Cooperative binding of an Ultrabithorax homeodomain protein to nearby and distant DNA sites.

Abstract

Cooperativity in binding of regulatory proteins to multiple DNA sites can heighten the sensitivity and specificity of the transcriptional response. We report here the cooperative DNA-binding properties of a developmentally active regulatory protein encoded by the Drosophila homeotic gene Ultrabithorax (Ubx). We show that naturally occurring binding sites for the Ubx-encoded protein contain clusters of multiple individual binding site sequences. Such sites can form complexes containing a dozen or more Ubx-encoded protein molecules, with simultaneous cooperative interactions between adjacent and distant DNA sites. The distant mode of interaction involves a DNA looping mechanism; both modes appear to enhance transcriptional activation in a simple yeast assay system. We found that cooperative binding is dependent on sequences outside the homeodomain, and we have identified regions predicted to form coiled coils carboxy terminal to the homeodomains of the Ubx-encoded protein and several other homeotic proteins. On the basis of our findings, we propose a multisite integrative model of homeotic protein action in which functional regulatory elements can be built from a few high-affinity sites, from many lower-affinity sites, or from sites of some intermediate number and affinity. An important corollary of this model is that even small differences in binding of homeotic proteins to individual sites could be summed to yield large overall differences in binding to multiple sites. This model is consistent with reports that homeodomain protein targets contain multiple individual binding site sequences distributed throughout sizable DNA regions. Also consistent is a recent report that sequences carboxy terminal to the Ubx homeodomain can contribute to segmental specificity.

HOXA9 regulates BRCA1 expression to modulate human breast tumor phenotype

“…Interestingly, site-directed mutagenesis of individual HOXA9 consensus binding sites did not significantly compromise BRCA1 promoter activity, suggesting cooperative release of tandem HOX consensus binding sites might be necessary to ablate HOXA9-dependent control of BRCA1 gene expression (Supplemental Figure 8). These observations are consistent with previous reports of promoter site cooperation and redundancy in other HOX-regulated genes (65)(66)(67). The findings indicate that DNA binding of HOXA9 directly regulates BRCA1 expression in breast cells.…”

Pbx proteins display hexapeptide-dependent cooperative DNA binding with a subset of Hox proteins.

“…However, the studies by Chan et al (1994) employed Ubx constructs that lacked the conserved hexapeptide motif and were conducted at protein concentrations at least several hundred-fold over those employed by us. Weak cooperativity was previously observed for Ubx alone on a multisite probe and proposed to be mediated by sequences carboxy-terminal to the Ubx homeo domain (Beachy et al 1993).…”

Kinetics of binding of Antp homeodomain to DNA analyzed by measurements of surface plasmon resonance

“…Thus, once one homeodomain had bound non-specifically to DNA, the second molecule could bind more rapidly to the DNA than the first molecule. Cooperative binding of a homeodomain to DNA has been reported previously [25]. Therefore, the values cited for k~ above should be considered the average values for mixed reactions.…”