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Alu repeats blocking and CEBPA overexpression in BLaER1 cells, CEBPA ChIP-Seq

ABSTRACT: Alu repeats blocking and CEBPA overexpression in BLaER1 cells, CEBPA ChIP-Seq

PROVIDER: PRJEB60232 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	ERR10934142.fastq.gz	Fastqsanger.gz
	ERR10934143.fastq.gz	Fastqsanger.gz
	ERR10934144.fastq.gz	Fastqsanger.gz
	ERR10934145.fastq.gz	Fastqsanger.gz
	ERR10934146.fastq.gz	Fastqsanger.gz

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Alu repeats blocking and CEBPA overexpression in BLaER1 cells, CEBPA ChIP-Seq

Project description:Weaker CEBPA binding in the human than in the mouse genome is a general trait of the human genome across multiple biological conditions. Alu repeats carry strong CEBPA binding motifs, which compete with regulatory regions for CEBPA binding. To directly test this hypothesis, we attempted to overcome Alu competition by using, first, a CRISPR-dCas9 system in BLaER1 cells (Rapino et al. 2013). By promoting the recruitment of the inactive Cas9 to Alu regions with specific gRNAs targeting Alu repeats containing strong CEBPA motifs we protected them, hampering CEBPA binding to these regions. We tested the effect of two different paired gRNA constructs in two replicates and one control paired gRNA in two replicates. Second, we also further overexpressed CEBPA in human BLaER1 cells to overcome Alu competition. We tested two doses of CEBPA overexpression in two replicates and one control experiment in two replicates.

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Project description:DNA methylation is theÂ major repression mechanismÂ for human retrotransposons, such as the Alu family. Here, we haveÂ derivedÂ methylation levels regarding 5238 loci belonging to twoÂ Alu subfamilies, AluYa5 and AluYb8, using High-Throughput Targeted Repeat Element Bisulfite Sequencing (HT-TREBS). The results indicate that ~90% of loci are repressed by high methylation levels. Of the remaining loci, many of these hypomethylated elements are found near gene promoters and show high levels of DNA methylation variation. WeÂ have characterizedÂ this variation in the context of tumorigenesis and inter-individual differences. Comparison of a primary breast tumor and its matched normal tissue revealed early DNA methylation changes in ~1% of AluYb8 elements in response to tumorigenesis. At the same time, AluYa5/Yb8 elements proximal to promoters also showed differences in methylation of up to one order of magnitude even between normalÂ individuals. Overall,Â the current study demonstrates that early loss of methylation occurs during tumorigenesis in a subset of young Alu elements, suggesting their potential clinical relevance. However, techniques such as deep-bisulfite-sequencing of individual loci using HT-TREBS are required to distinguish clinically relevant loci from the background observed forÂ AluYa5/Yb8 elements in general with regard to high levels of inter-individual variation in DNA methylation. HT-TREBS has been used with the Ion Torrent PGM platform to analyze the DNA methylation of 5238 AluYa5/Yb8 elements in a locus-specific manner in human skin-derived fibroblast cells, and a matched normal breast and primary tumor

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