Erythrocyte membrane protein genes serve as excellent models of complex gene locus structure and function, but their study has been complicated by both their large size and their complexity. To begin to understand the intricate interplay of transcription, dynamic chromatin architecture, transcription factor binding, and genomic organization in regulation of erythrocyte membrane protein genes, we performed chromatin immunoprecipitation (ChIP) coupled with microarray analysis and ChIP coupled with massively parallel DNA sequencing in both erythroid and nonerythroid cells. Unexpectedly, most regions of GATA-1 and NF-E2 binding were remote from gene promoters and transcriptional start sites, located primarily in introns. Cooccupancy with FOG-1, SCL, and MTA-2 was found at all regions of GATA-1 binding, with cooccupancy of SCL and MTA-2 also found at regions of NF-E2 binding. Cooccupancy of GATA-1 and NF-E2 was found frequently. A common signature of histone H3 trimethylation at lysine 4, GATA-1, NF-E2, FOG-1, SCL, and MTA-2 binding and consensus GATA-1-E-box binding motifs located 34 to 90 bp away from NF-E2 binding motifs was found frequently in erythroid cell-expressed genes. These results provide insights into our understanding of membrane protein gene regulation in erythropoiesis and the regulation of complex genetic loci in erythroid and nonerythroid cells and identify numerous candidate regions for mutations associated with membrane-linked hemolytic anemia.The erythrocyte membrane is a multifunctional, complex structure that provides the red cell the deformability and stability required to withstand its travels through macro-and microcirculation. It plays critical roles in erythropoiesis, including responding to erythropoietin, importing iron required for hemoglobin synthesis, and regulating cellular metabolism. Qualitative and quantitative disorders of erythrocyte membrane proteins have been associated with inherited abnormalities of red cell shape, including hereditary spherocytosis, hereditary elliptocytosis, and hereditary pyropoikilocytosis syndromes (65, 103). Despite biochemical and genetic linkage to specific erythrocyte membrane protein genes, e.g., ankyrin-1, ␣-or -spectrin, and band 3, mutations are found in the coding exons and promoter regions of only ϳ75% of cases studied. This suggests that the disease-causing mutation is located in critical regulatory regions outside the promoters and exons in a quarter of cases.Most erythrocyte membrane protein genes are large, comprised of Ͼ25 exons. They encode numerous diverse and complex isoforms, frequently generated by alternate splicing, alternate promoter usage, or alternate polyadenylation (18). In many cases, alternate promoters direct combinations of exons encoding diverse tissue-specific, cell type-specific, developmental-stage-specific, and/or differentiation stage-specific isoforms (6, 12, 13, 19, 21-24, 44, 52, 62, 78, 86, 108, 112-114). As such, erythrocyte membrane protein genes serve as excellent models of complex gene locus structure and fu...
