Genome-wide analyses of chromatin interactions after the loss of Pol I, Pol II, and Pol III

Abstract

Background The relationship between transcription and the 3D chromatin structure is debated. Multiple studies have shown that transcription affects global Cohesin binding and 3D genome structures. However, several other studies have indicated that inhibited transcription does not alter chromatin conformations. Results We provide the most comprehensive evidence to date to demonstrate that transcription plays a relatively modest role in organizing the local, small-scale chromatin structures in mammalian cells. We show degraded Pol I, Pol II, and Pol III proteins in mESCs cause few or no changes in large-scale 3D chromatin structures, selected RNA polymerases with a high abundance of binding sites or active promoter-associated interactions appear to be relatively more affected after the degradation, transcription inhibition alters local, small loop domains, as indicated by high-resolution chromatin interaction maps, and loops with bound Pol II but without Cohesin or CTCF are identified and found to be largely unchanged after transcription inhibition. Interestingly, Pol II depletion for a longer time significantly affects the chromatin accessibility and Cohesin occupancy, suggesting that RNA polymerases are capable of affecting the 3D genome indirectly. These direct and indirect effects explain the previous inconsistent findings on the influence of transcription inhibition on the 3D genome. Conclusions We conclude that Pol I, Pol II, and Pol III loss alters local, small-scale chromatin interactions in mammalian cells, suggesting that the 3D chromatin structures are pre-established and relatively stable.

Transcription shapes DNA replication initiation to preserve genome integrity

“…Since transcription inhibitors may have non-specific effects on DNA metabolism, we employed the auxin-inducible degron system to directly eliminate RNA polymerase II from mESCs in an acute manner as previously reported [ 49 ]. Briefly, the catalytic subunit of RNA polymerase II was tagged with a mAID tag, and indole-3-acetic acid (IAA) was added to induce degradation of the fusion protein.…”

rRNA Biogenesis Regulates Mouse 2C-like State by 3D Structure Reorganization of Peri-Nucleolar Heterochromatin

“…To further investigate the critical role of rRNA biogenesis in regulating the 2C program and the homeostasis between mES cells and 2C-like cells, we generated two rRNA biogenesis-inhibited mES cell lines: 1) a line with degraded Pol I protein (PRA1) by an auxin-inducible degron system 61 (Fig S5a), and 2) a snoRNA knockout line (SNORD113-114 gene cluster, a gift from Pengxu Qian) (Fig S5b), as snoRNAs are required for rRNA modification and biogenesis. Using these two cell lines, we performed DNA FISH experiments and found the similar molecular phenotypes of CX-5461-treated mES cells, i.e., the Dux locus and a representative locus within PNH region located further from the peri-nucleolar region (Fig.5a-5d).…”

At the Crossroad of Gene Regulation and Genome Organization: Potential Roles for ATP-Dependent Chromatin Remodelers in the Regulation of CTCF-Mediated 3D Architecture

“…While nucleosome-free regions are required for cohesin translocation [ 78 ], these may be indirectly formed as a consequence of nucleosome remodeling for transcription. Despite this established role for transcription in cohesin translocation, acute depletion of RNA polymerase II only alters local short-range contacts, not large-scale genome architecture or TAD structures in mESCs [ 91 ]. Therefore, cohesin establishment at TADs may require active recruitment of nucleosome remodelers in a manner independent of the transcriptional machinery.…”