Structure of the FoxM1 DNA-recognition domain bound to a promoter sequence

Abstract

FoxM1 is a member of the Forkhead family of transcription factors and is implicated in inducing cell proliferation and some forms of tumorigenesis. It binds promoter regions with a preference for tandem repeats of a consensus 'TAAACA' recognition sequence. The affinity of the isolated FoxM1 DNA-binding domain for this site is in the micromolar range, lower than observed for other Forkhead proteins. To explain these FoxM1 features, we determined the crystal structure of its DNA-binding domain in complex with a tandem recognition sequence. FoxM1 adopts the winged-helix fold, typical of the Forkhead family. Neither 'wing' of the fold however, makes significant contacts with the DNA, while the second, C-terminal, wing adopts an unusual ordered conformation across the back of the molecule. The lack of standard DNA-'wing' interactions may be a reason for FoxM1's relatively low affinity. The role of the 'wings' is possibly undertaken by other FoxM1 regions outside the DBD, that could interact with the target DNA directly or mediate interactions with other binding partners. Finally, we were unable to show a clear preference for tandem consensus site recognition in DNA-binding, transcription activation or bioinformatics analysis; FoxM1's moniker, 'Trident', is not supported by our data.

Figure 1.

DNA-binding properties of FoxM1-DBD as measured by fluorescence anisotropy. (A) Anisotropy measurements of 1 nM of TAMRA-labeled consensus sequence binding to different concentrations of FoxM1-DBD (residues 222–360). Error bars represent standard deviations of three different experimental measurements. (B) Competition assays in which increasing concentrations of non-labeled oligonucleotides derived from estrogen receptor alpha promoter elements are added allowing the determination of relative affinities expressed as EC50 values. (C) Anisotropy measurements using the TAMRA labeled consensus sequence as in (A), a sequence without the A(i + 4) consensus base and a non-consensus (random) sequence. (D) Competition assays in which increasing concentrations of various non-labeled palindromic oligonucleotides are added allowing the determination of relative affinities expressed as EC50 values.

Figure 2.

Crystal structure of the FoxM1 DNA binding domain. (A) cartoon representation of the macromolecules present in the asymmetric unit. The FoxM1 A molecule is shown with helices in red and β-strands in yellow and loop regions in green. The B molecule has the same coloring but with helices in purple. (B) A magnified representation of the FoxM1 A subunit bound to the major groove of the DNA. Secondary structural elements and the Nand C-termini are labeled. (C) The residues and bases involved in sequence-specific DNA-protein interactions are shown in stick-representation. Each residue and base is labeled, the chain identifiers of the DNA bases are also shown. (D) Close-up view of the protein-protein interface at the site of the palindromic psuedo 2-fold axis. Water molecules are shown in pink and magnesium ions in green.

Figure 3.

Interactions made between FoxM1 and its cognate DNA. A two-dimensional representation is shown that highlights the interactions made between FoxM1 and the DNA. The phosphate-sugar backbone for the oligonucleotide C-chain is on the left and Dchain on the right. Next to each backbone sugar is the single letter code of the respective base. Chemical moieties that are seen to be the correct distance in the crystal structure to form hydrogen bonds (solid line) or van-der-Waals interactions (brackets) with residues in the A protein subunit are indicated. Similar interactions are observed for the palandromicly-equivalent residues of the DNA and the B protein subunit.

Figure 4.

DNA bound Forkhead domains. Cartoon representation of the Fox proteins for which structures in complex with DNA are known. The first wing loop is highlighted in pink and the second in black. In Fox K2 and FoxP2 the fourth helix preceding wing2 is also highlighted. In the lower right a superposition between FoxM1 (coloured as in Figure 2) and FoxO3 is shown detailing the different conformations of the proteins Ctermini. The FoxM1 C-terminus is colored teal.

Figure 5.

Transcription activation assays and genomic search for Fox consensus sequences. (A) Luciferase reporter assays showing the FoxM1-mediated transcription rates from a Forkhead consensus promoter for wild type full-length protein or the L291Y mutant. As a control the western blot showing the cell-lysates expression levels of each protein is at the bottom of the figure. (B)The left panel shows a histogram of the number of forkhead consensus sites in either orientation found within the 3000 bp upstream of each gene in human genome colored in grey. Overlaid upon this are similar histograms that display the degree of conservation of such sequences within vertebrate genomes. The right panel depicts the results of an identical search made for tandem or palindromic forkhead consensus sequences and their degree of conservation. (C) Luciferase reporter assays where the FoxM1-mediated transcription rates from a forkhead consensus promoter for wild type full-length protein are competed with FoxM1 DC (1–600) or FoxM1-DBD (210–378). As a control the western blot showing the cell-lysates expression levels of each protein and a loading control is at the bottom of the figure. (D) Luciferase reporter assays where the FoxM1-mediated transcription rates from a forkhead consensus promoter for wild type full-length protein are competed with FoxM1-DBD or FoxO3a-DBD. Both the actual luciferase readout (blue bars) and the same reading normalized for relative protein expression (cyan bars) are shown for clarity. As a control the western blot showing the cell-lysates expression levels of each protein and a loading control is at the bottom of the figure.