Abstract
FoxA1-3 (formerly HNF3alpha, -beta, and -gamma), members of the FoxA subfamily of forkhead transcription factors, function as initial chromatin-binding and chromatin-remodeling factors in a variety of tissues, including liver and pancreas. Despite essential roles in development and metabolism, regulation of FoxA factors is not well understood. This study examines a potential role for acetylation in the regulation of FoxA chromatin binding and remodeling. Using in silico analysis, we have identified 11 putative p300 acetylation sites within FoxA1, five of which are located within wings 1 and 2 of its winged-helix DNA-binding domain. These polypeptide structures stabilize FoxA DNA and chromatin binding, and we have demonstrated that acetylation attenuates FoxA binding to DNA and diminishes its ability to remodel chromatin. FoxA acetylation is inhibited by chromatin binding. We propose a model whereby stable chromatin binding protects the FoxA DNA-binding domain from acetylation to preserve chromatin binding and remodeling by FoxA factors in the absence of extracellular cues.
Highlights
During early development, FoxA functions as a pioneer transcription factor, recognizing and binding to its sites within
Recent studies have demonstrated that stable nucleosome binding resides within the FoxA “winged-helix” DNA-binding domain shared with the linker histone H1 [23]
We have demonstrated that FoxA1 can be acetylated in vitro and in vivo (Fig. 1) and that acetylation attenuates binding of FoxA1 to its regulatory elements assembled within nucleosomal DNA (Fig. 4), curtailing its nucleosome-remodeling capabilities
Summary
Plasmid Construction and Mutagenesis—The bacterial expression plasmid encoding histidine-tagged full-length FoxA1 (pET28b-6xhis-FoxA) contains the mouse FoxA1 cDNA subcloned into the pET28b plasmid (Novagen, Gibbstown NJ) as described [8]. The indicated amounts of purified recombinant in vitro or mock-acetylated FoxA1 protein (wild-type or mutant) or in vitro translated wild-type, acetylation mimic, and acetylation mutant FoxA1 proteins were incubated with end-labeled eG site probe (7.8 ng for purified proteins and 24 ng for in vitro translated proteins) for 30 min at room temperature in binding buffer (10 mM Tris, pH 7.5, 1% Ficoll, 0.05 M NaCl, 0.33 mM MgCl2, 0.001 M dithiothreitol, and 130 ng/l bovine serum albumin). To examine the effect of acetylation on FoxA1 binding, binding reactions were carried out in a 20-l volume containing 5 ng of free DNA probe or 10 ng of nucleosomes (final concentration of 4 nM DNA or chromatin) and the indicated amounts of purified recombinant in vitro or mock-acetylated FoxA1 under final buffer conditions of 10 mM Tris, pH 7.5, 1% Ficoll, 1 mM MgCl2, 35 mM KCl, 5 mM dithiothreitol, 300 ng/l bovine serum albumin, and 4.8% glycerol at 21–25 °C for 1 h. The membrane was washed with 1ϫ phosphate-buffered saline, incubated for 1 h in ABC (Pierce) mixed in 1ϫ phosphate-buffered saline, and washed a final time before exposure to ECL Western blot detection reagent (GE Healthcare) and Kodak XAR film
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