Anti-CHAF1A monoclonal antibody, clone 2D3

Epigenetic silencing is mediated by families of factors that place, remove, read, and transmit repressive histone and DNA methylation marks on chromatin. How the roles for these functionally diverse factors are specified and integrated is the subject of intense study. To address these questions, HeLa cells harboring epigenetically silent green fluorescent protein reporter genes were interrogated with a small interference RNA library targeting 200 predicted epigenetic regulators, including potential activators, silencers, chromatin remodelers, and ancillary factors. Using this approach, individual, or combinatorial requirements for specific epigenetic silencing factors could be detected by measuring green fluorescent protein reactivation after small interference RNA-based factor knockdown. In our analyses, we identified a specific subset of 15 epigenetic factors that are candidates for participation in a functional epigenetic silencing network in human cells. These factors include histone deacetylase 1, de novo DNA methyltransferase 3A, components of the polycomb PRC1 complex (RING1 and HPH2), and the histone lysine methyltransferases KMT1E and KMT5C. Roles were also detected for two TRIM protein family members, the cohesin component Rad21, and the histone chaperone CHAF1A (CAF-1 p150). Remarkably, combinatorial knockdown of factors was not required for reactivation, indicating little functional redundancy. Consistent with this interpretation, knockdown of either KMT1E or CHAF1A resulted in a loss of multiple histone-repressive marks and concomitant gain of activation marks on the promoter during reactivation. These results reveal how functionally diverse factors may cooperate to maintain gene silencing during normal development or in disease. Furthermore, the findings suggest an avenue for discovery of new targets for epigenetic therapies.

Maintenance of a genome requires DNA repair integrated with chromatin remodeling. We have analyzed six transcriptome data sets and one data set on translational regulation of known DNA repair and remodeling genes in synchronized human cells. These data are available through our new database: www.dnarepairgenes.com. Genes that have similar transcription profiles in at least two of our data sets generally agree well with known protein profiles. In brief, long patch base excision repair (BER) is enriched for S phase genes, whereas short patch BER uses genes essentially equally expressed in all cell cycle phases. Furthermore, most genes related to DNA mismatch repair, Fanconi anemia and homologous recombination have their highest expression in the S phase. In contrast, genes specific for direct repair, nucleotide excision repair, as well as non-homologous end joining do not show cell cycle-related expression. Cell cycle regulated chromatin remodeling genes were most frequently confined to G1/S and S. These include e.g. genes for chromatin assembly factor 1 (CAF-1) major subunits CHAF1A and CHAF1B; the putative helicases HELLS and ATAD2 that both co-activate E2F transcription factors central in G1/S-transition and recruit DNA repair and chromatin-modifying proteins and DNA double strand break repair proteins; and RAD54L and FtAD54B involved in double strand break repair. TOP2A was consistently most highly expressed in G2, but also expressed in late S phase, supporting a role in regulating entry into mitosis. Translational regulation complements transcriptional regulation and appears to be a relatively common cell cycle regulatory mechanism for DNA repair genes. Our results identify cell cycle phases in which different pathways have highest activity, and demonstrate that periodically expressed genes in a pathway are frequently co-expressed. Furthermore, the data suggest that S phase expression and over-expression of some multifunctional chromatin remodeling proteins may set up feedback loops driving cancer cell proliferation. (C) 2015 The Authors. Published by Elsevier B.V.