Chromatin Structure and Transcription of the Human alpha Globin Locus in Erythroid and Non-Erythroid Environments.

The ζ, α-1 and α-2 are the major alpha like globin genes. The ζ gene is expressed in the embryonic stage, while α-1 and α-2 genes are expressed throughout the adult life. Although the alpha like globin genes are flanked by genes that are transcribed in many cell types, their expression is restricted to the erythroid cells. Since the alpha globin genes are situated amidst the actively transcribing genes, they are supposed to be in the open chromatin configuration, even when they are transcriptionally silent in non-erythroid cells. Hence, understanding the structure of the chromatin of the alpha globin locus in erythroid and non-erythroid cells is needed to delineate the cell type and developmental stage specific regulation of expression of these genes. In the present study, we have undertaken a comparative analysis of the chromatin structure of the alpha globin locus, recruitment of transcription factors, and the transcriptional activity of the locus in enrythroid and non-erythroid cells. We have taken advantage of the availability of genomic tiling microarrays that include 50 base oligonucleotides spaced at 38 base pair intervals throughout extended regions embedding and flanking the alpha globin cluster and performed ChIP-chip analysis. The data obtained from these studies suggest that in erythroid K562 cells, Histone 3 of the alpha globin locus is acetylated at Lys 9 and dimethylated at Lys4 throughout the locus. The trimethyl Lys 4 marker was present on the promoters of transcribed genes, but not on the active HS40 enhancer. However, Pol II and its phosphory-lated forms were present on both the actively transcribing genes and the HS40 enhancer. Among the transcription factors, NF-E2 was predominantly associated with the HS40 sequences while GATA-1 was present on the alpha like promoters as well as the HS40 enhancer. The insulator binding CTCF was detected at several flanking regions of the HS40 enhancer in K562 and HeLa cells. We speculate that differential interaction among CTCF sites may play a role in regulating the effects of the HS-40 enhancer. In erythroid K562 cells, a strong HS40 enhancer formed by the virtue of the recruitment of the enhancer factors can overcome blocking by the downstream flanking CTCF site and, in analogy to suggestions in studies of Drosophia insulating elements, this might be mediated by specific interactions between upstream and downstream insulators. In the non-erythroid cells, the alpha globin locus was hypoacetylated. Along with the absence of trimethylation of the Lys 4 marker for active transcription, the methylations at Lys 9, and Lys 27 that are associated with the inactive genes were also absent. We also observed a lack of Lys 36 marker associated with the body of the transcribing genes in HeLa cells. In contrast to these observations, we have detected a robust presence of Pol II and Brg1 on the entire locus. Surprisingly, we have detected significant amount of transcriptional activity associated with parts of the theta and zeta genes and intergenic regions in HeLa, NB4 and 06990 lymphoblastoid cells. Initial studies indicate the generation of spliced polyadenylated RNA of the alpha globin locus in HeLa cells. The transcription of the locus was not uniform, but it was localized to certain regions, suggesting that the alpha globin transcription is not just a uniform leaky transcription, but that there may be hitherto unappreciated transcriptional regulatory elements within the locus.