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LEO1 Is Required for Efficient Entry into Quiescence, Control of H3K9 Methylation and Gene Expression in Human Fibroblasts

(1) Background: The LEO1 (Left open reading frame 1) protein is a conserved subunit of the PAF1C complex (RNA polymerase II-associated factor 1 complex). PAF1C has well-established mechanistic functions in elongation of transcription and RNA processing. We previously showed, in fission yeast, that L...

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Autores principales: Laurent, Marc, Cordeddu, Lina, Zahedi, Yasaman, Ekwall, Karl
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2023
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10668985/
https://www.ncbi.nlm.nih.gov/pubmed/38002344
http://dx.doi.org/10.3390/biom13111662
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author Laurent, Marc
Cordeddu, Lina
Zahedi, Yasaman
Ekwall, Karl
author_facet Laurent, Marc
Cordeddu, Lina
Zahedi, Yasaman
Ekwall, Karl
author_sort Laurent, Marc
collection PubMed
description (1) Background: The LEO1 (Left open reading frame 1) protein is a conserved subunit of the PAF1C complex (RNA polymerase II-associated factor 1 complex). PAF1C has well-established mechanistic functions in elongation of transcription and RNA processing. We previously showed, in fission yeast, that LEO1 controls histone H3K9 methylation levels by affecting the turnover of histone H3 in chromatin, and that it is essential for the proper regulation of gene expression during cellular quiescence. Human fibroblasts enter a reversible quiescence state upon serum deprivation in the growth media. Here we investigate the function of LEO1 in human fibroblasts. (2) Methods: We knocked out the LEO1 gene using CRISPR/Cas9 methodology in human fibroblasts and verified that the LEO1 protein was undetectable by Western blot. We characterized the phenotype of the ΔLEO1 knockout cells with FACS analysis and cell growth assays. We used RNA-sequencing using spike-in controls to measure gene expression and spike-in controlled ChIP-sequencing experiments to measure the histone modification H3K9me2 genome-wide. (3) Results: Gene expression levels are altered in quiescent cells, however factors controlling chromatin and gene expression changes in quiescent human cells are largely unknown. The ΔLEO1 knockout fibroblasts are viable but have reduced metabolic activity compared to wild-type cells. ΔLEO1 cells showed a slower entry into quiescence and a different morphology compared to wild-type cells. Gene expression was generally reduced in quiescent wild-type cells. The downregulated genes included genes involved in cell proliferation. A small number of genes were upregulated in quiescent wild-type cells including several genes involved in ERK1/ERK2 and Wnt signaling. In quiescent ΔLEO1 cells, many genes were mis-regulated compared to wild-type cells. This included genes involved in Calcium ion transport and cell morphogenesis. Finally, spike-in controlled ChIP-sequencing experiments demonstrated that the histone modification H3K9me2 levels are globally increased in quiescent ΔLEO1 cells. (4) Conclusions: Thus, LEO1 is important for proper entry into cellular quiescence, control of H3K9me2 levels, and gene expression in human fibroblasts.
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spelling pubmed-106689852023-11-17 LEO1 Is Required for Efficient Entry into Quiescence, Control of H3K9 Methylation and Gene Expression in Human Fibroblasts Laurent, Marc Cordeddu, Lina Zahedi, Yasaman Ekwall, Karl Biomolecules Article (1) Background: The LEO1 (Left open reading frame 1) protein is a conserved subunit of the PAF1C complex (RNA polymerase II-associated factor 1 complex). PAF1C has well-established mechanistic functions in elongation of transcription and RNA processing. We previously showed, in fission yeast, that LEO1 controls histone H3K9 methylation levels by affecting the turnover of histone H3 in chromatin, and that it is essential for the proper regulation of gene expression during cellular quiescence. Human fibroblasts enter a reversible quiescence state upon serum deprivation in the growth media. Here we investigate the function of LEO1 in human fibroblasts. (2) Methods: We knocked out the LEO1 gene using CRISPR/Cas9 methodology in human fibroblasts and verified that the LEO1 protein was undetectable by Western blot. We characterized the phenotype of the ΔLEO1 knockout cells with FACS analysis and cell growth assays. We used RNA-sequencing using spike-in controls to measure gene expression and spike-in controlled ChIP-sequencing experiments to measure the histone modification H3K9me2 genome-wide. (3) Results: Gene expression levels are altered in quiescent cells, however factors controlling chromatin and gene expression changes in quiescent human cells are largely unknown. The ΔLEO1 knockout fibroblasts are viable but have reduced metabolic activity compared to wild-type cells. ΔLEO1 cells showed a slower entry into quiescence and a different morphology compared to wild-type cells. Gene expression was generally reduced in quiescent wild-type cells. The downregulated genes included genes involved in cell proliferation. A small number of genes were upregulated in quiescent wild-type cells including several genes involved in ERK1/ERK2 and Wnt signaling. In quiescent ΔLEO1 cells, many genes were mis-regulated compared to wild-type cells. This included genes involved in Calcium ion transport and cell morphogenesis. Finally, spike-in controlled ChIP-sequencing experiments demonstrated that the histone modification H3K9me2 levels are globally increased in quiescent ΔLEO1 cells. (4) Conclusions: Thus, LEO1 is important for proper entry into cellular quiescence, control of H3K9me2 levels, and gene expression in human fibroblasts. MDPI 2023-11-17 /pmc/articles/PMC10668985/ /pubmed/38002344 http://dx.doi.org/10.3390/biom13111662 Text en © 2023 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Laurent, Marc
Cordeddu, Lina
Zahedi, Yasaman
Ekwall, Karl
LEO1 Is Required for Efficient Entry into Quiescence, Control of H3K9 Methylation and Gene Expression in Human Fibroblasts
title LEO1 Is Required for Efficient Entry into Quiescence, Control of H3K9 Methylation and Gene Expression in Human Fibroblasts
title_full LEO1 Is Required for Efficient Entry into Quiescence, Control of H3K9 Methylation and Gene Expression in Human Fibroblasts
title_fullStr LEO1 Is Required for Efficient Entry into Quiescence, Control of H3K9 Methylation and Gene Expression in Human Fibroblasts
title_full_unstemmed LEO1 Is Required for Efficient Entry into Quiescence, Control of H3K9 Methylation and Gene Expression in Human Fibroblasts
title_short LEO1 Is Required for Efficient Entry into Quiescence, Control of H3K9 Methylation and Gene Expression in Human Fibroblasts
title_sort leo1 is required for efficient entry into quiescence, control of h3k9 methylation and gene expression in human fibroblasts
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10668985/
https://www.ncbi.nlm.nih.gov/pubmed/38002344
http://dx.doi.org/10.3390/biom13111662
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