Structural constraints in collaborative competition of transcription factors against the nucleosome

Abstract

Cooperativity in transcription factor (TF) binding is essential in eukaryotic gene regulation and arises through diverse mechanisms. Here, we focus on one mechanism, collaborative competition, which is of interest because it arises both automatically (with no requirement for TF coevolution) and spontaneously (with no requirement for ATP-dependent nucleosome remodeling factors). Previous experimental studies of collaborative competition analyzed cases in which target sites for pairs of cooperating TFs were contained within the same side of the nucleosome. Here, we utilize new assays to measure cooperativity in protein binding to pairs of nucleosomal DNA target sites. We focus on the cases that are of greatest in vivo relevance, in which one binding site is located close to the end of a nucleosome and the other binding site is located at diverse positions throughout the nucleosome. Our results reveal energetically significant positive (favorable) cooperativity for pairs of sites on the same side of the nucleosome but, for the cases examined, energetically insignificant cooperativity between sites on opposite sides of the nucleosome. These findings imply a special significance for TF binding sites that are spaced within one-half nucleosome length (74 bp) or less along the genome and may prove useful for prediction of cooperatively acting TFs genome wide.

Figure 1. DNA template for restriction enzyme digestion kinetic assay

(a) Schematic illustration of 147R DNA^; indicating locations of recognition sites for the four restriction enzymes and the DNA binding protein LexA. (b) Crystal structure of the nucleosome depicting the binding sites for LexA (red) and the restriction enzymes MspI (orange), HaeIII (magenta), AluI (blue) and StyI (green). Histone proteins are shown in ribbon representation (colored wheat).

Figure 2. Nucleosome-dependent cooperativity between LexA and StyI sites

Representative experiment probing accessibility at the StyI binding site in the absence and presence of saturating LexA. (a–d) Denaturing polyacrylamide gel analyses of StyI digestion reactions. Substrate (S) is converted to products (P1 and P2). Asterisk (*) indicates additional products resulting from StyI “star” activity. (a) < 0.5 nM naked DNA with 10 U/mL Sty1. (b) <0.5 nM naked DNA with 100 nM LexA and 10 U/mL StyI. (c) 5 nM nucleosomes with 5000 U/mL StyI. (d) 5 nM nucleosomes with 1 μM LexA and 5000 U/mL StyI. (e) Quantitative analysis of the digestion pattern resolved in the gels in panels a–d. Circles: naked DNA; squares: nucleosomes. Open datapoints: no LexA; filled datapoints: saturating LexA. The fraction of DNA uncut is plotted versus time. Data are fit to single exponential (a, b, d) or linear (c) functions. For nucleosomes, the zero time point is omitted from the analysis to exclude the digestion of any contaminating naked DNA, which is complete within the first minute of reaction (see Methods).

Figure 3. FRET assay for nucleosome-dependent cooperativity

Assay probes cooperativity between the LexA site and additional locations on the same side of the nucleosome. Shown are schematic illustrations of DNA templates and labeled nucleosome structures, indicating locations of the LexA binding site (red), fluorescence donor dye (Cy3, Cyan) and fluorescence acceptor dyes (Cy5, Magenta), and normalized emission spectra of the labeled nucleosomes titrated with increasing concentrations of LexA (with excitation of the Cy3 donor). All histone octamers had the engineered mutation H3C110A together with an additional unique cysteine at a different location on one or another of the four histones. (a) Cy3-1 nucleosomes: Cy3 attached at bp 1, Cy5 attached at H3V35C. (b) Cy3-35 nucleosomes: Cy3 attached at bp 35, Cy5 attached at H2BT112C. (c) Cy3-57 nucleosome: Cy3 attached at bp 57, Cy5 attached at H4L22C. (d) Quantitative analysis of FRET efficiency changes during LexA titrations for Cy3-1 (red circles), Cy3-35 (green squares), and Cy3-57 (blue triangles).

Figure 4. No nucleosome-dependent cooperativity between LexA and HaeIII sites

Representative experiment probing accessibility at the HaeIII binding site in the absence and presence of saturating LexA. (a–d) Denaturing polyacrylamide gel analyses of HaeIII digestion reactions. Substrate (S) is converted to products (P1 and P2) over time. (a) <0.5 nM naked DNA with 0.625 U/mL HaeIII. (b) <0.5 nM naked DNA with 100 nM LexA and 0.625 U/mL HaeIII. (c) 5 nM nucleosomes with 5000 U/mL HaeIII. (d) 5 nM nucleosomes with 1 μM LexA and 5000 U/mL HaeIII. (e) Quantitative analysis of the gels in panels (A–D). The fraction of DNA uncut is plotted versus time. Data are fit to single exponential (naked DNA) or linear (nucleosomal DNA) functions. For nucleosomes, the zero time point is omitted from data analysis (see Methods).

Figure 5. Position dependent cooperativity across the nucleosome

Shown are the coupling free energy between each restriction enzyme and LexA, for nucleosomes (solid bars) or naked DNA (hatched). Error bars: standard errors, for MspI (n=2), HaeIII (n=7), AluI (n=5) and Sty1 (n=3). Dashed horizontal lines represent the numerical value of kBT, the characteristic energy of thermal fluctuations, where KB is Boltzmann’s constant and T the absolute temperature.