The human β‐globin enhancer LCR HS2 plays a role in forming a TAD by activating chromatin structure at neighboring CTCF sites

The human β-globin locus control region (LCR) hypersensitive site 2 (HS2) is one of enhancers for transcription of the β-like globin genes in erythroid cells. Our previous study showed that the LCR HS2 has active chromatin structure before transcriptional induction of the β-globin gene, while another enhancer LCR HS3 is activated by the induction. To compare functional difference between them, we deleted each HS (ΔHS2 and ΔHS3) from the human β-globin locus in hybrid MEL/ch11 cells. Deletion of either HS2 or HS3 dramatically diminished the β-globin transcription and disrupted locus-wide histone H3K27ac and chromatin interaction between LCR HSs and gene. Surprisingly, ΔHS2 weakened interactions between CTCF sites forming the β-globin topologically associating domain (TAD), while ΔHS3 did not. CTCF occupancy and chromatin accessibility were reduced at the CTCF sites in the ΔHS2 locus. To further characterize the HS2, we deleted the maf-recognition elements for erythroid activator NF-E2 at HS2. This deletion decreased the β-globin transcription and enhancer-promoter interaction, but did not affect interactions between CTCF sites for the TAD. In light of these results, we propose that the HS2 has a role in forming a β-globin TAD by activating neighboring CTCF sites and this role is beyond typical enhancer activity.