Homodimerization of LDB1 is necessary for activation of the mouse β‐globin gene in vivo

A multiprotein complex that includes LDB1, TAL1, GATA‐1 and LMO2 proteins participates in long‐distant interaction in the β‐globin locus. The complex occupies the locus control region and the β‐globin promoter and mediates their proximity. Reduction of LDB1 using RNAi disrupts long‐distant interaction and blocks expression of the β‐globin gene. LDB1 protein has two defined domains that may participate in this process. The C‐terminal LIM‐interaction domain (LID) of LDB1 interacts with LMO2 which provides association of LDB1 with chromatin through DNA‐binding partners GATA‐1 and TAL1. It is known that the N‐terminal dimerisation domain (DD) of LDB1 participates in homodimerisation of the protein in vitro. We proposed that the DD domain of LDB1 plays a key role in long‐distant interaction between the LCR and β‐major gene promoter in vivo. Several deletions of LDB1 and a fusion protein that contains LMO2 and the DD domain of LDB1 were expressed in the background of endogenous LDB1 knock down MEL cells. These experiments demonstrated an important role for the LDB1 DD domain in β‐globin gene activation and LDB1 protein stability. Co‐immunoprecipitation of a truncated LDB1 protein consisting of only the DD domain with endogenous LDB1 confirms that homodimerisation of LDB1 in vivo occurs by interaction between DD domains. These results confirm the necessity of homodimerisation of LDB1 in the gene regulation processes.