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Assessing effect of CEBPA degradation on rRNA transcription machinery

ABSTRACT: We report the effect of degradation of CEBPA (a critical myeloid lineage transcription factor) on the occupancy of core rRNA transcription machinery on rDNA in mouse GMP cells. We generated a CEBPA-Degron line by tagging endogenous alleles of the Cebpa gene with the FKBPV degron domain, and degraded CEBPA-FKBPV-FLAG fusion protein using dTAGV-1 ligand. We used anti-FLAG pulldown to demonstrate binding of CEBPA protein to rDNA at a conserved motif within the 18S transcribed region. On degradation of CEBPA, we found that RPA194 (component of Pol I) and RRN3 occupancy on rDNA were reduced, while the occupancy of upstream factors TAF1B (component of SL-1) and UBTF were unchanged. In parallel, we also found that CEBPA degradation reduced nascent rRNA transcription, cellular ribosome abundance, and cell growth. Our work indicates that the cell-type-specific transcription factor CEBPA recruits the Pol I-RRN3 complex to ribosomal DNA to promote rRNA transcription.

ORGANISM(S): Mus musculus

PROVIDER: GSE193569 | GEO | 2022/01/15

REPOSITORIES: GEO

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BMH-21 inhibits RNA polymerase I transcription cycle and rDNA occupancy

Project description:The objective of this study was to determine the effect of a small-molecule Pol I inhibitor, BMH-21, on rRNA synthesis in vivo. NET-seq was performed to determine the Pol I occupancy after BMH-21 treatment, as compared to vehicle-treatment (phosphate buffer control). Our findings suggest that BMH-21 treatment reduces Pol I occupancy on the rDNA template. Additionally, BMH-21 induces repositioning of Pol I in AT-rich rDNA regions that are directly upstream from GC-rich regions. This study suggests that BMH-21 is a powerful inhibitor of transcription by Pol I, and gives a potential mechanism of action for this inhibitor in vivo.

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Determining the effect of RPA49 on RNA polymerase I occupancy in vivo

Project description:The central goal of this project was to determine the role of the RNA polymerase I (Pol I) subunits RPA34 and RPA49 in rRNA synthesis. Previous studies by various groups have demonstrated that these two subunits may play a role in transcription initiation and elongation by on Pol I occupancy in vivo, but this is still not clearly defined. Here, we deployed a high-resolution technique, native elongating transcript sequencing (NET-seq), to test the effects of RPA34 and RPA49 on Pol I occupancy in vivo. We found that when RPA34 was deleted, there was a significant reduction in Pol I occupancy across the rDNA template. Additionally, when RPA49 was deleted, Pol I occupancy was even further reduced across the template. Collectively, our findings suggest that perhaps RPA34 acts to stabilize RPA49 and these subunits may act primarily as transcription initiation factors for Pol I.

2023-05-22 | GSE232731 | GEO

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FGF2 antiproliferative stimulus triggers proteomic and transcriptional changes associated with nucleolar disorganization in K-ras-driven mouse tumor cells (Nucleoli TMT)

Project description:Chromatin plays a crucial role in the intermediation between cell signaling and gene expression. The nucleolus is sensitive to stress and can orchestrate a chain of cellular events in response to stress signals. Despite being a growth factor, FGF2 has anti-proliferative and tumor-suppressive functions in some cellular contexts. In this work, we investigated how the antiproliferative effect of FGF2 modulates chromatin, nucleolus, and rDNA-associated proteins. The chromatin and nucleolar proteome indicated that FGF2 stimulation modulates proteins related to transcription regulation, particularly rRNA expression, and chromatin remodeling proteins. Upon 24 hrs of FGF2 stimulation, the global transcriptional rate and nucleolus area increased in associationalong with intense nucleolar disorganization detected by fibrillarin dispersion and electron microscopy analyses. We confirmed that FGF2 stimulation induced immature rRNA accumulation by increasing rRNA transcription regardless of changes in ribosome profiling. The rDNA-associated protein analysis reinforced that FGF2 stimulus interferes with transcription and rRNA processing/modification, since the proteins Nolc1 and Tcof1 are were upregulated after FGF2 stimulation. Changes in rRNA expression may be crucial for triggering the antiproliferative effect induced by FGF2 since inhibiting RNA Pol I, responsible for rRNA expression, partially reversed the growth arrest induced by FGF2. Taken together, we demonstrate that the antiproliferative FGF2 stimulus triggers significant transcriptional changes, and modulation modulates of the main cell transcription site, the nucleolus, directly modulating the proteome of the rDNA loci.

2022-10-12 | MSV000090518 | MassIVE

FGF2 antiproliferative stimulus triggers proteomic and transcriptional changes associated with nucleolar disorganization in K-ras-driven mouse tumor cells (CLASP MudPIT data)

2022-10-12 | MSV000090516 | MassIVE

FGF2 antiproliferative stimulus triggers proteomic and transcriptional changes associated with nucleolar disorganization in K-ras-driven mouse tumor cells (Chromatin MudPIT data)

2022-10-11 | MSV000090511 | MassIVE

Assessing effect of CEBPA degradation on rRNA transcription machinery

Project description:Assessing effect of CEBPA degradation on rRNA transcription machinery

| PRJNA796604 | ENA

Glycogen synthase kinase (GSK) 3β phosphorylates and protects nuclear myosin 1c from proteasome-mediated degradation to activate rDNA transcription in early G1 cells

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2014-05-02 | E-GEOD-57153 | biostudies-arrayexpress

In beta-actin knockout mouse embryonic fibroblasts epigenetic reprogramming and chromatin modifications lead to rDNA transcription inactivation and defects in cell growth and proliferation

Project description:Actin and nuclear myosin 1 (NM1) are emerging as regulators of transcription and chromatin organization. Using a genome-wide approach we report here that β-actin binds both intergenic and genic regions across the entire mammalian genome, associated with both protein-coding and rRNA genes. Across the rDNA transcription unit the distribution of β-actin correlated with NM1 and the other subunits of the B-WICH complex, WSTF and SNF2h. In β-actin-/- mouse embryonic fibroblasts (MEFs), we found that rRNA synthesis levels are down-regulated concomitantly with drops in Pol I and NM1 occupancies across the rRNA gene. In knock-in experiments, reintroduction of wild-type β-actin but not mutated forms with polymerization defects, rescued rRNA synthesis underscoring the direct role for β-actin in Pol I transcription and the importance of polymerization during the transcription process. The rRNA defects in the β-actin-/-MEFs are accompanied by epigenetic reprogramming that leads to up-regulation of the repressive mark H3K4me1. In addition, a high resolution chromatin accessibility assay showed that the absence of β-actin leads to a more compact chromatin at specific locations, including promoter-proximal enhancer (T0 sequence) and Sal boxes (T1-T10), disturbing binding of the transcription termination factor 1 (TTF1). We propose a novel mechanism where the polymerase-associated β-actin synergizes with NM1 to coordinate the establishment of permissive chromatin with the correct rDNA topology for Pol I transcription enhancement.

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