Down-regulation of TDT transcription in CD4(+)CD8(+) thymocytes by Ikaros proteins in direct competition with an Ets activator

Ikaros is a unique regulator of lymphopoiesis that associates with pericent romeric heterochromatin and has been implicated in heritable gene inactivat ion. Binding and competition experiments demonstrate that Ikaros dimers com pete with an Ets activator for occupancy of the lymphocyte-specific TdT pro moter. Mutations that selectively disrupt Ikaros binding to an integrated T dT promoter had no effect on promoter function in a CD4(+)CD8(+) thymocyte line. However, these mutations abolished down-regulation on differentiation , providing evidence that Ikaros plays a direct role in repression. Reduced access to restriction enzyme cleavage suggested that chromatin alterations accompany down-regulation. The Ikaros-dependent down-regulation event and the observed chromatin alterations appear to precede pericentromeric reposi tioning. Current models propose that the functions of Ikaros should be disr upted by a small isoform that retains the dimerization domain and lacks the DNA-binding domain. Surprisingly, in the CD4(+)CD8(+) thymocyte line, over expression of a small Ikaros isoform had no effect on differentiation or on the pericentromeric targeting and DNA-binding properties of Ikaros. Rather , the small isoform assembled into multimeric complexes with DNA-bound Ikar os at the pericentromeric foci. The capacity for in vivo multimer formation suggests that interactions between Ikaros dimers bound to the TdT promoter and those bound to pericentromeric repeat sequences may contribute to the pericentromeric repositioning of the inactive gene.