Human embryonic stem cells (hESCs) are unique pluripotent cells capable of self-renewal and differentiation into all three germ layers. To date, more cell surface markers capable of reliably identifying hESCs are needed. The epithelial cell adhesion molecule (EpCAM) is a type I transmembrane glycoprotein expressed in several progenitor cell populations and cancers. It has been used to enrich cells with tumor-initiating activity in xenograft transplantation studies. Here, we comprehensively profile the expression of EpCAM by immunofluorescence microscopy, Western blotting, and flow cytometry using an anti-EpCAM monoclonal antibody (mAb) OC98-1. We found EpCAM to be highly and selectively expressed by undifferentiated rather than differentiated hESCs. The protein and transcript level of EpCAM rapidly diminished as soon as hESC had differentiated. This silencing was closely and exclusively associated with the radical transformation of histone modification at the EpCAM promoter. Moreover, we demonstrated that the dynamic pattern of lysine 27 trimethylation of histone 3 was conferred by the interplay of SUZ12 and JMJD3, both of which were involved in maintaining hESC pluripotency. In addition, we used chromatin immunoprecipitation analysis to elucidate the direct regulation by EpCAM of several reprogramming genes, including c-MYC, OCT-4, NANOG, SOX2, and KLF4, to help maintain the undifferentiation of hESCs. Collectively, our results suggest that EpCAM might be used as a surface marker for hESC. The expression of EpCAM may be regulated by epigenetic mechanisms, and it is strongly associated with the maintenance of the undifferentiated state of hESCs.

The influence of mono-ubiquitylation of histone H2B (H2Bub) on transcription via nucleosome reassembly has been widely documented. Recently, it has also been shown that H2Bub promotes recovery from replication stress; however, the underling molecular mechanism remains unclear. Here, we show that H2B ubiquitylation coordinates activation of the intra-S replication checkpoint and chromatin re-assembly, in order to limit fork progression and DNA damage in the presence of replication stress. In particular, we show that the absence of H2Bub affects replication dynamics (enhanced fork progression and reduced origin firing), leading to γH2A accumulation and increased hydroxyurea sensitivity. Further genetic analysis indicates a role for H2Bub in transducing Rad53 phosphorylation. Concomitantly, we found that a change in replication dynamics is not due to a change in dNTP level, but is mediated by reduced Rad53 activation and destabilization of the RecQ helicase Sgs1 at the fork. Furthermore, we demonstrate that H2Bub facilitates the dissociation of the histone chaperone Asf1 from Rad53, and nucleosome reassembly behind the fork is compromised in cells lacking H2Bub. Taken together, these results indicate that the regulation of H2B ubiquitylation is a key event in the maintenance of genome stability, through coordination of intra-S checkpoint activation, chromatin assembly and replication fork progression. Eukaryotic DNA is organized into nucleosomes, which are the fundamental repeating units of chromatin. Coordination of chromatin structure is required for efficient and accurate DNA replication. Aberrant DNA replication results in mutations and chromosome rearrangements that may be associated with human disorders. Therefore, cellular surveillance mechanisms have evolved to counteract potential threats to DNA replication. These mechanisms include checkpoints and specialized enzymatic activities that prevent the replication and segregation of defective DNA molecules. We employed a genome-wide approach to investigate how chromatin structure affects DNA replication under stress. We report that coordination of chromatin assembly and checkpoint activity by a histone modification, H2B ubiquitylation (H2Bub), is critical for the cell response to HU-induced replication stress. In cells with a mutation that abolishes H2Bub, replication progression is enhanced, and the forks are more susceptible to damage by environmental insults. The replication proteins on replicating DNA are akin to a train on the tracks, and movement of this train is carefully controlled. Our data indicate that H2Bub helps organize DNA in the nuclei during DNA replication; this process plays a similar role to the brakes on a train, serving to slow down replication, and maintaining stable progression of replication under environmental stress.

DNA lesion bypass is mediated by DNA damage tolerance (DDT) pathways and homologous recombination (HR). The DDT pathways, which involve translesion synthesis and template switching (TS), are activated by the ubiquitylation (ub) of PCNA through components of the RAD6-RAD18 pathway, whereas the HR pathway is independent of RAD18. However, it is unclear how these processes are coordinated within the context of chromatin. Here we show that Bre1, an ubiquitin ligase specific for histone H2B, is recruited to chromatin in a manner coupled to replication of damaged DNA. In the absence of Bre1 or H2Bub, cells exhibit accumulation of unrepaired DNA lesions. Consequently, the damaged forks become unstable and resistant to repair. We provide physical, genetic, and cytological evidence that H2Bub contributes toward both Rad18-dependent TS and replication fork repair by HR. Using an inducible system of DNA damage bypass, we further show that H2Bub is required for the regulation of DDT after genome duplication. We propose that Bre1-H2Bub facilitates fork recovery and gap-filling repair by controlling chromatin dynamics in response to replicative DNA damage. Significance DNA damage-bypass mechanisms that facilitate the resolution of replication blocks in proliferating cells during and after S phase are important for the defense against damage-induced mutations, genome instability, and cancer. Lesion bypass, mediated either by the ubiquitylation of the replication factor proliferating cell nuclear antigen or by homologous recombination, takes place in the context of chromatin. However, the implications of nucleosome dynamics and chromosome packaging in the efficiency of replication-associated damage processing are still largely unknown. Our physical, genetic, and cytological studies suggest that ubiquitylation of histone H2B facilitates the replicative bypass of fork-stalling DNA lesions by contributing to both DNA damage tolerance and homologous recombination during and after replication, thus revealing a direct link between chromatin architecture and lesion bypass.

Transcription-replication conflicts (TRCs) occur when intensive transcriptional activity compromises replication fork stability, potentially leading to gene mutations. Transcription-deposited H3K4 methylation (H3K4me) is associated with regions that are susceptible to TRCs; however, the interplay between H3K4me and TRCs is unknown. Here we show that H3K4me aggravates TRC-induced replication failure in checkpoint-defective cells, and the presence of methylated H3K4 slows down ongoing replication. Both S-phase checkpoint activity and H3K4me are crucial for faithful DNA synthesis under replication stress, especially in highly transcribed regions where the presence of H3K4me is highest and TRCs most often occur. H3K4me mitigates TRCs by decelerating ongoing replication, analogous to how speed bumps slow down cars. These findings establish the concept that H3K4me defines the transcriptional status of a genomic region and defends the genome from TRC-mediated replication stress and instability.

Neurons and astrocytes are generated from common neural precursors, yet neurogenesis precedes astrocyte formation during embryogenesis. The mechanisms of neural development underlying suppression and de-suppression of differentiation- related genes for cell fate specifications are not well understood. By using an in vitro system in which NTera-2 cells were induced to differentiate into an astrocyte-like lineage, we revealed a novel role for Sin3A in maintaining the suppression of GFAP in NTera-2 cells. Sin3A coupled with MeCP2 bound to the GFAP promoter and their occupancies were correlated with repression of GFAP transcription. The repression by Sin3A and MeCP2 may be an essential mechanism underlying the inhibition of cell differentiation. Upon commitment toward an astrocyte-like lineage, Sin3A- MeCP2 departed from the promoter and activated STAT3 simultaneously bound to the promoter and exon 1 of GFAP; meanwhile, olig2 was exported from nuclei to the cytoplasm. This suggested that a three-dimensional or higher-order structure was provoked by STAT3 binding between the promoter and proximal coding regions. STAT3 then recruited CBP/p300 to exon 1 and targeted the promoter for histone H3K9 and H3K14 acetylation. The CBP/p300-mediated histone modification further facilitates chromatin remodeling, thereby enhancing H3K4 trimethylation and recruitment of RNA polymerase II to activate GFAP gene transcription. These results provide evidence that exchange of repressor and activator complexes and epigenetic modifications are critical strategies for cellular differentiation and lineage-specific gene expression.

Histones are subject to several post-translational modifications, which act to regulate gene expression and other processes on the DNA template. One such modification is the addition of a single ubiquitin moiety, which has been reported to influence chromatin dynamics and exhibit cross-talk with other histone modifications. Mono-ubiquitylation of H2B has been reported in eukaryotes as divergent as budding yeast, flies and humans, and is linked to transcriptional activation and gene silencing. Furthermore, ubiquitylation of H2A is also important for transcriptional repression in higher eukaryotes, and both histones play key roles in DNA repair. In this review, we will give an overview of the enzymes important for ubiquitylation and deubiquitylation of the various histone species, before examining the role of ubiquitylated histones in shaping the chromatin landscape and thus controlling the accessibility of DNA to effector proteins, through putative roles in promoting histone-histone interactions and stabilizing the structure of nucleosomes. We will finally discuss other processes reported to involve ubiquitylation of histones, including DNA repair, recombination and mRNA processing, underlining the diverse actions of these modifications.