Nucleosome-binding affinity as a primary determinant of the nuclear mobility of the pioneer transcription factor FoxA - PubMed (original) (raw)

Nucleosome-binding affinity as a primary determinant of the nuclear mobility of the pioneer transcription factor FoxA

Takashi Sekiya et al. Genes Dev. 2009.

Abstract

FoxA proteins are pioneer transcription factors, among the first to bind chromatin domains in development and enable gene activity. The Fox DNA-binding domain structurally resembles linker histone and binds nucleosomes stably. Using fluorescence recovery after photobleaching, we found that FoxA1 and FoxA2 move much more slowly in nuclei than other transcription factor types, including c-Myc, GATA-4, NF-1, and HMGB1. We find that slower nuclear mobility correlates with high nonspecific nucleosome binding, and point mutations that disrupt nonspecific binding markedly increase nuclear mobility. FoxA's distinct nuclear mobility is consistent with its pioneer activity in chromatin.

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Figures

Figure 1.

Low nuclear mobility correlates with high nonspecific nucleosome binding. (A, left) FRAP of indicated GFP fusion proteins. FoxA1 recovery was the slowest among the five TFs. Values represent means from at least 10 cells; standard deviations shown. The first time points were 0.24–0.5 sec after the final bleach pulse and set as time zero. Half-recovery times (_t_1/2) are shown. Note that the blue triangles depicting points for GATA-4 are partially obscured by the yellow dots for c-Myc. (Right) Nuclear localization of GFP fusion proteins used in the FRAP assay. (B) SDS-PAGE of 3 μg of purified proteins used in C. (C) EMSA with increasing concentrations of purified recombinant proteins shown in B and reconstituted NCP147 and 5S rDNA mononucleosomes. FoxA1 showed the strongest affinity to both mononucleosomes. All shifted complexes are factors on nucleosomes; nucleosome preps were virtually free of pure DNA and complexes of factors with free DNA migrated differently (see Supplemental Fig. S2A). (D) Summary of the results from FRAP and EMSA assays.

Figure 2.

Markedly slow movement of FoxA1 across the nucleus. (A) Half-area FRAP assay of c-Myc. (Left) Quantification of the fluorescence recoveries of c-Myc after bleaching half the nuclear area. Average fluorescence intensities in the bleached (blue circles) and unbleached (red circles) region were plotted over time. Values are averages from at least 10 cells. (Right) Fluorescent images of c-Myc during the FRAP process. White bars indicate the border between the bleached and unbleached areas. Times after bleaching are indicated. (B) Half-area FRAP assay of FoxA1, as in A. (C) The average fluorescence intensities of FoxA1 and c-Myc in each area were quantified and plotted. (Blue squares) Area 1; (red) area 2; (yellow) area 3; (green) total bleached region. (D) Different movement models for c-Myc and FoxA1. (Left) c-Myc interaction with chromatin is less stable, with more repeated association and dissociation allowing more extensive diffusion in the nucleoplasm. (Right) The higher chromatin affinity of FoxA1 keeps it more closely associated with chromatin, causing slower overall movement.

Figure 3.

Specific and nonspecific binding of FoxA to mononucleosomes. (A) Schematic of NCP147 derivatives with an integrated eH FoxA-bds at indicated base pair positions with respect to the dyad axis (D). (B) EMSA of NCP147 and its derivative D + 2 mononucleosomes with FoxA1. The FoxA1–probe complex position is shown. (C) Quantification of the EMSA in B. The ratio of shifted/unshifted mononucleosomes at 2 nM FoxA1 is shown. Data are shown with the standard deviation (SD); n = 3; (*) P < 0.05; (**) P < 0.01, Student's _t_-test. (D) Modeling of the FoxA-DBD and the D + 2 mononucleosome complex, by the program LSQMAN (see the Supplemental Material). The FoxA recognition site (TGTTTGC) is shown in dark blue; light blue shows the 13-bp fragment from the published FoxA–DNA structure (Clark et al. 1993). The hypothetical position of FoxA (based on superposition of the DNA part of the FoxA–DNA structure with the nucleosomal DNA) is shown in magenta. No clashes between the nucleosome and FoxA were observed in this or any other of the tested configurations, except with the very end of nucleosomal DNA. The nucleosomal dyad axis is indicated (Φ).

Figure 4.

Sequence nonspecific interaction with DNA and nucleosomes is a major determinant of FoxA1 movement in nucleus. (A) Molecular modeling of the FoxA–DNA complex (Clark et al. 1993) using RASMOL. DNA in ball and stick, FoxA in gray wireframe, and amino acids mutated in spacefill. Amino acids are N216 (red), H220 (blue), R262 (orange), R265 (pink), S242 (green), and W244 (cyan). (B,C,E,F) EMSA with increasing concentrations of purified recombinant proteins of FoxA1 and the free N1-A DNA (B,C) and mononucleosome (E,F) probes indicated. Data from B, C, and E, F, where there is >90% free probe and quantifiable shifting, are plotted in D and G, respectively. N1-A DNAs or mononucleosomes either contained a FoxA-bds (solid bars), or lacked a FoxA-bds site (N1-A/eGeH mut, open bars). The data show that NH-mut is diminished in sequence-specific binding but retain nonspecific binding, and that RR-mut is diminished in overall nonspecific binding but retain some specific binding. (H, left) Fluorescence recovery kinetics of GFP fusion FoxA1 proteins indicated. The data show that loss of nonspecific binding (RR-mut) elicits a more dramatic increase in nuclear mobility than loss of specific binding (NH-mut).

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Nucleosome-binding affinity as a primary determinant of the nuclear mobility of the pioneer transcription factor FoxA - PubMed (original) (raw)