Identification of a Functional Network of Human Epigenetic Silencing Factors

Abstract

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.Epigenetic processes control the binary on-off states of specific gene sets, thereby creating heritable transcription patterns that drive development and maintain cellular identity. It is now well established that chromatin-based mechanisms underlie such epigenetic control, largely mediated by intricate chemical marks that are placed or removed by chromatin-modifying enzymes (1-8).The prominent epigenetic regulatory marks on eukaryotic chromatin are histone modifications and DNA cytosine methylation (5meCpG), which are placed by enzyme complexes containing members of the histone modifying and DNA methyltransferase (DNMT) 4 families, respectively. The histone tails that protrude from nucleosomes are thus decorated by a variety of position-specific "histone code" marks, including acetyl, methyl, and ubiquitin lysine modifications. In contrast, DNAbased epigenetic regulation is limited to DNA methylation. Histone marks may be either activating or repressive, whereas the 5meCpG DNA mark is strictly repressive. The presence or absence of these chromatin marks provide cues for recruitment of downstream protein effectors that positively or negatively affect transcription or may also d...

Human factors and pathways essential for mediating epigenetic gene silencing

“…The reporter cell population harbors chromosomally dispersed, epigenetically silent GFP genes, thus favoring detection of multiple classes of silencing mechanisms, rather than idiosyncratic, locus-specific mechanisms. This reporter cell system was used previously to carry out an siRNA knockdown screen with a biased epigenetic factor set (200 genes/factors) 23 . The GFP signal after target gene knockdown was used as readout, providing a quantitative measure of reporter gene reactivation (Fig.…”

Assembly of Helper-Dependent Adenovirus DNA into Chromatin Promotes Efficient Gene Expression

“…Thus, the cell can epigenetically modulate expression from the vector through changes in histone modification, leading to inhibition of gene expression from certain invading DNAs. Epigenetic silencing is not unique to Ad and has been observed for both integrated (54,55) and episomal (11,57,72) vectors. The fact that hdAd containing eukaryotic-derived stuffer DNA escapes epigenetic shutdown likely reflects its strong resemblance to cellular DNA (e.g., identical GC and CpG dinucleotide contents, correct nucleosome placement and phasing, etc.…”

Selective Recognition of H3.1K36 Dimethylation/H4K16 Acetylation Facilitates the Regulation of All-trans-retinoic Acid (ATRA)-responsive Genes by Putative Chromatin Reader ZMYND8

“…Manner-ZMYND8 is a component of the coregulator complex network having an extensive interaction with transcription machinery (5,38). Recently ZMYND8 has been established to be a component of the NuRD chromatin remodeling complex (39) and can influence gene transcription (5).…”