High-resolution dynamic mapping of histone-DNA interactions in a nucleosome

Abstract

The nature of the nucleosomal barrier that regulates access to the underlying DNA during many cellular processes is not fully understood. Here we present a detailed map of histone-DNA interactions along the DNA sequence to near base pair accuracy by mechanically unzipping single molecules of DNA, each containing a single nucleosome. This interaction map revealed a distinct approximately 5-bp periodicity that was enveloped by three broad regions of strong interactions, with the strongest occurring at the dyad and the other two about +/-40-bp from the dyad. Unzipping up to the dyad allowed recovery of a canonical nucleosome upon relaxation of the DNA, but unzipping beyond the dyad resulted in removal of the histone octamer from its initial DNA sequence. These findings have important implications for how RNA polymerase and other DNA-based enzymes may gain access to DNA associated with a nucleosome.

Figure 1

Nucleosome disruptions under a constant unzipping force. (a) Experimental configuration. A DNA molecule was mechanically unzipped through a nucleosome uniquely positioned at a 601 sequence. (b) Representative traces for unzipping under a constant applied force (~ 28 pN). Two traces are shown: one from forward unzipping (black) and one from reverse unzipping (red). Both traces were low pass filtered from the raw traces (grey) to 60 Hz. The unzipping fork paused at specific locations, which are evident from both the traces (left) and their corresponding dwell time histograms (right).

Figure 2

Histone-DNA interaction map within a nucleosome core particle. Top Panel: Crystal structure of the nucleosome core particle, where dots indicate regions where interactions between DNA and one of the core histones are likely to occur. The two halves of the nucleosome are shown separately for clarity. Bottom Panel: A histone-DNA interaction map constructed from the averaged dwell time histograms of the unzipping fork at constant force (~ 28 pN). Individual traces were low-pass filtered to 60 Hz and their dwell time histograms were binned to 1 bp. A total of 27 traces from the forward template and 30 traces from the reverse template were used for the construction. Each peak corresponds to an individual histone-DNA interaction and the heights are indicative of their relative strengths. Three regions of strong interactions are indicated: one located at the dyad (region 2) and two located off-dyad (regions 1 and 3). Colored boxes indicate predictions from the crystal structure where individual histone binding motifs are expected to interact with DNA. The H3 N-terminal alpha helices (αN) and the histone loops (L1, L2) and alpha helices (α1) that compose the L1L2 and α1α1 DNA-binding sites observed in the crystal structure are also indicated.

Figure 3

Nucleosome disruptions under a constant loading rate. (a) Representative traces for unzipping under a constant loading rate (8 pN s⁻¹). Two traces are shown: one from forward unzipping (black) and one from reverse unzipping (red). For clarity, the naked DNA signature before and after each nucleosome disruption event is not shown. The unzipping fork again paused at specific locations, which are evident from both the traces (top) and their corresponding dwell time histograms (bottom). (b) The average dwell time histograms of the unzipping fork under a constant loading rate. Individual traces such as those shown above were low-pass filtered to 60 Hz and their dwell time histograms were binned to 1 bp. A total of 36 traces from each direction were used for the construction. Other notations are the same as those of Figure 2.

Figure 4

Mechanical unzipping (left) to mimic motor enzyme progression into a nucleosome (right). (a) DNA was unzipped with a loading rate clamp (8 pN s⁻¹) until the unzipping force reached ~ 20 pN, which typically occurred within the 1^st region of interactions (green curve). The unzipping force was then held at this force for 10 s, resulting in a horizontal force line due to the hopping of the unzipping fork among different positions within the first region. These steps mimic a motor invasion into the 1^st region of interactions and subsequent pausing within the region (right). The tension in the DNA was then relaxed for ~ 3 s and the state of the nucleosome was determined by unzipping a second time (orange curve). (b) Similar to b, except that the unzipping force was held at ~ 21 pN immediately after the unzipping fork entered the dyad region of interactions. These steps mimic motor invasion into the dyad region of interactions before pausing (right). (c) Similar to b, except that DNA was unzipped past the dyad region of interactions. This mimics motor invasion past the dyad (right).