Systematic mapping of occluded genes by cell fusion reveals prevalence and stability of cis-mediated silencing in somatic cells

Abstract

Both diffusible factors acting in trans and chromatin components acting in cis are implicated in gene regulation, but the extent to which either process causally determines a cell's transcriptional identity is unclear. We recently used cell fusion to define a class of silent genes termed ''cis-silenced'' (or ''occluded'') genes, which remain silent even in the presence of transacting transcriptional activators. We further showed that occlusion of lineage-inappropriate genes plays a critical role in maintaining the transcriptional identities of somatic cells. Here, we present, for the first time, a comprehensive map of occluded genes in somatic cells. Specifically, we mapped occluded genes in mouse fibroblasts via fusion to a dozen different rat cell types followed by whole-transcriptome profiling. We found that occluded genes are highly prevalent and stable in somatic cells, representing a sizeable fraction of silent genes. Occluded genes are also highly enriched for important developmental regulators of alternative lineages, consistent with the role of occlusion in safeguarding cell identities. Alongside this map, we also present whole-genome maps of DNA methylation and eight other chromatin marks. These maps uncover a complex relationship between chromatin state and occlusion. Furthermore, we found that DNA methylation functions as the memory of occlusion in a subset of occluded genes, while histone deacetylation contributes to the implementation but not memory of occlusion. Our data suggest that the identities of individual cell types are defined largely by the occlusion status of their genomes. The comprehensive reference maps reported here provide the foundation for future studies aimed at understanding the role of occlusion in development and disease.

In vivo myomaker‐mediated heterologous fusion and nuclear reprogramming

“…Our analysis of nuclear reprogramming in vivo indicates that reprogramming is not universally activated because we did not biochemically detect dystrophin expression from nonmuscle nuclei in mdx 4cv mice, but we did observe activation of the Myll locus. This hypothesis is consistent with work correlating the lack of reprogramming of a particular gene with its genomic distance, defined as the transcriptional start site to the end of the 3' untranslated region (50). Given that dystrophin is one of the longest genes in the genome and is likely under the control of numerous genomic regulatory elements, the requirements for activation of dystrophin transcription may be too challenging through reprogramming.…”

Molecular features of cellular reprogramming and development

“…Reprogramming is initiated in the heterokaryon phase, during which both nuclei remain discrete, and includes global epigenetic remodelling that precedes the activation of pluripotent loci 216,217 . In the late heterokaryon phase, select loci are activated through a process that may require DNA replication [218][219][220] . The somatic and pluripotent nuclei fuse after cell division, and additional genes are then reprogrammed to consolidate the pluripotent network within the somatic genome 218,220 .…”

“…In the late heterokaryon phase, select loci are activated through a process that may require DNA replication [218][219][220] . The somatic and pluripotent nuclei fuse after cell division, and additional genes are then reprogrammed to consolidate the pluripotent network within the somatic genome 218,220 . During direct reprogramming, OCT4, SRY-box 2 (SOX2), Krüppel-like factor 4 (KLF4) and MYC (OSKM) are introduced into somatic cells, which respond by increasing proliferation and undergoing local changes to their epigenome.…”

Gene Resistance to Transcriptional Reprogramming following Nuclear Transfer Is Directly Mediated by Multiple Chromatin-Repressive Pathways

“…(G–K) Shown is the (G) DNase I sensitivity (Yue et al., 2014), (H) H3K4me3 (Kaneda et al., 2011), (I) H3K27me3 (Kaneda et al., 2011), (J) H2Aub (this study), and (K) H3K9me3 (Bulut-Karslioglu et al., 2014) metaplot analysis ±5 kB around the TSSs of genes showing resistance in OOC-NT (red, from this study), mouse egg-NT (orange, from Matoba et al., 2014), transcription factor-induced reprogramming (purple, from Soufi et al., 2012), and cell fusion (gray, from Looney et al., 2014). …”