EXPERIMENT_TITLE: GSM4093597: HaCaT_unstimulated_HiC; Homo sapiens; Hi-C
EXPERIMENT_DESIGN_LIBRARY_DESCRIPTOR_LIBRARY_CONSTRUCTION_PROTOC: Cells were thawed on ice and re-suspended in 50ml ice-cold lysis buffer (10mM Tris-HCl pH 8, 10mM NaCl, 0.2% Igepal CA-630, one protease inhibitor cocktail tablet). Cells were lysed on ice for a total of 30 min, with 2x10 strokes of a dounce homogeniser with a 5 minute break between douncing. The nuclei were pelleted and permeabilised and the chromatin was digested with HindIII. Ends were marked with biotin followed by in-nucleus ligation, decrosslinking and biotin pulldown according to the previously established Hi-C protocol (Dryden et al., 2014; P. Martin et al., 2015) Sequencing libraries were generated with 6-8 cycles of PCR using Truseq single indexes (Illumina) and Phusion polymerase (NEB)
STUDY_DESCRIPTOR_STUDY_ABSTRACT: Background: Genome-wide association studies (GWAS) have uncovered many genetic risk loci for psoriasis, yet many remain uncharacterised in terms of the causal gene and their biological mechanism in disease. This is largely a result of the findings that over 90% of GWAS variants map outside of protein-coding DNA, and instead are enriched in cell type and stimulation-specific gene regulatory regions. Results: Here, we use a disease-focused Capture Hi-C (CHi-C) experiment to link psoriasis-associated variants with their target genes in psoriasis-relevant cell lines (HaCaT keratinocytes and My-La CD8+ T cells). We confirm previously assigned genes, suggest novel candidates and provide evidence for complexity at psoriasis GWAS loci. For one locus, uniquely, we combine further epigenomic evidence to demonstrate how a psoriasis-associated region forms a functional interaction with the distant (>500 kb) KLF4 gene. This interaction occurs between the gene and active enhancers in HaCaT cells, but not in My-La cells. We go on to investigate this long-distance interaction further with Cas9 fusion protein-mediated chromatin modification (CRISPR activation) coupled with RNA-seq, demonstrating how activation of the psoriasis associated enhancer upregulates known genes in the KLF4 pathway, specific to skin cells and apoptosis. Conclusions: Taken together, our study design provides a robust pipeline to follow up GWAS disease associated variants and implicate the causal genes, cell types, direction of effect and consequences, which are vital next steps for the functional translation of genetic findings into clinical benefit. Overall design: Capture Hi-C was performed targeting psoriasis GWAS loci in relevant cell lines (HaCaT keratinocyte - unstimulated or stimulated with IFNg - and My-La CD8+ T cells) in order to uncover gene targets of psoriasis-associated SNPs. 3' mRNA-seq was performed to determine target gene expression in the same cell lines. To further explore an interaction between the psoriasis locus at 9q31 and the KLF4 gene, HiChIP for H3K27ac was performed in the same cell lines and confirmed H3K27ac-mediated interactions bewteen the psoriasis locus and the KLF4 promoter in HaCaT cells, but not in My-La cells. A CRISPRa experiment was conducted in HaCaT dCas9 P300 cells, using a pool of sgRNA targeting a selection of the psoriasis-associated SNPs in 9q31, and further RNA-seq was performed to determine differential gene expression against control cell lines expressing a non-specific sgRNA.
SAMPLE_TITLE: GEO accession GSM4093597 is currently private and is scheduled to be released on Sep 23, 2022.
Pool_Member_@attributes_sample_title: GEO accession GSM4093597 is currently private and is scheduled to be released on Sep 23, 2022.
RUN_SET_RUN_Pool_Member_@attributes_sample_title: GEO accession GSM4093597 is currently private and is scheduled to be released on Sep 23, 2022.
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