Polycomb repressive complex two (PRC2) has been implicated in embryonic stem (ES) cell pluripotency; however, the mechanistic roles of this complex are unclear. It was assumed that ES cells contain PRC2 with the same subunit composition as that identified in HeLa cells and Drosophila embryos. Here, we report that PRC2 in mouse ES cells contains at least three additional subunits: JARID2, MTF2, and a novel protein denoted esPRC2p48. JARID2, MTF2, and esPRC2p48 are highly expressed in mouse ES cells compared to differentiated cells. Importantly, knockdowns of JARID2, MTF2, or esPRC2p48 alter the level of PRC2-mediated H3K27 methylation and result in the expression of differentiation-associated genes in ES cells. Interestingly, expression of JARID2, MTF2, and esPRC2p48 together, but not individually, enhances Oct4/Sox2/Klf4-mediated reprograming of mouse embryonic fibroblasts (MEFs) into induced pluripotent stem cells, whereas knockdown or knockout of JARID2, MTF2, or esPRC2p48 significantly inhibits reprograming. JARID2, MTF2, and esPRC2p48 modulate H3K27 methylation and facilitate repression of lineage-associated gene expression when transduced into MEFs, and synergistically stimulate the histone methyl-transferase activity of PRC2 in vitro. Therefore, these studies identify JARID2, MTF2, and esPRC2p48 as important regulatory subunits of PRC2 in ES cells and reveal critical functions of these subunits in modulating PRC2’s activity and gene expression both in ES cells and during somatic cell reprograming.

Post-translational histone modifications play important roles in regulating chromatin structure and function. Histone H2B ubiquitination and deubiquitination have been implicated in transcriptional regulation, but the function of H2B deubiquitination is not well defined, particularly in higher eukaryotes. Here we report the purification of ubiquitin-specific peptidase 49 (USP49) as a histone H2B-specific deubiquitinase and demonstrate that H2B deubiquitination by USP49 is required for efficient cotranscriptional splicing of a large set of exons. USP49 forms a complex with RuvB-like1 (RVB1) and SUG1 and specifically deubiquitinates histone H2B in vitro and in vivo. USP49 knockdown results in small changes in gene expression but affects the abundance of >9000 isoforms. Exons down-regulated in USP49 knockdown cells show both elevated levels of alternative splicing and a general decrease in splicing efficiency. Importantly, USP49 is relatively enriched at this set of exons. USP49 knockdown increased H2B ubiquitination (uH2B) levels at these exons as well as upstream 39 and downstream 59 intronic splicing elements. Change in H2B ubiquitination level, as modulated by USP49, regulates U1A and U2B association with chromatin and binding to nascent pre-mRNA. Although H3 levels are relatively stable after USP49 depletion, H2B levels at these exons are dramatically increased, suggesting that uH2B may enhance nucleosome stability. Therefore, this study identifies USP49 as a histone H2B-specific deubiquitinase and uncovers a critical role for H2B deubiquitination in cotranscriptional pre-mRNA processing events.

No abstract

Posttranslational histone modifications play an important role in regulating chromatin based nuclear processes including transcription. Of these modifications, histone ubiquitination is among the least understood. Histone ubiquitination predominately targets histones H2A and H2B. While ubiquitination of H2B is evolutionarily conserved from budding yeast to mammals, ubiquitination of H2A has not been detected in budding yeast, worms, or plants. Until recently, studies of histone ubiquitination lagged far behind the study of other histone modifications, largely because antibodies specific for ubiquitinated histones are difficult to generate. Despite this obstacle, the identification of the enzymatic machineries involved in histone ubiquitination, together with the successful use of a combination of genetic and immunoblot approaches to detect ubiquitinated histones, have helped to reveal important regulatory roles for this modification in transcriptional initiation and elongation, cell cycle progression, and DNA damage response. With the aid of the recently developed ubiquitinated histone-specific antibodies, an intriguing link between histone ubiquitination and cancer development has been established. While the enzymes involved in H2B ubiquitination were identified first in budding yeast and subsequently in higher organisms based on gene homology, the identification of the enzymatic machineries involved in H2A ubiquitination largely depended on a biochemical purification approach. The unbiased search for ubiquitin ligases targeting histones also led to the identification of a H3 and H4 ubiquitin ligase. Here we detail a protocol for the biochemical approach to identify histone ubiquitin ligase(s) from HeLa cells. Similar approaches have been successfully used to identify histone methyltransferases, histone demethylases, chromatin remodeling factors, and general transcription factors. So long as an in vitro enzymatic assay can be established, the approach we describe can be easily adapted to identify other histone and non-histone modifying enzymes.

Posttranslational histone modifications play important roles in regulating chromatin structure and function (Martin and Zhang, Nat Rev Mol Cell Biol 6:838-849, 2005; Jenuwein and Allis, Science 293:1074-1080, 2001). One example of such modifications is histone ubiquitination, which occurs predominately on H2A and H2B. Recent studies have highlighted important regulatory roles of H2A ubiquitination in Polycomb group proteins-mediated gene silencing (Wang et al., Nature 431:873-878, 2004; Joo et al., Nature 449:1068-1072, 2007) and H2B ubiquitination in transcription, H3 methylation, and DNA methylation (Zhang, Genes Dev 17:2733-2740, 2003; Sun and Allis, Nature 418:104-108, 2002; Sridhar et al., Nature 447:735-738, 2007). Here we describe methods for in vitro histone ubiquitination and deubiquitination assays. We also describe approaches to investigate the in vivo function of a putative histone ubiquitin ligase and deubiquitinase. These experimental procedures are largely based on our studies in mammalian cells. These methods should provide useful tools for studying this bulky histone modification.