EXPERIMENT_TITLE: GSM5145773: H2_HiC; Homo sapiens; Hi-C
EXPERIMENT_DESIGN_LIBRARY_DESCRIPTOR_LIBRARY_CONSTRUCTION_PROTOC: 5e6 cells were crosslinked with 1% formaldehyde for 10 minutes at room temperature. The reaction was quenched for 5 minutes at room temperature with a final concentration of 200 mM glycine. Fixed cells were lysed in 1mL cell lysis buffer (10mM Tris-HCl pH 8, 10mM NaCl, 0.2% NP-40/Igepal) for 30 minutes on ice. The cell suspension was pelleted at 2500 x g for 5 minutes at 4oC and washed once with lysis buffer. The cell pellet was resuspended in 0.5% SDS and incubated at 62oC for 10 minutes. 10% Triton X-100 was used to quench the SDS and incubated at 37oC for 15 minutes. 100U of MboI was used to digest the chromatin overnigt at 37oC with shaking. Following heat inactivation of MboI, restriction fragment overhangs where marked with biotin. Ligation of marked ends was performed using T4 ligase at room temperature for 4 hours. Ligated DNA was sheared using a Covaris M220 with the power settings of peak incident power of 50, duty cycle 200, 200 cycles/burst, for 90 seconds. Sheared DNA was purified using AMPure XP beads (Beckman Coulter, A63881). Biotin-labelled DNA was isolated using Dynabeads MyOne Streptavidin C1 beads Libraries were prepared using standard reagents from New England BioLabs for Illumina sequencing
STUDY_DESCRIPTOR_STUDY_ABSTRACT: Naturally occurring mutations in the Î³-globin promoters can result in hereditary persistence of fetal hemoglobin (HPFH), a benign condition that ameliorates the severity of Î²-hemoglobinopathies through increased post-natal fetal hemoglobin (HbF) expression. Mutations in the Î³-globin promoters result in loss or de novo recruitment of transcription factors, yet the cis­-regulatory elements involved in Î³-globin transcription in the context of HPFH are not clearly defined. We demonstrate that the -115 cluster of HPFH mutations require GATA1 binding to at -186 in cooperation with NF-Y at the proximal CCAAT box at -85 for Î³-globin expression. We show that multiple HPFH mutations increase HbF in an erythroid cell line and result in GATA1 binding at -186. Mutation of the -186 GATA motif in HPFH erythroid cell lines and erythroid-differentiated CD34+ cells resulted in loss of GATA1 binding and reduction in fetal hemoglobin. NF-Y ChIP-seq in HPFH erythroid cell lines demonstrated binding to the proximal CCAAT box at -85 was also found to be critical for HPFH-mediated Î³-globin expression. Together, the -186 GATA motif and -85 proximal CCAAT box function in an additive and independent manner. Our results reveal detailed evidence for common, positive acting cis-regulatory elements that may provide more general mechanisms for erythroid gene expression. Overall design: ChIP-seq experiments were performed in HUDEP-2 cells harboring mutations in the ?-globin promoter for GATA1 and NF-YB. Input samples were used as control ATAC-seq experiments were performed in undifferentiated HUDEP-2 cells harboring mutations in the -115 CCAAT box of the ?-globin promoter. Hi-C experiments were performed in WT and single HBG allele HUDEP-2 cells
SAMPLE_TITLE: H2_HiC
SAMPLE_SAMPLE_ATTRIBUTES_SAMPLE_ATTRIBUTE: [{"TAG": "source_name", "VALUE": "H2_HiC"}, {"TAG": "cell line", "VALUE": "Hudep2"}]
Pool_Member_@attributes_sample_title: H2_HiC
RUN_SET_RUN_Pool_Member_@attributes_sample_title: H2_HiC
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