5'HS5 of the human beta-globin locus control region is dispensable for the formation of the beta-globin active chromatin hub.

Hypersensitive site 5 (5'HS5) of the beta-globin Locus Control Region functions as a developmental stage-specific border in erythroid cells. Here, we have analyzed the role of 5'HS5 in the three dimensional organization of the beta-gene locus using the Chromatin Conformation Capture (3C) technique. The results show that when 5'HS5 is deleted from the locus, both remote and internal regulatory elements are still able to interact with each other in a three-dimensional configuration termed the Active Chromatin Hub. Thus, the absence of 5'HS5 does not have an appreciable effect on the three dimensional organization of the beta-globin locus. This rules out models in which 5'HS5 nucleates interactions with remote and/or internal regulatory elements. We also determined the binding of CTCF, the only defined insulator protein in mammalian cells, to 5'HS5 by using chromatin immunoprecipitation (ChIP) assays. We detect low levels of CTCF binding to 5'HS5 in primitive erythroid cells, in which it functions as a border element. Surprisingly, we also observe binding levels of CTCF to 5'HS5 in definitive erythroid cells. Thus, binding of CTCF to 5'HS5 per se does not render it a functional border element. This is consistent with the previous data suggesting that CTCF has dual functionality.