Sekido R, Murai K, Kamachi Y, Kondoh H
Institute for Molecular and Cellular Biology, Osaka University, Suita, Japan.
Genes Cells. 1997 Dec;2(12):771-83. doi: 10.1046/j.1365-2443.1997.1570355.x.
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. DeltaEF1 is a repressor protein of delta1-crystallin minimal enhancer DC5 binding at the CACCT site, and inhibits activator deltaEF3 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. deltaEF1 also binds to the E2-box sequence CACCTG, and represses E2-box-dependent enhancers.
The mechanism of the repressor action of deltaEF1 was investigated by examining various deletion mutants of deltaEF1 for their activity to repress delta1-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 deltaEF1, dependent on the NR domain, was also demonstrated in a situation where the binding sites of deltaEF1 and deltaEF3 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 delta(Int)F form, having both N-fin and C-fin, indicated that a single DNA binding domain is assembled from two zinc finger clusters.
Two mechanisms are involved in the repressor action of deltaEF1. 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.
激活因子与抑制因子之间的拮抗作用对于许多细胞特异性增强子的调控至关重要，在此过程中，激活因子和抑制因子在结合同一位点时相互竞争。DeltaEF1是一种抑制蛋白，可与delta1-晶体蛋白最小增强子DC5在CACCT位点结合，并抑制激活因子deltaEF3与重叠位点的结合。它有两个锌指结构簇，分别靠近N端和C端，即N-fin和C-fin，中间还有一个同源结构域。DeltaEF1也可与E2-box序列CACCTG结合，并抑制E2-box依赖的增强子。
通过检测DeltaEF1的各种缺失突变体对包含DC5序列和一个额外激活位点的delta1-晶体蛋白增强子片段HN的抑制活性，研究了DeltaEF1的抑制作用机制。发现两个锌指结构簇对于DNA结合和抑制都是必需的，但同源结构域并非如此。此外，靠近N端的NR结构域对于完全抑制是必需的。当NR结构域与Gal4 DNA结合结构域融合时，显示出活性抑制作用。在DeltaEF1和DeltaEF3的结合位点分离的情况下，也证明了DeltaEF1依赖于NR结构域的活性抑制作用。分离形式的N-fin和C-fin分别与5'-(T/C)ACCTG-3'和5'-(t/C)ACCT-3'序列结合，而同源结构域未显示出DNA结合活性。对同时具有N-fin和C-fin的delta(Int)F形式的DNA结合分析表明，单个DNA结合结构域由两个锌指结构簇组装而成。
DeltaEF1的抑制作用涉及两种机制。第一，与激活因子的结合位点竞争，这依赖于两个锌指结构簇的完整性；第二，使增强子沉默的活性抑制作用，这归因于NR结构域。
