beta-Globin locus control region HS2 and HS3 interact structurally and functionally

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Abstract
The overall structure of the DNase I hypersensitive sites (HSs) that comprise the β‐globin locus control region (LCR) is highly conserved among mammals, implying that the HSs have conserved functions. However, it is not well understood how the LCR HSs, either individually or collectively, activate transcription. We analyzed the interactions of HS2, HS3 and HS4 with the human ϵ‐ and β‐globin genes in chromatinized episomes in fetal/embryonic K562 cells. Only HS2 activates transcription of the ϵ‐globin gene, while all three HSs activate the β‐globin gene. HS3 stimulates the β‐globin gene constitutively, but HS2 and HS4 transactivation requires expression of the transcription factor EKLF, which is not present in K562 cells but is required for β‐globin expression in vivo. To begin addressing how the individual HSs may interact with one another in a complex, we linked the β‐globin gene to both the HS2 and HS3. HS2 and HS3 together resulted in synergistic stimulation of β‐globin transcription. Unexpectedly, mutated, inactive forms of HS2 impeded the activation of the β‐globin gene by HS3. Thus, there appear to be distinct interactions among the HSs and between the HSs and the globin genes. These preferential, non‐exclusive interactions may underlie an important structural and functional cooperativity among the regulatory sequences of the β‐globin locus in vivo.