Abstract
Forkhead factors are important regulators of animal development and homeostasis. They are among the earliest to bind quiescent genes, which they activate in conjunction with other transcription factors. Many liver-specific genes are under the control of FoxA2, a liver-enriched forkhead protein. Here we confirmed by chromatin immunoprecipitation that FoxA2 is one of the factors bound to the promoter-proximal enhancer of the gene encoding apolipoprotein AI (a component of high density lipoprotein) and that it functions in synergy with the nuclear receptor hepatocyte nuclear factor-4alpha. Furthermore, toward identifying additional cofactors that could potentially regulate FoxA2 activity, we identified DNA-dependent protein kinase (DNA-PK) as a FoxA2-associated factor upon affinity purification of epitope-tagged FoxA2. We show that FoxA2, found to be a phosphoprotein in vivo, is also an efficient substrate for DNA-PK, which targets serine 283. This residue is contained within a conserved serine-glutamine phosphorylation signal for DNA-PK, located within the C-terminal third of the polypeptide, just distal to its winged-helix DNA binding domain. We establish that this residue is critical for FoxA2 function because FoxA2 bearing a mutation at this site is severely compromised in its ability to activate a reporter gene under the control of its cognate DNA-binding site (apoAI site B). Complementary experiments rule out that this mutation compromises the ability of FoxA2 to either translocate to the nucleus or to bind site B. We therefore conclude that DNA-PK-dependent phosphorylation of FoxA2 plays a critical role in its transcriptional activation function per se.
Highlights
Aiguader, 88 E-08003 Barcelona, Spain. 2 To whom correspondence should be addressed: Laboratory of Biochemistry and Molecular Biology, Rockefeller University, 1230 York Ave., New York, NY 10065
We first assessed by chromatin immunoprecipitation (ChIP) experiments using antibodies against HNF-4␣ and FoxA2 and whether these factors are localized to the Apolipoprotein AI (apoAI) enhancer in cell types that express apoAI
Given an emerging role of the superfamily of forkhead transcription factors as pioneer factors in developmental pathways through alterations in chromatin configuration, these results have important implications for how the activity of these factors might in turn be regulated
Summary
Epitope Tagging and Protein Purification from Mammalian Cells—Rat FoxA2 cDNA was subcloned into VP5 [24], a pIRESneo-1-derived (Clontech) vector, which allows expression as a FLAG-fused protein (f:FoxA2). Nuclear extract from a f:FoxA2-expressing clone was subjected to affinity purification over M2-agarose, as described previously [24]. ChIP assays were performed on extracts of 293T cells that had been cross-linked with formaldehyde 48 h after transfection with the B3XAI.LUC reporter and vectors expressing FoxA2 derivatives (see below). The beads were incubated for 4 h at 25 °C with HeLa nuclear extract (Dignam et al [27]; 5 mg of total protein) or a derived chromatographic fraction, in buffer (BC100) containing 20 mM Tris-HCl, pH 7.4, 100 mM KCl, 0.1 mM EDTA, 20% glycerol supplemented with 0.1% Nonidet P-40. Whole cell extracts from HepG2 and 293T were prepared in lysis buffer containing 0.5 M NaCl and 1% Nonidet P-40 as described previously [24]. Images were collected using a ϫ40 objective on an Olympus BX51 epifluorescent microscope and DP-BSW Olympus image acquisition software
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