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Transcription of ribosomal RNA (rRNA) by RNA Polymerase I (Pol I) is often upregulated in cancer to facilitate rapid cell growth and proliferation, and has emerged as a potential target for chemotherapeutic agents. BMH-21 and Pt(II) chemotherapeutic agent oxaliplatin are well documented as inhibitors of Pol I activity, however the underlying mechanisms for this inhibition are not completely understood. Here, we applied chromatin immunoprecipitation sequencing (ChIP-seq) techniques and immunofluorescence imaging to probe the influence of oxaliplatin and BMH-21 on Pol I machinery. We demonstrate oxaliplatin and BMH-21 induce early nucleolar stress leading to the formation of “nucleolar caps” containing Pol I and upstream binding factor (UBF) which corresponds with broad reductions in ribosomal DNA (rDNA) occupancy of Pol I. Distinct occupancy patterns for the two compounds are revealed in ChIP-seq experiments. Taken together, our findings suggest that in vivo, oxaliplatin does not induce Pol I inhibition via interrupting a specific step in Pol I transcription, while treatment with BMH-21 induced unique polymerase stalling at the promoter and terminator regions of the human ribosomal RNA gene.
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Transcription inhibition suppresses nuclear blebbing and rupture independent of nuclear rigidity
Chromatin is an essential component of nuclear mechanical response and shape that maintains nuclear compartmentalization and function. However, major genomic functions, such as transcription activity, might also impact cell nuclear shape via blebbing and rupture through their effects on chromatin structure and dynamics. To test this idea, we inhibited transcription with several RNA polymerase II inhibitors in wild type cells and perturbed cells that present increased nuclear blebbing. Transcription inhibition suppresses nuclear blebbing for several cell types, nuclear perturbations, and transcription inhibitors. Furthermore, transcription inhibition suppresses nuclear bleb formation, bleb stabilization, and bleb-based nuclear ruptures. Interestingly, transcription inhibition does not alter either H3K9 histone modification state, nuclear rigidity, or actin compression and contraction, which typically control nuclear blebbing. Polymer simulations suggest that RNA pol II motor activity within chromatin could drive chromatin motions that deform the nuclear periphery. Our data provide evidence that transcription inhibition suppresses nuclear blebbing and rupture, separate and distinct from chromatin rigidity.
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- Award ID(s):
- 2204312
- PAR ID:
- 10465654
- Publisher / Repository:
- The Company of Biologists
- Date Published:
- Journal Name:
- Journal of Cell Science
- ISSN:
- 0021-9533
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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