OCT4 interprets and enhances nucleosome flexibility

Abstract

Pioneer transcription factors are proteins that induce cellular identity transitions by binding to inaccessible regions of DNA in nuclear chromatin. They contribute to chromatin opening and recruit other factors to regulatory DNA elements. The structural features and dynamics modulating their interaction with nucleosomes are still unresolved. From a combination of experiments and molecular simulations, we reveal here how the pioneer factor and master regulator of pluripotency, Oct4, interprets and enhances nucleosome structural flexibility. The magnitude of Oct4's impact on nucleosome dynamics depends on the binding site position and the mobility of the unstructured tails of nucleosomal histone proteins. Oct4 uses both its DNA binding domains to propagate and stabilize open nucleosome conformations, one for specific sequence recognition and the other for nonspecific interactions with nearby regions of DNA. Our findings provide a structural basis for the versatility of transcription factors in engaging with nucleosomes and have implications for understanding how pioneer factors induce chromatin dynamics.

Figure 1.

Oct4 binds to native nucleosomes at different positions with either DNA binding subdomain. (A, B) Schematics of the consensus OCT4 binding site, LIN28B (A) and ESRRB (B) genomic nucleosomes and mutants showing the positioning and sequences of the Oct4 binding sites. The mutants are named with a ‘m’ preceded by the number of the binding sites mutated and followed by the names of these sites. The mutated sites are shown in brackets if there are more than three. The POU_S, POU_HD, and Sox2 binding sites are in orange, teal, and red, respectively. Arrows indicate the binding orientation. The POU_S binds in a 5’-ATGC-3’ orientation to free DNA, whereas the POU_HD in a 5’-T(A)AAT-3’ orientation with the N-terminal tail recognizing the first half and the globular part the second half. (C, D) Structural views of LIN28B (C) and ESRRB (D) with the histone core in gray cartoons, DNA in white surface, binding sites in the corresponding colors, and histone tails in blue (H3), green (H4), orange (H2AN), red (H2AC), and purple (H2B). These representations and coloring are kept throughout the manuscript. (E, F) EMSAs of purified Oct4 with free DNA (left) and reconstituted nucleosomes (right). The third gel in 1E shows a competition assay using excess specific and nonspecific competitor. Filled horizontal arrowheads indicate nucleosomes or nucleosome-protein complexes and empty arrowheads, free DNA or DNA–protein complexes.

Figure 2.

Nucleosome dynamics influence Oct4 binding. (A, B) LIN28B. (C, D) ESRRB. EMSAs of purified Oct4 with untreated (left) or crosslinked (right) nucleosomes (A, C). Filled arrowheads signal nucleosomes or nucleosome-protein complexes and empty arrowheads point to free DNA. The bar graphs display the mean and standard deviation of densitometry relative to starting nucleosome amounts (B, D). n = 3 per condition, * P= 0.0344,** P= 0.0045, *** P= 0.0001. (E) Densitometry of Oct4 competition EMSAs using LIN28B (green) or ESRRB (blue) WT nucleosomes (mean values with the standard deviations as error bars). For ESRRB n = 4, for LIN28B n = 5. (F) Same experiment as in panel E but comparing LIN28B WT and 4m nucleosomes (for 4m n = 6).

Figure 3.

Oct4 binding modifies nucleosome breathing. (A–C) Oct4-LIN28B. (D–F) Oct4-ESRRB. Representative structures were selected from two independent simulations of each complex (A, B, D, E). In Oct4–LIN28B, Oct4 is bound to the HD^–7 binding site, in reverse orientation (A, B). The configuration of Oct4 is either canonical (A) or generated from a simulation of apo Oct4 (B). In Oct4-ESRRB, Oct4 is bound to the S^+5.5 site (D, E). Both starting configurations of Oct4 were taken from the simulation of apo Oct4. (see Materials and Methods). The structures were selected to correspond to the most sampled conformation in the following 2D histograms. The 2D histogram depict the γ₁/γ₂ conformational sampling in two simulations (2 μs aggregate time) of the free nucleosome (black) and two simulations (2 μs aggregate time) of Oct4-nucleosome complexes (green scatter plot with blue and yellow contours for the individual simulations) (C, F). The motions described by γ₁ and γ₂ are indicated with double headed arrows. The arrows in the square insets indicate the direction of the nucleosome opening. The ‘*’ labels the sequence specific bound subdomain (also in subsequent figures).

Figure 4.

Oct4 modifies the histone tail-DNA interaction profiles.(A–C) LIN28B, free or with Oct4 bound to HD^–7 in reverse orientation. (D–F) ESRRB, free or with Oct4 bound to S^+5.5. Representative structures of the free nucleosome and Oct4-nucleosome complexes are shown together with the R_g histograms (A, D) to illustrate the nucleosome conformations sampled. The histone tail–DNA interaction profiles show the number of stable contacts between the tails in the proximity of the Oct4 binding site and the DNA in the simulations of free nucleosomes (B, E) and Oct4–nucleosome complexes (C, F). A contact was defined as stable if it was present in more than the 75% of the 1 μs simulation time. The dotted line marks the position of the dyad

Figure 5.

Oct4 stabilizes and enhances histone tail mediated nucleosome opening (A, B) Oct4–ESRRB complex with the nucleosome closed. (C, D) Oct4–ESRRB complex with the nucleosome open. Representative structures from the S^5.5₂ (A) and S^5.5₁ (C) simulations are shown together with the corresponding time series of R_g (orange) and the number of contacts between the H3 tail and the inner (cyan) and outer (blue) gyres of the DNA (B, D). In gray/black are data from a simulation started after 1 μs of S^5.5₁ after removing Oct4. (E) ESRRB nucleosome opening in simulations biased to prevent interactions between the H3 and H2AC tails near the Oct4 binding site and the outer DNA gyre. Time series of R_g are shown from simulations started after 1 μs of S^5.5₂ with (orange) or without Oct4 (grey) and after 1 μs of ESRRB₁ (green) (see Methods). (F) ESRRB nucleosome opening in simulations started with the H3 and H2AC tails remodelled using a representative configuration selected from the open nucleosome conformation found in S^5.5₁. Time series of R_g from three simulations are shown: S^5.5_T1 (orange), (S^5.5_T1–Oct4) started after 1 μs of S^5.5_T1 after removing Oct4 (grey), and a 1 μs unbiased simulation (green) started after 800 ns of S^5.5_T1 and 250 ns in which a bias was added (S^5.5_T1-b1) to move the POU_HD in between the two DNA gyres (see Materials and Methods). (F, G) ESRRB nucleosome opening in simulations started after removing the histone tails (tail-less ESRRB-TL nucleosome). Histograms of R_g (G) and γ₁/γ₂ angles (H) were calculated from 4 μs ensembles (two independent simulations, each 2 μs long). The ensemble of the free ESRRB-TL (black) is compared with two ensembles of Oct4-ESRRB-TL complexes (dark and light orange in (G), green in (H)) which differ in the configuration Oct4 used to start the simulations (Table 1). Two contours are shown in the 2D histograms corresponding to 1 and 100 counts.

Figure 6.

Oct4 modifies nucleosome dynamics by sequence specific DNA recognition with one subdomain and nonspecific DNA interactions with the other subdomain. (A) Sampling of the nucleosome surface by the Oct4 subdomains in 10 μs aggregate simulation time of the Oct4-ESRRB complex. (B) Number of contacts between the POU_S (red, orange) or the POU_HD (blue, cyan) with DNA bases (sequence specific, upper plots) or DNA backbone (nonspecific, lower plots) of the inner gyre (red, blue) or the outer gyre (orange, cyan) in two of the simulations shown in (A) (S^+5.5₁ and S^+5.5₂). (C) Same as (A) in 4 μs aggregate simulation time of the Oct4-ESRRB complex started with the H3 and H2AC tails remodelled using a configuration selected from the open nucleosome conformation found in S^+5.5₁. (D) Same data as in (B) but from the simulations S^+5.5_T1,T2.

Figure 7.

Summary of Oct4 mediated nucleosome dynamics The Oct4 POU_S and POU_HD subdomains are shown as orange and teal circles, respectively. Larger circles with straight lines represent nucleosomes and their linker DNA as viewed from the top in closed, partially open, or fully open configurations. The red points DNA indicate Oct4’s sequence specific binding half-sites. Coiled lines represent histone tails. Dotted arrows indicate hypothetical transitions.