Joint regulation of the MAP1B promoter by HNF3β/Foxa2 and Engrailed is the result of a highly conserved mechanism for direct interaction of homeoproteins and Fox transcription factors

Abstract

The MAP1B (Mtap1b) promoter presents two evolutionary conserved overlapping homeoproteins and Hepatocyte nuclear factor 3β (HNF3β/Foxa2) cognate binding sites (defining putative homeoprotein/Fox sites, HF1 and HF2). Accordingly, the promoter domain containing HF1 and HF2 is recognized by cerebellum nuclear extracts containing Engrailed and Foxa2 and has regulatory functions in primary cultures of embryonic mesmetencephalic nerve cells. Transfection experiments further demonstrate that Engrailed and Foxa2 interact physiologically in a dose-dependent manner: Foxa2 antagonizes the Engrailed-driven regulation of the MAP1B promoter, and vice versa. This led us to investigate if Engrailed and Foxa2 interact directly. Direct interaction was confirmed by pull-down experiments, and the regions participating in this interaction were identified. In Foxa2 the interacting domain is the Forkhead box DNA-binding domain. In Engrailed, two independent interacting domains exist: the homeodomain and a region that includes the Pbx-binding domain. Finally, Foxa2 not only binds Engrailed but also Lim1, Gsc and Hoxa5 homeoproteins and in the four cases Foxa2 binds at least the homeodomain. Based on the involvement of conserved domains in both classes of proteins, it is proposed that the interaction between Forkhead box transcription factors and homeoproteins is a general phenomenon.

Evolution of a derived protein–protein interaction between HoxA11 and Foxo1a in mammals caused by changes in intramolecular regulation

“…In vitro translated protein reactions were divided between single-protein (e.g., HoxA11 only) and double-protein (e.g., HoxA11, Foxo1a) reactions and diluted to 500 μL using modified BF1 buffer (20 mM Tris HCl (pH 7.4), 100 mM NaCl, 1 mM EDTA, 10% glycerol, 0.01% Triton X-100, 0.5 mM DTT) (33). Samples were incubated overnight with anti-Flag beads; they were then washed and visualized as described above.…”

Crucial roles ofFoxa2in mouse anterior–posterior axis polarization via regulation of anterior visceral endoderm-specific genes

“…The FOXA2 protein was produced employing an in vitro translational system (Promega, Madison, WI) as described (32). Binding reaction was conducted as reported (33). The competition assay included two molecular ratios of labeled oligonucleotides to unlabelled oligonucleotides, of 1:50 or 1:100.…”

A phylogenetically conserved cis-regulatory module in theMsx2promoter is sufficient for BMP-dependent transcription in murine andDrosophilaembryos

“…Whether Fox proteins can bind this sequence, and, if so, whether such binding is disrupted by mutations in the TTAATT site are unanswered questions. It is intriguing that Fox proteins can form heterodimers with homeodomain-containing proteins (Foucher et al, 2003), and that the Fox protein Fast1 can interact with Smads and modulate the activity of TGFβ-responsive promoters (Chen et al, 1996;Chen et al, 1997;Weisberg et al, 1998).…”