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Integration of a scalable parallel reporter genes for qRT-PCR based enhancer screening with CRISPR dCas9 activation and supershift EMSA identifies impaired BACH1 repression of ST8SIA1 to contribute to the increased risk for periodontitis in smokers

ABSTRACT: Integration of a scalable parallel reporter genes for qRT-PCR based enhancer screening with CRISPR dCas9 activation and supershift EMSA identifies impaired BACH1 repression of ST8SIA1 to contribute to the increased risk for periodontitis in smokers

PROVIDER: PRJNA673904 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR12967717.fastq.gz	Fastqsanger.gz
	SRR12967718.fastq.gz	Fastqsanger.gz
	SRR12967719.fastq.gz	Fastqsanger.gz
	SRR12967720.fastq.gz	Fastqsanger.gz
	SRR12967721.fastq.gz	Fastqsanger.gz

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Integration of a scalable parallel reporter genes for qRT-PCR based enhancer screening with CRISPR dCas9 activation and supershift EMSA identifies impaired BACH1 repression of ST8SIA1 to contribute to the increased risk for periodontitis in smokers

Project description:Genome-wide association studies identified many loci associated with human diseases, but assigning causal alleles remains difficult. Likewise, generating biological meaning underlying a statistical association is challenging. To address these problems, we developed a scalable reporter gene system for the simultaneous transfection and parallel quantification of enhancer activity. Additionally, we characterized a genetic association at the gene ST8SIA1 that increases the risk for periodontitis in smokers. We cloned unique DNA barcode sequences to a reporter gene. Transcript levels of the barcodes and firefly luminescence of the reporter was compared in HeLa cells for several known enhancers. We mapped the associated SNPs to DNA elements with predictive features of regulatory function on gene expression from ENCODE. We tested whether associated alleles changed predicted transcription factor binding sites and proved transcription factor binding using antibody electrophoretic mobility shift assays. Using CRISPR dCas9 activation, we pinpointed the target gene of the association. Using RNA-Sequencing, we identified gene sets that are associated with ST8SIA1. Two associated repressor elements bind the transcription factor BACH1 with the putative causative variant rs2012722 decreasing BACH1 binding by 40%. ST8SIA1 is the likely target gene of the association, regulating the cell cycle arrest and integrin cell surface signaling. We found the periodontitis risk gene ABCA1 as the most significantly up-regulated gene. In conclusion, we developed a low cost, easy to use parallel reporter gene system that facilitates enhancer screening, and described an experimental pipeline for straightforward identification and characterization of causal variants and of their target genes.

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Project description:BTB and CNC homology 1 (BACH1) is a heme-binding transcription factor repressing the transcription from a subset of MAF recognition elements (MAREs) at low intracellular heme levels. Upon heme binding, BACH1 is released from the MAREs, resulting in increased expression of antioxidant response genes. To systematically address the gene regulatory networks involving BACH1, we combined chromatin immunoprecipitation-sequencing (ChIP-seq) analysis of BACH1 target genes in HEK 293 cells with knock-down of BACH1 using three independent types of small interfering RNAs followed by transcriptome profiling using microarrays. The 59 BACH1 target genes identified by ChIP-seq were found highly enriched in genes showing expression changes after BACH1 knock-down, demonstrating the impact of BACH1 repression on transcription. In addition to known and new BACH1 targets involved in heme degradation (HMOX1, FTL, FTH1, ME1, SLC48A1) and redox regulation (GCLC, GCLM, SLC7A11), we also discovered BACH1 target genes effecting cell cycle and apoptosis pathways (ITPR2, CALM1, SQSTM1, TFE3, EWSR1, CDK6, BCL2L11, MAFG) as well as subcellular transport processes (CLSTN1, PSAP, MAPT, vault RNA). The newly identified impact of BACH1 on genes involved in neurodegenerative processes and proliferation provides an interesting basis for future dissection of BACH1-mediated gene repression in neurodegeneration and virus-induced cancerogenesis. Examination of BACH1 binding in HEK 293T cells by chromatin immunoprecipitation-sequencing (CHIP-seq) with input DNA as control.

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