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Effect of depletion of CTR9 on gene expression during early osteogenic differentiation of human primary mesenchymal stem cells

ABSTRACT: Effect of depletion of CTR9 on gene expression during early osteogenic differentiation of human primary mesenchymal stem cells

PROVIDER: PRJNA880314 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR21553603.fastq.gz	Fastqsanger.gz
	SRR21553604.fastq.gz	Fastqsanger.gz
	SRR21553605.fastq.gz	Fastqsanger.gz
	SRR21553606.fastq.gz	Fastqsanger.gz
	SRR21553607.fastq.gz	Fastqsanger.gz

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Effect of depletion of CTR9 on gene expression during early osteogenic differentiation of human primary mesenchymal stem cells

Project description:Cell-fate determination of human mesenchymal stem/stromal cells (hMSCs) is precisely regulated by lineage-specific transcription factors and epigenetic enzymes. We found that CTR9, a key scaffold subunit of Polymerase Associated Factor Complex (PAFc), selectively regulates hMSC differentiation to osteoblasts and chondrocytes, but not to adipocytes. An in vivo ectopic osteogenesis assay confirmed the essentiality of CTR9 in hMSC-derived bone formation. CTR9 counteracts the activity of EZH2, the epigenetic enzyme that deposits H3K27me3, in hMSCs. Accordingly, CTR9 knockdown (1) hMSCs gain H3K27me3 mark, and the osteogenic differentiation defects of CTR9 KD hMSCs can be partially rescued by treatment with EZH2 inhibitors. Transcriptome analyses identified Bone Morphology Protein-2 (BMP-2) as a downstream effector of CTR9. BMP-2 secretion, membrane anchorage, as well as the BMP-SMAD pathway were impaired in CTR9 KD MSCs, and the effects were rescued by BMP-2 supplementation. This study uncovers an epigenetic mechanism engaging CTR9-H3K27me3-BMP-2 axis to regulate osteochondral lineage differentiation of hMSCs. To investigate the function of CTR9 in the gene regulation of early osteogenic committment , we established human MSCs in which CTR9 gene has been knocked down by two individual shRNAs (shControl vs shCTR9#3/#5）.

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Project description:CTR9 is the scaffold subunit in PAFc (Polymerase associated factor complex), a multifunctional complex employed in multiple steps of RNA Pol II-mediated transcription. CTR9/PAFc is well known as an evolutionarily conserved elongation factor regulating gene activation via coupling with histone modifications enzymes. However, little is known about its function to restrain repressive histone markers. Using inducible and stable CTR9 knockdown breast cancer cell lines, we discovered that the amount of H3K27me3 is strictly controlled by CTR9. Quantitative profiling on histone modifications revealed a striking increase of H3K27me3 level upon loss of CTR9. Moreover, loss of CTR9 leads to genome-wide expansion of H3K27me3, as well as increased recruitment of PRC2 on chromatin, which can be reversed by CTR9 restoration. Moreover, CTR9 depletion triggers a PRC2 subtype switch from less active PRC2.2 to PRC2.1 with higher methyltransferase activity. As a consequence, CTR9 depletion generates vulnerability that renders breast cancer cells hypersensitive to PRC2 inhibitors. Our findings that CTR9 demarcates PRC2-mediated H3K27me3 levels and genomic distribution provide a unique mechanism of transition from transcriptionally active to repressive chromatin states and shed light on the biological functions of CTR9 in development and cancer.

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