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Hypersensitive site 5 (5′HS5) of the  β-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  β-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  β-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.

5′HS5 of the Human β-globin Locus Control Region Is Dispensable for the Formation of the β-globin Active Chromatin Hub