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LEO1 Is Required for Efficient Entry into Quiescence, Control of H3K9 methylation and Gene Expression in Human Fibroblasts [RNA-Seq]

ABSTRACT: Abstract: (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 have previously shown, in fission yeast, that LEO1 is controlling histone H3K9 methylation levels by affecting the turnover of histone H3 in chromatin, and that it is essential for 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 DLEO1 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 DLEO1 knockout fibroblasts are viable but have reduced metabolic activity compared to wild type cells. DLEO1 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 DLEO1 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 DLEO1 cells. (4) Conclusions: Thus, LEO1 is important for proper entry into cellular quiescence, control H3K9me2 levels and gene expression in human fibroblasts.

ORGANISM(S): Homo sapiens

PROVIDER: GSE247831 | GEO | 2023/11/29

REPOSITORIES: GEO

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Project description:Abstract: (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 have previously shown, in fission yeast, that LEO1 is controlling histone H3K9 methylation levels by affecting the turnover of histone H3 in chromatin, and that it is essential for 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 DLEO1 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 DLEO1 knockout fibroblasts are viable but have reduced metabolic activity compared to wild type cells. DLEO1 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 DLEO1 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 DLEO1 cells. (4) Conclusions: Thus, LEO1 is important for proper entry into cellular quiescence, control H3K9me2 levels and gene expression in human fibroblasts.

Project description:Background: Although quiescence—reversible cell-cycle arrest—is a key part in the life history and fate of many mammalian cell types, the mechanisms of gene regulation in quiescent cells are poorly understood. We sought to clarify the role of microRNAs as regulators of the cellular functions of quiescent human fibroblasts. Results: Using microarrays, we discovered that the expression of the majority of profiled microRNAs differed between proliferating and quiescent fibroblasts. Fibroblasts induced into quiescence by contact inhibition or serum starvation had similar microRNA profiles, indicating common changes induced by distinct quiescence signals. By analyzing the gene expression patterns of microRNA target genes with quiescence, we discovered a strong regulatory function for miR-29, which is downregulated with quiescence. Using microarrays and immunoblotting, we confirmed that miR-29 targets genes encoding collagen and other extracellular matrix proteins and that those target genes are induced in quiescence. In addition, overexpression of miR-29 resulted in more rapid cell cycle re-entry from quiescence. We also found that let-7 and miR-125 were upregulated in quiescent cells. Overexpression of either one alone resulted in slower cell cycle re-entry from quiescence, while the combination of both together slowed cell cycle re-entry even further. Conclusions: microRNAs regulate key aspects of fibroblast quiescence including the proliferative state of the cells as well as their gene expression profiles, in particular, the induction of extracellular matrix proteins in quiescent fibroblasts. microRNAs in human neonatal dermal fibroblasts in 3 cell cycle conditions (proliferating, 4 days serum starved, 7 days contact inhibited) were analyzed by one color microarray. 3 separate fibroblast cell lines from different human donors were analyzed for each condition, with two separate labeling and hybridization samples for each cell line. In total, 18 samples were hybridized to the microRNA microarray.

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LEO1 Is Required for Efficient Entry into Quiescence, Control of H3K9 methylation and Gene Expression in Human Fibroblasts [RNA-Seq]

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