Analysis of transcription factor interactions with the β‐globin gene locus throughout the cell cycle

Expression of the β‐globin genes is regulated by erythroid cell and developmental stage‐specific mechanisms. Prior studies have found several factors and cofactors that associate with the upstream DNaseI hypersensitive sites (HSs) of the locus control region (LCR) and with individual globin gene promoters. Among the factors associated with the LCR HSs and the globin gene promoters are RNA polymerase II (Pol II) and the basal transcription factor TFIIB. In this study, we explored the association, disassociation and reassociation of numerous proteins to five regions within the gene locus throughout the cell cycle in a human erythroleukemia cell line (K562), in which the γ‐globin genes are highly expressed. Specifically, we analyzed the interactions of TFIIB, TBP, CBP, and Pol II with LCR hypersensitive site 2 (HS2), the Gγ‐gene promoter, a region 3′ to the Aγ‐globin gene, the β‐globin gene promoter, and the gene encoding Erythroid Krüppel‐like Factor (EKLF) as a control. Interactions were analyzed by chromatin immunoprecipitation (ChIP) followed by real‐time PCR in unsynchronized cells, cells synchronized in the G 1 /S‐phase transition and in M‐phase. The data reveal dynamic interaction patterns of proteins with the globin gene locus at various cell cycle stages and suggest that Pol II remains associated with the 5′ region of the γ‐globin gene promoter in M‐phase.