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The beta-globin locus control region (LCR) is able to enhance the expression of all globin genes throughout the course of development. However, the chromatin structure of the LCR at the different developmental stages is not well defined. We report DNase I and micrococcal nuclease hypersensitivity, chromatin immunoprecipitation analyses for histones H2A, H2B, H3, and H4, and 3C (chromatin conformation capture) assays of the normal and mutant beta-globin loci, which demonstrate that nucleosomes at the DNase I hypersensitive sites of the LCR could be either depleted or retained depending on the stages of development. Furthermore, MNase sensitivity and 3C assays suggest that the LCR chromatin is more open in embryonic erythroblasts than in definitive erythroblasts at the primary- and secondary-structure levels; however, the LCR chromatin is packaged more tightly in embryonic erythroblasts than in definitive erythroblasts at the tertiary chromatin level. Our study provides the first evidence that the occupancy of nucleosomes at a DNase I hypersensitive site is a developmental stage-related event and that embryonic and adult cells possess distinct chromatin structures of the LCR.

To delineate the relationship between epigenetic modifications and hemoglobin switching, we compared the pattern of histone acetylation and pol II binding across the beta-globin locus at fetal and adult stages of human development. To make this comparison possible, we introduced an external control into experimental samples in chromatin immunoprecipitation (ChIP) assays. Using this common standard, we found that the locus control region (LCR) was acetylated to the same level at all stages, whereas acetylation levels at the individual gene regions correlated with the state of transcription. In the active genes, the promoters were less acetylated compared with the coding regions. Furthermore, all globin promoters were acetylated to a similar level irrespective of the state of transcription. However, after correction for the loss of nucleosomes, the level of acetylation per histone at the active gamma and beta promoters was 5- to 7-fold greater than that at the inactive epsilon promoter. Although the histone acetylation level within the LCR was developmentally stable, pol II binding in fetal erythroblasts was 2- to 3-fold greater than that in adult erythroblasts. These results demonstrate that dynamic changes in histone acetylation and pol II take place as the human beta-globin gene region undergoes its developmental switches.

High-level transcription of the globin genes requires the enhancement by a distant element, the locus control region (LCR). Such long-range regulation in vivo involves spatial interaction between transcriptional elements, with intervening chromatin looping out. It has been proposed that the clustering of the HS sites of the LCR, the active globin genes, as well as the remote 5' hypersensitive sites (HSs) (HS-60/-62 in mouse, HS-110 in human) and 3'HS1 forms a specific spatial chromatin structure, termed active chromatin hub (ACH). Here we report the effects of the HS3 deletions of the LCR on the spatial chromatin structure of the beta-globin locus as revealed by the chromatin conformation capture (3C) technology. The small HS3 core deletion (0.23 kb), but not the large HS3 deletion (2.3 kb), disrupted the spatial interactions among all the HS sites of the LCR, the beta-globin gene and 3'HS1. We have previously demonstrated that the large HS3 deletion barely impairs the structure of the LCR holocomplex, while the structure is significantly disrupted by the HS3 core deletion. Taken together, these results suggest that the formation of the ACH is dependent on a largely intact LCR structure. We propose that the ACH indeed is an extension of the LCR holocomplex.