EXPERIMENT_TITLE: GSM4672364: H3K27ac HiChIP in human aortic valve interstitial cells; Homo sapiens; Hi-C
EXPERIMENT_DESIGN_LIBRARY_DESCRIPTOR_LIBRARY_CONSTRUCTION_PROTOC: Human aortic valve interstitial cells were isolated from control non-mineralized aortic valves obtained from patients undergoing heart transplantation. The protocol was approved by the local ethical committee and informed consent was obtained from the subjects. Aortic leaflets were cut into pieces and incubated in 0.3% type I collagenase (Invitrogen, Thermo Fisher Scientific, ON, Canada) at 37°C for 45 minutes, then filtered through a 70 µm mesh, centrifuged for 5 minutes at 1,500 rpm and resuspended in complete media (DMEM, 10% FBS with L-glutamine and sodium pyruvate). Cells were used between passages 3 to 7. H3K27ac HiChIP was performed from about 1 million of cells as described previously (Mumbach et al., 2016, Nat Methods). Library was generated in 50 µL of a PCR master mix (25 µL of Phusion HF 2X (New England BioLabs, ON, Canada), 1 µL of each Nextera Ad1_noMX and Nextera Ad2.X at 12.5 µM, and 23 µL of water). The following PCR program was performed: 72 °C for 5 min, 98 °C for 1 min, then cycle at 98 °C for 15 s, 63 °C for 30 s, and 72 °C for 1 min. Cycle number was set to 10 considering the amount of DNA. Library was placed on a magnet and eluted into a new tube before purification with clean up & concentrator kit (Zymo Research, Cedarlane, Canada). Library size selection (300-700 bp) was performed by cutting a 2% agarose gel slice and DNA purified with PureLink Quick Gel Extraction (Thermo Fisher, ON, Canada). Library quality was assessed on a BioAnalyser (Agilent, ON, Canada) using Agilent High Sensitivity DNA kit (Agilent, ON, Canada). Paired-end sequencing with 500 million reads was performed with NovaSeq 6000 S4 (UC San Diego IGM Genomics Center). HiChIP
STUDY_DESCRIPTOR_STUDY_ABSTRACT: Genome-wide association studies identified a strong signal for non coding variants at the 1p21.2 locus associated with calcific aortic valve stenosis (CAVS). Regulation at the locus and impact on the biology of the aortic valve is presently unknown. Integrative mapping of genetic association data was performed. The locus was evaluated by 3D genome mapping, analysis of atomic resolution data, DNA-binding assay and CRISPR activation (CRISPRa). Weighted gene co-expression network analysis (WGCNA) was performed to determine regulatory network in CAVS. The functional impact of the locus was assessed in isolated VICs. Fine-grained mapping at 1p21.2 risk locus identified rs6702619 as being located in open chromatin and a distant-acting super-enhancer including chromatin interaction with the promoter of PALMD in VICs. Atomic-level data showed that the risk variant modified base readout and DNA shape, which prevented the recruitment of NFATC2, a transcription factor involved in heart valve morphogenesis, and lowered the expression of PALMD. CRISPRa confirmed that rs6702619 exerts a control on the expression of PALMD. WGCNA performed in 233 calcified aortic valves identified a co-expression network encompassing PALMD, which was enriched in actin-based process. In LC-MS/MS and co-immunoprecipitation assay, actin was identified as a protein interacting with PALMD. Lower expression of PALMD in VICs promoted the polymerization of actin, the activation of myocardin-related transcription factor and fibrogenesis. Risk allele at rs6702619 disrupts a NFATC2 binding site and decreases enhancer-mediated expression of PALMD, which results in actin polymerization, a myofibroblast-like phenotype in VICs and fibrogenesis, a key underpinning process in the pathogenesis of CAVS. Overall design: H3K27ac HiChIP in human aortic valve interstitial cells.
SAMPLE_TITLE: H3K27ac HiChIP in human aortic valve interstitial cells
SAMPLE_SAMPLE_ATTRIBUTES_SAMPLE_ATTRIBUTE: [{"TAG": "source_name", "VALUE": "Human aortic valve interstitial cells from non-mineralized aortic valves"}, {"TAG": "cell type", "VALUE": "aortic valve interstitial cells"}, {"TAG": "antibody", "VALUE": "H3K27ac"}]
Pool_Member_@attributes_sample_title: H3K27ac HiChIP in human aortic valve interstitial cells
RUN_SET_RUN_Pool_Member_@attributes_sample_title: H3K27ac HiChIP in human aortic valve interstitial cells
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