HMGB1 restores a dynamic chromatin environment in the presence of linker histone by deforming nucleosomal DNA

Abstract

The essential architectural protein HMGB1 increases accessibility of nucleosomal DNA and counteracts the effects of linker histone H1. However, HMGB1 is less abundant than H1 and binds nucleosomes more weakly raising the question of how HMGB1 effectively competes with H1. Here, we show that HMGB1 rescues H1's inhibition of nucleosomal DNA accessibility without displacing H1. HMGB1 also increases the dynamics of condensed, H1-bound chromatin. Cryo-EM shows that HMGB1 binds at internal locations on a nucleosome and locally distorts the DNA. These sites, which are away from the binding site of H1, explain how HMGB1 and H1 co-occupy a nucleosome. Our findings lead to a model where HMGB1 counteracts the activity of H1 by distorting nucleosomal DNA and by contacting the H1 C-terminal tail. Compared to direct competition, nucleosome co-occupancy by HMGB1 and H1 allows a greater diversity of dynamic chromatin states and may be generalizable to other chromatin regulators.

Figure 1.. HMGB1 grants access to nucleosomal DNA

(A) A schematic of autoinhibition of HMGB1’s DNA binding A and B-boxes by the negatively charged C-terminal tail. (B) A schematic of the restriction enzyme accessibility (REA) assay showing how nucleosomal DNA cutting by the restriction enzyme PstI occurs when nucleosomal DNA transiently unwraps from the octamer core. The green star represents the fluorescent label on the end of the DNA. (C) Representative gels of time courses of REA on 10/10-Pst137 nucleosomes with (bottom) and without (top) the presence of 2.5μM HMGB1. PstI cleavage results in the appearance of the lower, cut band over time. (D) Quantification of three replicate experiments of (B). The fraction of DNA that remains uncut is plotted as a function of time. The data for all three replicates are fit by a two-step exponential decay function. (E, F and G) Top panels show quantification of three replicate REA experiments on 0/10-Pst137 (E), 10/0-Pst137 (F) and Core-Pst137 (G) with (teal) and without (black) HMGB1. The fraction of DNA that remains uncut is plotted as a function of time. The data for all three replicates are fit by a two-step exponential decay function. Bottom panels show representative gels of time courses of REA on nucleosomes with (bottom) and without (top) the presence of 2.5μM HMGB1 or 25μM HMGB1 for Core Pst137.

Figure 2.. HMGB1 distorts nucleosomal DNA at multiple sites

(A) Cryo-EM density map of the A box of HMGB1 bound to a 0/10 nucleosome at SHL −2 at 2.9Å resolution. (B) Atomic model built using the density in (A). (C) A comparison of the DNA bound by HMBG1 at SHL −2 (gray) to the unbound DNA at SHL +2 (teal), showing the widening of the minor grove by the HMG box. (D) An additional cryo-EM class revealed by cryoDRGN showing extra density for HMGB1 bound at SHL −6.

Figure 3.. HMGB1 selectively stabilizes a conformation of nucleosomes with fast-cutting nucleosomal DNA

(A) Representative time-course gels of REA experiments on 10/10-Pst137 nucleosomes as a function of varying HMGB1 concentrations (65.5nM, 164nM, 410nM, 1.024μM, 2.56μM, 6.4μM, and 16μM). (B) Quantification of REA experiments in (A). Each time course is fit by a two-step exponential decay. (C) Rate constants (k_fast and k_slow) for experiments in (A) are shown as a function of HMGB1 concentration. Error bars represent the standard deviation for three separate replicates. (D) The fraction of nucleosomes that are described by k_fast is plotted as a function of HMGB1 concentration. Error bars represent the standard deviation for three separate replicates. Curve represents a fit by an equation derived from the model in (E). An overall half-maximal concentration of HMGB1 for its effect is 1.42μM (K_1/2). Inset shows the same plot with a logarithmic x-axis. (E) A schematic of a model showing a slow and a fast-cutting population of nucleosomes that interconvert slowly on timescale of PstI cutting and are bound by HMGB1 with different affinities. K_D^s is 2.4μM while K_D^f is 650nM. K_D’s are derived from the equations in Materials and Methods.

Figure 4.. HMGB1 and H1 compete to tune nucleosomal DNA accessibility

(A) Representative time-course gels of REA experiments in the presence of indicated concentrations of H1 and/or HMGB1. (B) The fraction of fast cutting nucleosomes is shown as a function of HMGB1 concentration. Before HMGB1 is added, nucleosomes are either bound by no H1 (black), 15nM H1 (coral), or 15nM H1-ΔCTE (green). Curve represents a fit by an equation derived from the model in Figure 3E. (C) Representative REA time courses of 40/40-Pst137 nucleosomes with or without H1 and/or HMGB1. (D) The fraction of fast cutting nucleosomes is shown as a function of HMGB1 concentration. 40/40-Pst137 nucleosomes were pre-incubated with 20nM H1 before HMGB1 was added. Curve represents a fit by an equation derived from the model in Figure 3E. K_1/2 is 112.6nM, K_D^s is 126.3nM and K_d^f is 21.25nM.

Figure 5.. HMGB1 increases accessibility of DNA within chromatin

(A) Heat maps of SAMOSA-ChAAT experiments showing nucleosome footprints on a 12x601 array with a range of HMGB1 concentrations. Purple represents DNA that is accessible as assessed by the presence of adenine methylation. Grey represents DNA that is inaccessible as assessed by the lack of methylation and is interpreted as a nucleosome footprint. (B) The average methylation across every 601 sequence within the array is combined to show an average nucleosome footprint within the array. (C) Box and whiskers plot of nucleosome footprint size is plotted as a function of HMGB1 concentration. Solid line represents the median footprint size, while the height of the box represents the interquartile range, and the error bars represent the farthest data point within 1.5x the interquartile range from the box. (D) Heat maps of SAMOSA-ChAAT experiments showing nucleosome footprints on a 12x601 array with H1 and a range of HMGB1 concentrations. Purple and grey represent accessible and inaccessible DNA, respectively as in (A). (E) The average methylation across every 601 sequence within the array is combined to show an average nucleosome footprint within the array. (F) Box and whiskers plot of nucleosome footprint size is plotted as a function of HMGB1 concentration. Solid line represents the median footprint size, while the height of the box represents the interquartile range, and the error bars represent the farthest data point within 1.5x the interquartile range from the box.

Figure 6.. HMGB1 increases the turnover dynamics of H1 and chromatin

(A) Representative microscopy images of chromatin condensates. 30nM Alexa647-12x601 arrays are mixed with 360nM Alexa555-H1 and varying concentrations of HMGB1. Scale bar represents 10μm. (B) Violin plots of the quantification of the average Alexa555-H1 fluorescence intensity within chromatin condensates. All values are normalized to chromatin and H1 alone with no HMGB1 added. Solid and dotted lines represent the median and interquartile values respectively. Values represent an average of three experimental replicates with n>10 condensates per replicate. (C) Quantification of the fluorescence recovery after photobleaching of Alexa555-H1 within chromatin condensates. Values are normalized from 0 to 1 corresponding to post and pre-bleach respectively. Points represent an average of three experimental replicates with n>10 condensates per replicate. Error bars represent standard deviation of all condensates within the three replicates. Data are fit to a one-phase exponentially association. (D and E) Violin plots of the quantification of the fits to the FRAP data in (C). The rate constant for recovery (k_obs) and the plateau of recovery (Mobile Fraction) are plotted for the four different conditions in (D) and (E) respectively. Solid and dotted lines represent the median and interquartile values respectively. (F) Quantification of the fluorescence recovery after photobleaching of Alexa647-12x601 arrays within condensates. Values are normalized from 0 to 1 corresponding to post and pre-bleach respectively. Points represent a single experimental replicate with n>10 condensates. Error bars represent standard deviation of all condensates. Data are fit to a one-phase exponentially association. (G and H) Violin plots of the quantification of the fits to the FRAP data in (F). The rate constant for recovery (k_obs) and the plateau of recovery (Mobile Fraction) are plotted for the five different conditions in (G) and (H) respectively. Solid and dotted lines represent the median and interquartile values respectively.

Figure 7.. HMGB1 and H1 compete to regulate chromatin dynamics across scales

(A) HMGB1 promotes unwrapping through interaction with the H3 tail. This replaces the H3 tail’s interaction with nucleosomal DNA and relieves autoinhibition of HMGB1, allowing the HMG boxes to bind and unwrap nucleosomal DNA. (B) When longer flanking DNA is present, HMGB1 is able to rescue H1 inhibition of DNA unwrapping. The extra flanking DNA allows additional HMGB1 molecules to interact, which loosens H1’s interaction with the flanking DNA, allowing HMGB1 to rescue DNA unwrapping. (C) HMGB1’s loosening of H1’s interaction with flanking DNA makes both H1 and chromatin itself more dynamic within condensed chromatin.