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Activation of γ-globin gene expression by GATA1 and NF-Y in hereditary persistence of fetal hemoglobin

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.

ORGANISM(S): Homo sapiens

PROVIDER: GSE152338 | GEO | 2021/04/22

REPOSITORIES: GEO

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Project description:Hereditary Persistence of Fetal Hemoglobin (HPFH) is characterized by persistent high levels of fetal hemoglobin (HbF) in adults. Several contributory factors, both genetic and environmental, have been identified, but others remain elusive. Ten of twenty-seven members from a Maltese family presented with HPFH. A genome-wide SNP scan followed by linkage analysis revealed a candidate region on chromosome 19p13.12-13. Sequencing identified a nonsense mutation in the KLF1 gene, p.K288X, ablating the DNA binding domain of this key erythroid transcriptional regulator. Only HPFH family members were heterozygote carriers of this mutation. Expression profiling on primary erythroid progenitors revealed down-regulation of KLF1 target genes in HPFH samples. Functional assays demonstrated that, in addition to its established role in adult globin expression, KLF1 is a critical activator of the BCL11A gene, encoding a suppressor of HbF expression. These observations provide a rationale for the effects of KLF1 haploinsufficiency on HbF levels. To identify differentially expressed genes, RNA was isolated from erythroid progenitors (HEPs) cultured from peripheral blood of four HPFH (KLF1 p.K288X/wt) and four non-HPFH family members (wt/wt) and used for genome-wide expression analysis. A minimum of 1.5E6 HEPs were harvested at day12 of culture and RNA was extracted with Trizol reagent (Sigma) and purified using the RNeasy Mini Kit (Qiagen, Crawley, UK), including an on-column DNaseI digestion, according to the manufacturer’s instructions. RNA yield was determined using the 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). 8-10 micr-g of total RNA was analysed by microarrays using cells from day 12 of culture. Quality of the total RNA samples and the resulting cRNA was assessed on the Bioanalyzer. Fragmented biotinylated cRNA was prepared and 15μg hybridized to HG-U133 plus 2 GeneChips, according to the manufacturer’s protocols (Affymetrix). The data was normalized by Robust Multi-Array average (RMA) algorithm.

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Activation of γ-globin gene expression by GATA1 and NF-Y in hereditary persistence of fetal hemoglobin

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