell debris was removed by successive filtration through Whatman paper No. 1 and Millipore filters (0.45 ,um). The ethanol extract was taken to dryness in vacuo at 35°C, redissolved in 4 ml of 50% (v/v) ethanol, and centrifuged at 23,500g for 20 min. The supernatant was condensed in vacuo to 100 Ml with a speed vac concentrator (Savant). The sample was then analyzed directly by HPLC. A Beckman Model 110 dual pump HPLC system with a prepacked column of reversed-phase C18 (Ultrasphere ODS 5 gm, 4.6 x 250 mm; Altex) was used. The aqueous buffer consisted of 0.2 M acetic acid, adjusted to pH 3.5 with TEA. Samples were eluted with a 635 www.plantphysiol.org on May 10, 2018 -Published by Downloaded from