Nucleosomal elements that control the topography of the barrier to transcription

Abstract

The nucleosome represents a mechanical barrier to transcription that operates as a general regulator of gene expression. We investigate how each nucleosomal component-the histone tails, the specific histone-DNA contacts, and the DNA sequence-contributes to the strength of the barrier. Removal of the tails favors progression of RNA polymerase II into the entry region of the nucleosome by locally increasing the wrapping-unwrapping rates of the DNA around histones. In contrast, point mutations that affect histone-DNA contacts at the dyad abolish the barrier to transcription in the central region by decreasing the local wrapping rate. Moreover, we show that the nucleosome amplifies sequence-dependent transcriptional pausing, an effect mediated through the structure of the nascent RNA. Each of these nucleosomal elements controls transcription elongation by affecting distinctly the density and duration of polymerase pauses, thus providing multiple and alternative mechanisms for control of gene expression by chromatin remodeling and transcription factors.

Figure 1. Transcription through modified nucleosomes

(A) (Left panel) Experimental setup for single-molecule transcription experiments. Two laser beams (red) are used to trap anti-digoxygenin (AD) and streptavidin (SA) coated beads. A DNA tether is formed between a Pol II and the upstream DNA. The blue arrow shows the direction of transcription. (Right panel) Cartoon schematic of the histone-DNA contacts on nucleosome is shown as color coded rectangle. Asterisks are the positions of Sin H4 (cyan) and Sin H3 (purple) mutations. The position of Pol II as a function of time during single-molecule transcription of (B) bare DNA (black) and unmodified nucleosomes (red), (C) tailless (blue) and mock-acetylated (green) nucleosomes, (D) Sin Sin H4 (cyan) and H3 (purple) mutant nucleosomes. Traces where Pol II passed the nucleosome positioning sequence (NPS, shaded yellow) are shown on the left and traces that arrested at the nucleosome are on the right. Inserts show the percentages of Pol II molecules that transcribed the entire NPS. For additional information, see Table S1 and Figure S2.

Figure 2. Tails affect pausing in the nucleosome entry region

(A), (C) Pause density as a function the position of Pol II’s active center on the template for tailless and acetylated nucleosomes. The nucleosome entry/exit regions are shaded yellow, and the central region is shaded gray. (B), (D) Pause durations in the entry, central and exit regions of these nucleosomes. The pause-free velocity of tail modified nucleosomes are shown in Figure S3.

Figure 3. Sin mutants destabilized at the dyad

(A), (C) Pause density as a function the position of Pol II’s active center on the template for Sin H4 and SinH3 nucleosomes. (B), (D) Pause durations in the entry, central and exit regions of these nucleosomes. The pause-free velocity of Sin mutants are shown in Figure S4.

Figure 4. Nucleosome dynamics in the absence of Pol II

(A) Experimental setup for single-molecule nucleosome dynamics experiments. The symbols are the same as in Figure 1. (B) Force-extension curves of the nucleosome in 40 mM KCl (left) and 300 mM KCl (right). The pulling curve is in black, and the relaxation one is in blue. The unwrapping events corresponding to outer and inner rips are indicated with arrows. (C) An example of nucleosome transition events between the wrapped state and the unwrapped one. (D) Summary of changes for the nucleosome wrapping and unwrapping rates. For more information about the unwrapping and rewrapping rates of the nucleosome, see Table S2.

Figure 5. Nucleosome wrapping equilibrium during transcription

(A) Kinetic scheme of transcription through the nucleosome. The parameters involved are: the elongation rate (k_e), intrinsic diffusion rate of the polymerase during a pause (k₀), the barrier to backtracking (ΔG), and the rates of nucleosome unwrapping (k_u), and wrapping (k_w). The labels of the states indicate the number of base pairs Pol II backtracked and the state of the nucleosome: unwrapped (u) or wrapped (w). (B) Fitted values of local wrapping equilibrium constant of the nucleosome (K_w= k_w/k_u) for the three regions. (C) Elements that control the wrapping equilibrium for the three regions. (D) Magnitude of the nascent RNA barrier to backtracking in the three regions. For additional information, see Table S3 and Figure S5.