Comparative analysis of linker histone H1, MeCP2, and HMGD1 on nucleosome stability and target site accessibility

Abstract

Chromatin architectural proteins (CAPs) bind the entry/exit DNA of nucleosomes and linker DNA to form higher order chromatin structures with distinct transcriptional outcomes. How CAPs mediate nucleosome dynamics is not well understood. We hypothesize that CAPs regulate DNA target site accessibility through alteration of the rate of spontaneous dissociation of DNA from nucleosomes. We investigated the effects of histone H1, high mobility group D1 (HMGD1), and methyl CpG binding protein 2 (MeCP2), on the biophysical properties of nucleosomes and chromatin. We show that MeCP2, like the repressive histone H1, traps the nucleosome in a more compact mononucleosome structure. Furthermore, histone H1 and MeCP2 hinder model transcription factor Gal4 from binding to its cognate DNA site within the nucleosomal DNA. These results demonstrate that MeCP2 behaves like a repressor even in the absence of methylation. Additionally, MeCP2 behaves similarly to histone H1 and HMGD1 in creating a higher-order chromatin structure, which is susceptible to chromatin remodeling by ISWI. Overall, we show that CAP binding results in unique changes to nucleosome structure and dynamics.

Figure 1

Nucleosomal construct used in FRET study (a) DNA construct used in our FRET studies; contains a 601 NPS, the Cy3 fluorophore (position 43), a Gal4 binding site shown as a blue bar (position 48–76), and an extra 50 bp linker DNA at the entry/exit ends of the 601 NPS. (b) Structure of Cy3-Cy5 labeled nucleosome construct adapted from, showing the labels in relation to the DNA and histone octamer.

Figure 2. CAP-nucleosome binding.

CAP-chromatosomes were created by titrating 0–250 nM of (a) histone H1, (b) MeCP2, or (c) HMGD1 into 5 nM labeled nucleosomes. Once binding equilibrium was reached, samples were run on a 5% native acrylamide gel to view CAP-chromatosome formation.

Figure 3. Histone H1 and MeCP2 influence nucleosome breathing.

CAPs were titrated into 5 nM Cy3-Cy5 paired FRET mononucleosomes to measure changes in nucleosome breathing dynamics due to CAP binding. The CAP-chromatosome was excited with 510 nm light and the emission spectrum collected from 550–750 nm. FRET efficiency was calculated using the Ratio_A method. Arrows indicate direction of changes in signal with Cy3 and Cy5. (a) Emission spectra and (b) the corresponding increase in FRET efficiency for select concentrations of the Drosophila histone H1 titration into labeled nucleosomes. (c) Emission spectra and (d) the corresponding changes in FRET efficiency for select concentrations of the MeCP2 titration into labeled nucleosomes. (e) Emission spectra and (f) the corresponding changes in FRET efficiency for select concentrations of HMGD1 titration into labeled nucleosomes. Normalized spectral graphs (to Cy3 emission) for select concentrations of CAPs are in Supplementary Fig. 3 to visualize the change in Cy5 emission. (g) The FRET efficiency measurements were plotted on a semi-log graph based on CAP concentration and fit to a Hill binding curve (shown are curves fitted with or without Hill coefficients). Due to the lack of measurable change in FRET efficiency upon HMGD1 binding, no Hill curve was drawn. Note: Error bars for all graphs are the standard deviation of 3 replicates.

Figure 4. Histone H1- and MeCP2-chromatosomes hinder Gal4 from binding its target site within mononucleosomes.

(a) Fluorescence emission spectra of Cy3-Cy5 labeled mononucleosomes with increasing concentrations of Gal4 (Normalized to Cy3 emission spectral graphs for select concentrations are in Supplemental Fig. 4). As Gal4 binds to its target site, the Cy5 emission decreases leading to a decrease in FRET efficiency (b). (c) The normalized FRET efficiency measurements (complete spectra graphs are in Supplemental Fig. 3) of a Gal4 titration into mononucleosomes not stabilized by CAP, bound with 15 nM histone H1, or 25 nM MeCP2 were plotted on a semi-log plot based on Gal4 concentration. Note: the concentrations for CAPs used, were based on their pre-determined S_1/2 values (Table 1). Values represent averages of duplicate or triplicate experiments and error bars depict the standard deviations of the mean.

Figure 5. CAP-mediated higher order chromatin structures are susceptible to chromatin remodeling by ISWI.

(a) Scheme of the 3055 bp DNA construct containing a 17-mer array of 601 NPS separated by 30 bp linker DNA. The array DNA comprised 17 repeats of a NPS harboring the Widom-601 nucleosome positioning sequence (dashed line). Numbers indicate positions of restriction enzyme sites with respect to the nucleosome boundary. (b) Remodeled and non-remodeled (ISWI or no ISWI) chromatin samples were digested with BamHI. ImageQuant imaging software was used to measure the intensity of the full and digested array DNA. These values were used to determine the percent uncut. (c) After ISWI remodeling, chromatin samples were digested with PstI and imaged on an agarose gel. Digestion amount quantified using band intensity, accounting for non-saturated arrays and were compared to No CAP chromatin. Error bars are the mean ± standard deviation.