EXPERIMENT_TITLE: GSM8185639: WT_hCNCC_PC_HiC; Homo sapiens; Hi-C
EXPERIMENT_DESIGN_LIBRARY_DESCRIPTOR_LIBRARY_CONSTRUCTION_PROTOC: Cells were first fixed with 2% formaldehyde for 10 minutes at ambient temperature, followed by quenching with 0.2M glycine for 5 minutes. Post-fixation, cells were lysed, and endogenous nucleases were inactivated with 0.3% SDS. Chromatin was digested using 100U of HindIII (New England Biolabs), labeled with biotin-14-dCTP (Invitrogen), and then ligated using 50U of T4 DNA ligase (New England Biolabs). After reversing the cross-links, DNA was isolated using the QIAamp DNA Mini Kit (Qiagen), in strict accordance with the manufacturer's instructions. Subsequent steps involved fragmenting the DNA to 300-600 bp, end-repair, A-tailing, and adapter ligation using SureSelect adaptors. Biotinylated fragments were isolated through streptavidin-affinity pull-down and PCR amplified. Promoter-specific Capture Hi-C was performed using the SureSelect XT Library Prep Kit ILM (Agilent Technologies), which included a custom-designed biotinylated RNA bait library and paired-end blockers. Post-capture PCR enrichment was followed by final purification of the libraries with AMPure XP beads (Beckman Coulter). Library quality was evaluated using Bioanalyzer profiles (Agilent Technologies), and high-throughput sequencing was performed on the Illumina NovaSeq 6000 platform. promoter capture HiC
STUDY_DESCRIPTOR_STUDY_ABSTRACT: Enhancers, through the combinatorial action of transcription factors (TFs), dictate both the spatial specificity and the levels of gene expression, and their aberrations can result in diseases. The association between HMX1 and its downstream enhancer with ear deformities has been previously documented. However, the pathogenic variations and molecular mechanisms underlying the bilateral constricted ear (BCE) malformations remain unclear. This study identifies a copy number variation (CNV) encompassing three enhancers that induces BCE. These enhancers, collectively termed the positional identity hierarchical enhancer cluster (PI-HEC), co-regulate HMX1, each displaying unique activity-location-structure characteristics. A thorough exploration of this regulatory locus reveals that specific motif clusters within the PI-HEC variably modulate its activity and specificity, with the high mobility group (HMG) box combined with Coordinator and homeodomain (HD)-TFs notably influencing them respectively. Our findings based on various types of mouse models, reveal that both aberrant Hmx1 expression in the fibroblasts of the basal pinna, originating from neural crest cells, and ectopic expression in the distal pinna structures contribute to the abnormal development of the outer ear, including the cartilage, muscle, and epidermis tissues. This study deepens our understanding of mammalian ear morphogenesis and sheds light on the complexity of gene expression regulation by enhancers and specific sequence motifs. Overall design: Promoter capture HiC (PC-HiC) data of H9 derived cranial neural crest cell
SAMPLE_TITLE: WT_hCNCC_PC_HiC
SAMPLE_SAMPLE_ATTRIBUTES_SAMPLE_ATTRIBUTE: [{"TAG": "source_name", "VALUE": "H9-derived cranial neural crest cells"}, {"TAG": "cell line", "VALUE": "H9-derived cranial neural crest cells"}, {"TAG": "cell type", "VALUE": "Stem cells"}, {"TAG": "geo_loc_name", "VALUE": "missing"}, {"TAG": "collection_date", "VALUE": "missing"}]
Pool_Member_@attributes_sample_title: WT_hCNCC_PC_HiC
RUN_SET_RUN_Pool_Member_@attributes_sample_title: WT_hCNCC_PC_HiC
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