The phosphorylation of histone variant H2AX at DNA double-strand breaks is believed to be critical for recognition and repair of DNA damage. However, little is known about the molecular mechanism regulating the exchange of variant H2AX with conventional H2A in the context of the nucleosome. Here, we isolate the H2AX-associated factors, which include FACT (Spt16/SSRP1), DNA-PK, and PARP1 from a human cell line. Our analyses demonstrate that the H2AX-associated factors efficiently promote both integration and dissociation of H2AX and this exchange reaction is mainly catalyzed by FACT among the purified factors. The phosphorylation of H2AX by DNA-PK facilitates the exchange of nucleosomal H2AX by inducing conformational changes of the nucleosome. In contrast, poly-ADP-ribosylation of Spt16 by PARP1 significantly inhibits FACT activities for H2AX exchange. Thus, these data establish FACT as the major regulator involved in H2AX exchange process that is modulated by H2AX phosphorylation and Spt16 ADP-ribosylation.

Summary DNA-bound transcription factors recruit many coactivator proteins to remodel chromatin and activate transcription. The Mediator complex is believed to recruit RNA polymerase II to most protein-encoding genes. It is generally assumed that interaction of Mediator subunits with DNA-binding transcription factors is responsible for Mediator recruitment to promoters. However, we report here that Mediator recruitment by nuclear receptors (NR) requires a new coactivator protein, CCAR1 (cell cycle and apoptosis regulator 1). CCAR1 associates with components of the Mediator and p160 coactivator complexes and is recruited to endogenous NR target genes in response to the appropriate hormone. Reduction of endogenous CCAR1 levels inhibited hormone-induced expression of endogenous NR target genes, hormone-induced recruitment of Mediator components and RNA polymerase II to target gene promoters, and estrogen-dependent growth of breast cancer cells. Thus, CCAR1 regulates expression of key proliferation inducing genes. CCAR1 also functions as a p53 coactivator, suggesting a broader role in transcriptional regulation.

SUMMARY Histone modifications play important roles in the regulation of gene expression and chromatin organization. VprBP has been implicated in transcriptionally silent chromatin formation and cell cycle regulation, but the molecular basis underlying such effects remains unclear. Here we report that VprBP possesses an intrinsic protein kinase activity and is capable of phosphorylating histone H2A on threonine 120 (H2AT120p) in a nucleosomal context. VprBP is localized to a large set of tumor suppressor genes and blocks their transcription, in a manner that is dependent on its kinase activity toward H2AT120. The functional significance of VprBP-mediated H2AT120p is further underscored by the fact that RNAi knockdown and small-molecule inhibition of VprBP reactivate growth regulatory genes and impede tumor growth. Our findings establish VprBP as a major kinase responsible for H2AT120p in cancer cells and suggest that VprBP inhibition could be a new strategy for the development of anticancer therapeutics.

Linker histone H1 has been generally viewed as a global repressor of transcription by preventing the access of transcription factors to sites in chromatin. However, recent studies suggest that H1 can interact with other regulatory factors for its action as a negative modulator of specific genes. To investigate these aspects, we established a human cell line expressing H1.2, one of the H1 subtypes, for the purification of H1-interacting proteins. Our results showed that H1.2 can stably associate with sets of cofactors and ribosomal proteins that can significantly repress p53-dependent, p300-mediated chromatin transcription. This repressive action of H1.2 complex involves direct interaction of H1.2 with p53, which in turn blocks p300-mediated acetylation of chromatin. YB1 and PUR␣, two factors present in the H1.2 complex, together with H1.2 can closely recapitulate the repressive action of the entire H1.2 complex in transcription. Chromatin immunoprecipitation and RNA interference analyses further confirmed that the recruitment of YB1, PUR␣, and H1.2 to the p53 target gene Bax is required for repression of p53-induced transcription. Therefore, these results reveal a previously unrecognized function of H1 as a transcriptional repressor as well as the underlying mechanism involving specific sets of factors in this repression process.Histones are the major protein components to compact genomic DNA into the limited volume of the nucleus as a highly organized chromatin structure. The basic element of chromatin is the nucleosome, which consists of 146 base pairs of DNA wrapped around an octameric core of histones containing two molecules each of H2A, H2B, H3, and H4 (1-4). This repeating unit of chromatin is associated with another type of histone called linker histone H1 to achieve an additional level of compaction, making genes inaccessible to transcription factors and preventing their expression (5-9). Mammalian cells have at least eight histone H1 subtypes including H1.1 through H1.5 and somatic cell-specific H1o as well as germ cell-specific H1t and H1oo, all consisting of a highly conserved globular domain and less conserved N-and C-terminal domains (6,8,10). The existence of multiple H1 subtypes and the diversity of their amino acid sequences raise the possibility that individual subtypes have nonredundant functions in various cellular processes. In addition, the expression of each H1 subtype depends on the tissue, phase of the cell cycle, and developmental stage, further suggesting the specific contribution of linker histone subtypes for regulation of various cellular processes (6,8,11).Although most studies have focused on the contribution of H1 as a structural component of the nucleosome, it is becoming apparent that H1 also acts as a repressor for specific gene transcription (12)(13)(14)(15). This repressive capacity of H1 on transcription appears to be accomplished by its localization at particular chromosomal domains with specific transcription regulators. Msx1 recruits a linker histone H1 to the MyoD gene,...

BackgroundThe incorporation of histone variants into nucleosomes is one of the main strategies that the cell uses to regulate the structure and function of chromatin. Histone H2A.Z is an evolutionarily conserved histone H2A variant that is preferentially localized within nucleosomes at the transcriptional start site (TSS). H2A.Z reorganizes the local chromatin structure and recruits the transcriptional machinery for gene activation. High expression of H2A.Z has been reported in several types of cancers and is causally linked to genomic instability and tumorigenesis. However, it is not entirely clear how H2A.Z overexpression in cancer cells establishes aberrant chromatin states and promotes gene expression.ResultsThrough integration of genome-wide H2A.Z ChIP-seq data with microarray data, we demonstrate that H2A.Z is enriched around the TSS of cell cycle regulatory genes in bladder cancer cells, and this enrichment is correlated with the elevated expression of cancer-promoting genes. RNAi-mediated knockdown of H2A.Z in the cancer cells causes transcriptional suppression of multiple cell cycle regulatory genes with a distinct decrease in cell proliferation. H2A.Z nucleosomes around the TSS have higher levels of H3K4me2/me3, which coincides with the recruitment of two chromatin factors, WDR5 and BPTF. The observed recruitment is functional, as the active states of H2A.Z target genes are largely erased by suppressing the expression of WDR5 or BPTF, effects resembling H2A.Z knockdown.ConclusionsWe conclude that H2A.Z is overexpressed in bladder cancer cells and contributes to cancer-related transcription pathways. We also provide evidence in support of the engagement of H3K4me2/me3 and WDR5/BPTF in H2A.Z-induced cancer pathogenesis. Further studies are warranted to understand how H2A.Z overexpression contributes to the recruitment of the full repertoire of transcription machinery to target genes in bladder cancer cells.