High-resolution and High-accuracy Topographic and Transcriptional Maps of the Nucleosome Barrier

Abstract

Nucleosomes represent mechanical and energetic barriers that RNA Polymerase II (Pol II) must overcome during transcription. A high-resolution description of the barrier topography, its modulation by epigenetic modifications, and their effects on Pol II nucleosome crossing dynamics, is still missing. Here, we obtain topographic and transcriptional (Pol II residence time) maps of canonical, H2A.Z, and monoubiquitinated H2B (uH2B) nucleosomes at near base-pair resolution and accuracy. Pol II crossing dynamics are complex, displaying pauses at specific loci, backtracking, and nucleosome hopping between wrapped states. While H2A.Z widens the barrier, uH2B heightens it, and both modifications greatly lengthen Pol II crossing time. Using the dwell times of Pol II at each nucleosomal position we extract the energetics of the barrier. The orthogonal barrier modifications of H2A.Z and uH2B, and their effects on Pol II dynamics rationalize their observed enrichment in +1 nucleosomes and suggest a mechanism for selective control of gene expression. Highlights A single-molecule unzipping assay mimics DNA unwinding by Pol II and maps the topography of human canonical, H2A.Z and uH2B nucleosome barriers at high resolution Real-time dynamics and full molecular trajectories of Pol II crossing the nucleosomal barrier reveal the transcriptional landscape of the barrier at high accuracy H2A.Z enhances the width and uH2B the height of the barrier A unified mechanical model links position-dependent dwell times of Pol II on the nucleosome with energetics of the barrier

Dynamics of transcription elongation are finely tuned by dozens of regulatory factors

“…Thus, Rpb4 prevents Pol II from pausing at the 3' regions of genes that may protect from premature termination before the canonical 3' cleavage site is transcribed. Similarly, more 3' pause sites are found in the ubp8∆ strain, consistent with the global increase in this strain of H2B ubiquidylation, a mark that increases the nucleosomal barrier to Pol II and is coincident with Pol II pausing at transcription termination sites (Bonnet et al, 2014;Chen et al, 2019;Harlen et al, 2016). Together, these data show how the chromatin landscape and transcriptional regulatory network of the cell dictate sites of Pol II pausing that in turn controls where and for how long Pol II pauses during elongation.…”

“…dst1∆ pause sites clustered far away from those in wild-type cells, consistent with the backtracking role of Dst1 that leads to downstream-shifted pause sites (Churchman and Weissman, 2011;Noe Gonzalez et al, 2021). H2B ubiquitidation increases the nucleosomal barrier to Pol II (Chen et al, 2019), so alterations to histone ubiquitination might lead to new pause sites. Interestingly, pause sites after the loss of Rad6, Ubp8, Paf1 and Cdc73 all cluster together.…”

The H2A.Z-nucleosome code in mammals: emerging functions

“…H2A.Z.1 also regulates the negative elongation factor (NELF) turnover and the reloading of the preinitiation complex [57]. This robust study combining genomic techniques and live cell high-resolution microscopy supports that H2A.Z.1 slows RNAPII pause-release rate and is further supported by biophysical data that H2A.Z nucleosome widens the transcriptional barrier and lengthens the crossing of RNAPII compared with canonical nucleosomes [62].…”

“…In this section, we will explore the emerging functions in transcriptional regulation associated directly with H2A.Z. The role of H2A.Z in RNA polymerase II (RNAPII) pausing and elongation It has been suggested that H2A.Z plays a critical role in RNAPII initiation and elongation; however, opposing results are also reported (Figure 3) [57,[59][60][61][62]. After RNAPII enters early elongation, it pauses about 20-60 bp downstream from the TSS; this 'pausing' effect is increasingly recognised as an important step in regulating gene expression [63].…”

Primary Role of the Nucleosome

“…Passage of Pol II is nevertheless possible because of transient unwrapping of DNA at the ends of the nucleosome (Polach and Widom, 1995;Prunell and Kornberg, 1978); the transcribing polymerase invades an unwrapped end and proceeds, by successive unwrapping events, through the nucleosome. Passage of Pol II is able to fully displace the nucleosome (Lorch et al, 1987;Kulaeva et al, 2013;Chen et al, 2019).…”