Chromatin Structure and Transcription of the Human Alpha Globin Locus during Erythroid Differentation

Abstract The human alpha globin genes are controlled by DNase hypersensitive sites (HS) HS-4, HS-8, HS-10, HS-33 and HS-40 upstream of the ζ gene. Among these, HS40 functions as a strong enhancer of the alpha like genes. The alpha globin genes are situated amidst actively transcribing genes, but are transcriptionally silent in non-erythroid cells including hematopoietic progenitor cells We have undertaken an analysis of the chromatin structure of the alpha globin locus, recruitment of transcription factors, and the transcriptional activity of the locus in CD34+ hematopoietic progenitor cells and upon their differentiation into erythroid cells. Chromatin immunoprecipitation (ChIP) followed by PCR analysis of all the regulatory and structural segments of the α-globin locus were performed using antibodies against chemically modified tails of histone H3, the insulator binding factor CTCF, transcription factors such as GATA-1 and NF-E2, and Pol II. Both H3Me2K4 and H3AcK9 modifications were present at HS48 and HS33 in CD34+ cells and substantially increase when these cells are differentiated into erythroid lineage. At the HS40 region, these modifications were present at a low level in CD34+ cells and did not change during erythroid differentiation. Among the α-like gene promoters, we find these modifications at the Mu and theta gene promoters in CD34+ cells and they increase during erythropoiesis. These modifications were absent at the zeta gene promoter consistent with the inactivity of this gene during definitive erythropoiesis. Overall the dominant HS40 enhancer possesses moderate levels of H3Me2K4 and H3AcK9 modifications, and its cognate major a-globin promoter is devoid of these modifications in CD34+ cells even when these cells are differentiated into erythroid lineage. The entire α-globin locus including the HS enhancer regions and a-like gene promoters did not contain the unphosphorylated (initiation) form of Pol II recruitment in CD34+ cells. When these cells differentiated into the erythroid lineage, Pol II was recruited at the HS40 and HS48 regions and at the Mu and theta promoters. Rearrangement of the CTCF binding sites at the α-globin locus occurs during differentiation of CD34+ cells into the erythroid lineage. In CD34+ cells, as in HeLa cells, the α-globin genes are flanked by multiple CTCF binding events at the 5′ and 3′ ends of the locus. At the 5′ end of the locus, the HS40 and HS48 sequences were surrounded by four CTCF binding sites at HS33, HS46, HS55 and HS90. At the 3′ end of the locus CTCF was observed at the theta globin promoter and at the 3′ end of the theta globin gene. Upon differentiation of the CD34+ cells into the erythroid pathway, CTCF recruitment is significantly reduced at HS90 and HS46 sequences, while the sites at HS55 and HS33 show increased CTCF binding. Thus, in contrast to the CD34+ cells, the HS40 and HS48 sequences are y flanked by two CTCF recruitment sites in erythroid cells. Such a differential placement of CTCF binding sites suggests that differential interaction among CTCF sites may regulate the effects of the HS-40 enhancer. In erythroid cells, a strong HS40 enhancer formed by virtue of the recruitment of the enhancer factors can overcome blocking by the downstream flanking CTCF site and this might be mediated by specific interactions between the two flanking insulators. The CTCF binding at the 3′ end of the theta globin gene is abolished during erythropoiesis of CD34+ cells. However, the recruitment of CTCF at the theta globin promoter is unchanged suggesting that the theta globin may be insulated by the influence of the α-globin enhancer sequences. We have detected transcripts from parts of the theta and zeta genes and intergenic regions in HeLa, NB4 and 06990 lymphoblastoid cells and primary erythroid cells in culture. The transcription of the locus was localized to certain regions, suggesting that there may be unappreciated transcriptional regulatory elements within the locus.