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Next Generation Sequencing of sh-control and sh-KDM10A/KDM10B double knockdown HeLa Cells Transcriptomes

ABSTRACT: Next Generation Sequencing of sh-control and sh-KDM10A/KDM10B double knockdown HeLa Cells Transcriptomes

PROVIDER: PRJNA560886 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR10003852_1.fastq.gz	Fastqsanger.gz
	SRR10003852_2.fastq.gz	Fastqsanger.gz
	SRR10003853_1.fastq.gz	Fastqsanger.gz
	SRR10003853_2.fastq.gz	Fastqsanger.gz
	SRR10003854_1.fastq.gz	Fastqsanger.gz

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Next Generation Sequencing of sh-control and sh-KDM10A/KDM10B double knockdown HeLa Cells Transcriptomes

Project description:Histone methyl groups can be removed by histone demethylases. Although LSD1 and JmjC domain-containing proteins have been identified to be histone demethylases, enzymes responsible for many histone methylation states or sites are not identified. Here, we performed a high-content cell-based screening of a cDNA library containing 2,500 nuclear proteins and identified KDM10A as a histone demethylase specific for histone H4 lysine 20 (H4K20). Ectopic expression of KDM10A reduced the global levels of H4K20me1/2/3 in 293T cells. In vitro, KDM10A specifically demethylated H4K20me1/2/3 and generated formaldehyde. The enzymatic activity required Fe(II) and α-ketoglutarate as cofactors. Domain mapping indicated that the demethylase activity required its UBA domain. We also demonstrated that KDM10B, a protein homologous to KDM10A, had H4K20 demethylase activity in vitro and in vivo. ChIP-seq reveals that KDM10A/B demethylated H4K20 methylation at specific genomic loci in vivo. We further demonstrated that KDM10A/B activated the transcription of coding genes by demethylating H4K20me1 in the gene body, and the transcription of repetitive elements by demethylating H4K20me3 in intergenic regions. NMR analyses demonstrated that an HxxxE motif in the UBA domain is crucial for iron binding, mutation of these two residues abolished iron binding and the demethylase activity. Thus, we identified a family of UBA domain-containing proteins as histone demethylases.

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Project description:PHF8 is an H3K9me2 demethylase, interacts with H3K4me3 and RNA Polymerase II, is enriched at thousands of transcription start sites and can act as a transcriptional co-activator. In the following experiment we wanted to analyze the impact of PHF8-knockdown on transcript levels in HeLa cells. Three independent replicate knockdown experiments with PHF8-shA and control NT-sh were performed (6 samples in total). The two samples of each replicate experiment were hybridized in two color dye swap experiments (6 arrays in total).

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