Extensive changes in posttranslational histone modifications accompany the rewiring of the transcriptional program during stem cell differentiation. However, the mechanisms controlling the changes in specific chromatin modifications and their function during differentiation remain only poorly understood. We show that histone H2B monoubiquitination (H2Bub1) significantly increases during differentiation of human mesenchymal stem cells (hMSCs) and various lineage-committed precursor cells and in diverse organisms. Furthermore, the H2B ubiquitin ligase RNF40 is required for the induction of differentiation markers and transcriptional reprogramming of hMSCs. This function is dependent upon CDK9 and the WAC adaptor protein, which are required for H2B monoubiquitination. Finally, we show that RNF40 is required for the resolution of the H3K4me3/H3K27me3 bivalent poised state on lineage-specific genes during the transition from an inactive to an active chromatin conformation. Thus, these data indicate that H2Bub1 is required for maintaining multipotency of hMSCs and plays a central role in controlling stem cell differentiation.
SUMMARY The MDM2 oncoprotein ubiquitinates and antagonizes p53 but may also carry out p53-independent functions. Here we report that MDM2 is required for the efficient generation of induced pluripotent stem cells (iPSCs) from murine embryonic fibroblasts, in the absence of p53. Similarly, MDM2 depletion in the context of p53 deficiency also promoted the differentiation of human mesenchymal stem cells and diminished clonogenic survival of cancer cells. Most of the MDM2-controlled genes also responded to the inactivation of the Polycomb Repressor Complex 2 (PRC2) and its catalytic component EZH2. MDM2 physically associated with EZH2 on chromatin, enhancing the trimethylation of histone 3 at lysine 27 and the ubiquitination of histone 2A at lysine 119 (H2AK119) at its target genes. Removing MDM2 simultaneously with the H2AK119 E3 ligase Ring1B/RNF2 further induced these genes and synthetically arrested cell proliferation. In conclusion, MDM2 supports the Polycomb-mediated repression of line-age-specific genes, independent of p53.
Sirtuin 2 (SIRT2) is a sirtuin family deacetylase that directs acetylome signaling, protects genome integrity, and is a murine tumor suppressor. We show that SIRT2 directs replication stress responses by regulating the activity of cyclin-dependent kinase 9 (CDK9), a protein required for recovery from replication arrest. SIRT2 deficiency results in replication stress sensitivity, impairment in recovery from replication arrest, spontaneous accumulation of replication protein A to foci and chromatin, and a G2/M checkpoint deficit. SIRT2 interacts with and deacetylates CDK9 at lysine 48 in response to replication stress in a manner that is partially dependent on ataxia telangiectasia and Rad3 related (ATR) but not cyclin T or K, thereby stimulating CDK9 kinase activity and promoting recovery from replication arrest. Moreover, wild-type, but not acetylated CDK9, alleviates the replication stress response impairment of SIRT2 deficiency. Collectively, our results define a function for SIRT2 in regulating checkpoint pathways that respond to replication stress through deacetylation of CDK9, providing insight into how SIRT2 maintains genome integrity and a unique mechanism by which SIRT2 may function, at least in part, as a tumor suppressor protein.DNA damage | cell cycle checkpoint | genome maintenance M embers of the sirtuin NAD + -dependent deacetylase family regulate multiple biological processes, including genome maintenance, aging, tumorigenesis, differentiation, and metabolism (1-3). There are seven mammalian homologs of yeast silent information regulator 2 (Sir2). Whereas sirtuin 1 (SIRT1) and sirtuin 6 (SIRT6) have been linked directly to DNA repair or DNA damage response (DDR) signaling pathways (4-12), the role for other sirtuin family members, including SIRT2, is less clear. Mice deficient in Sirt2 develop breast, liver, and other cancers (13,14), and SIRT2 expression is decreased in human breast and liver cancers (14), suggesting that SIRT2 is a tumor suppressor protein (TSP). In support of a role for SIRT2 in the DDR, Sirt2 deficiency in mouse embryo fibroblasts (MEFs) results in a mitotic checkpoint deficit, increased DNA damage, and genomic instability (13). In addition, SIRT2 deficiency in chicken DT40 cells induces hypersensitivity to cisplatin (15). SIRT2 also deacetylates histone H3 lysine 56, a marker for packaging of newly replicated and repaired DNA into chromatin (16), implying that SIRT2 may function in the maintenance of genome integrity during DNA replication.The replication stress response (RSR) is a subset of the DDR signaling network that recognizes challenges to DNA replication and mobilizes diverse DNA repair and cell cycle checkpoint pathways. The RSR is critical for the prevention of cancer by acting as a barrier against genomic instability and tumorigenesis (17,18). At the apex of the RSR signaling cascade is the ataxiatelangiectasia and Rad3-related protein (ATR) checkpoint kinase (19). ATR function is essential to stabilize stalled replication forks and promote recovery. Disruption of ...
Unlike other metazoan mRNAs, replication-dependent histone gene transcripts are not polyadenylated but instead have a conserved stem-loop structure at their 3′ end. Our previous work has shown that under certain conditions replication-dependent histone genes can produce alternative transcripts that are polyadenylated at the 3′ end and, in some cases, spliced. A number of microarray studies examining the expression of polyadenylated mRNAs identified changes in the levels of histone transcripts e.g. during differentiation and tumorigenesis. However, it remains unknown which histone genes produce polyadenylated transcripts and which conditions regulate this process. In the present study we examined the expression and polyadenylation of the human histone H2B gene complement in various cell lines. We demonstrate that H2B genes display a distinct expression pattern that is varies between different cell lines. Further we show that the fraction of polyadenylated HIST1H2BD and HIST1H2AC transcripts is increased during differentiation of human mesenchymal stem cells (hMSCs) and human fetal osteoblast (hFOB 1.19). Furthermore, we observed an increased fraction of polyadenylated transcripts produced from the histone genes in cells following ionizing radiation. Finally, we show that polyadenylated transcripts are transported to the cytoplasm and found on polyribosomes. Thus, we propose that the production of polyadenylated histone mRNAs from replication-dependent histone genes is a regulated process induced under specific cellular circumstances.
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