Two mechanisms in the action of repressor δEF1: binding site competition with an activator and active repression

Background: Counteraction between activators and repressors is crucial for the regulation of a number of cell-specific enhancers, where an activator and a repressor are mutually competitive in binding to the same site. δEF1 is a repressor protein of δ1-crystallin minimal enhancer DC5 binding at the CACCT site, and inhibits activator δEF3 from binding to the overlapped site. It has two zinc finger clusters N-fin and C-fin, close to N- and C-termini, respectively, and a homeodomain in the middle. δEF1 also binds to theE2-box sequence CACCTG, and represses E2-box-dependent enhancers. 

Results: The mechanism of the repressor action of δEF1 was investigated by examining various deletion mutants of δEF1 for their activity to repress δ1-crystallin enhancer fragment HN which contained DC5 sequence and an additional activator site. Both zinc finger clusters were found to be essential for DNA binding and repression, but the homeodomain was not. In addition, the NR domain close to the N-terminus was required for full repression. The NR domain showed active repression when fused to the Gal4 DNA binding domain. Active repression by δEF1, dependent on the NR domain, was also demonstrated in a situation where the binding sites of δEF1 and δEF3 were separated. N-fin and C-fin in their isolated forms bind the 5′-(T/C)ACCTG-3′ and 5′-(t/C)ACCT-3′ sequences, respectively, while the homeodomain showed no DNA binding activity. An analysis of DNA binding of the ΔIntF form, having both N-fin and C-fin, indicated that a single DNA binding domain is assembled from two zinc finger clusters. 

Conclusion: Two mechanisms are involved in the repressor action of δEF1. First, a binding site competition with an activator which depends on the integrity of both zinc finger clusters, and second, an active repression to silence an enhancer which is attributed to the NR domain.