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Figure 6—figure supplement 1. Crossing Time, Crossing Probability and Pause-free Velocity of Pol II during Transcription through NPS DNA or Nucleosomes. ; (A–E) Histograms of crossing time of Pol II transcription through bare NPS DNA (A), xWT (B), hWT (C), H2A.Z (D) and uH2B (E) nucleosomes. (F) Relative percentage of Pol II molecules that are arrested or crossed during transcription through bare NPS DNA or nucleosomes. (G) Pol II arrest positions within the NPS. The positions are normalized to the beginning of the NPS. Each dot is a single arresting event. The percentages of arresting before or after dyad are shown below the dots. (H) Pause-free velocity of Pol II molecules before, inside and after NPS during transcription through bare NPS DNA, and xWT, hWT, H2A.Z and uH2B nucleosomes. Only traces that reached the stall site at the end of the template are considered. Pause-free velocities are calculated in three fastest regions (to partially correct for velocity differences due to sequence variations) up to100 bp before, inside, and up to 100 bp after NPS.

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Figure 6. Transcriptional Maps of the Nucleosome Reveal that H2A.Z Enhances the Width and uH2B the Height of the Barrier. ; (A) Median residence time histograms of Pol II transcription through bare NPS DNA (black), xWT (orange), hWT (red), H2A.Z (blue) and uH2B (green) nucleosomes. Bar width is 1 bp and major peak positions are labeled (in bp) above the corresponding peaks. NPS entry, dyad, NPS exit are marked with blue dashed lines. The polar plots on the right are the corresponding transcriptional maps of the nucleosome, formed by projecting the residence time histogram onto the surface of nucleosomal DNA. The top axis (red) indicates corresponding positions of the first half of nucleosome expressed as superhelical locations (SHL). n = 35, 23, 26, 21, 31, respectively for NPS DNA, xWT, hWT, H2A.Z and uH2B nucleosomes. (B) Crossing time (total time Pol II takes to cross the entire nucleosome region) distributions plotted using the complementary cumulative distribution function (CCDF, fraction of events longer than a given crossing time). Crossing times of Bare NPS DNA, Xenopus WT (xWT), human WT (hWT), uH2B and H2A.Z nucleosomes are plotted in black, orange, red, green and blue, respectively. See also Figure 6—figure supplement 1 on statistics of the crossing time, crossing probability, pause-free velocity and arrest position.

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Figure 7—figure supplement 1. Fitting Nucleosome Energy Profiles Based on Pol II Dwell Times. ; (A–C) Mean dwell times (colored dotted lines) calculated from best-fit mechanical model (see Figure 7D–E) whose corresponding nucleosome binding energies (insets, colored lines) are shown for (A) hWT, (B) H2A.Z, and (C) uH2B nucleosomes. The black dashed lines of the inset are binding energy data obtained from unzipping under equilibrium conditions (hopping, see Figure 3D inset), and black dashed lines of the main plots are dwell times calculated from these energy landscapes. The hopping data covers a narrow region of sequence, and therefore allows prediction of Pol II pausing only at the start of the NPS. (D) The experimental (black lines) and calculated mean dwell time (dotted line in magenta) for Xenopus WT (xWT) nucleosome, with the energy landscape extracted from the experimental mean dwell time using the same procedure as for hWT, H2A.Z, and uH2B (Figure 7D). Inset compares hWT (red) and xWT (magenta) energy landscapes.

2019

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Figure 3—figure supplement 1. Hopping of the Unzipping Fork Near the Proximal Dimer Region of the Nucleosome. ; (A, B) Unzipping traces of human WT nucleosome (A) and bare NPS DNA (B). The slow hopping event near the proximal dimer region of WT nucleosome is indicated with a dashed blue square box; no similar slow hopping was observed in the corresponding region during unzipping of bare NPS DNA. Insets are the zoomed-in view of the dashed square boxes. Raw force-extension unzipping curves of hWT nucleosome (orange dots) and bare DNA (black dots) near the hopping region are plotted at maximum bandwidth (800 Hz), with the asterisk highlighting the nucleosome-specific slow hopping event (top right inset in B). Unzipped bp is normalized to the beginning of the second NPS. Rezipping traces are not shown for clarity. (C) Aligned individual force-extension curves (thin colored curves) and mean force-extension curve (thick black curve), for bare DNA. (D) Energy of DNA unzipping for each base pair, calculated from mean force-extension curve. (E) Comparison of experimental mean force-extension curve (blue) to the force-extension calculated from the extracted DNA unzipping energy (red). (F) Force-extension traces obtained at fixed trap separations with WT nucleosome. Color indicates increasing trap separation (purple to red), with number indicating the trap separation in nm. (G) DNA unzipping energy for each base pair, calculated from equilibrium hopping data at multiple fixed trap separations as in (F). (H) Comparison of experimental mean force-extension curve for bare DNA (black) and DNA with a WT nucleosome (red) to the force-extension curve predicted by the apparent DNA unzipping energy from equilibrium hopping data for the WT nucleosome (cyan).

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Figure 1. Dual-trap Optical Tweezers Single-molecule Unzipping Assay Unwinds Nucleosomal DNA and Maps Histone-DNA Interactions. ; (A) Geometry of the single-molecule unzipping assay. Dashed arrows denote directions of trap movement (20 nm/s) during unzipping (red arrow) or rezipping (black arrow). Two DNA handles connect to the template DNA, which consists of two tandem NPS repeats and an end hairpin. Diagram illustrates nucleosome unzipping, with the second NPS replaced with a pre-assembled mononucleosome. For simplicity, linkers and restriction sites flanking the NPS are not shown. (B, C) Unzipping (red) and rezipping (black) traces of bare NPS DNA (B) and a single WT human nucleosome (C). The presence of the nucleosome on the second NPS causes characteristic high force (20–40 pN) transitions that correspond to disruption of histone-DNA contacts. The unzipped basepairs (bp) are normalized to the beginning of the second NPS. The nucleosome rezipping trace matches that of bare NPS DNA, indicating complete histone removal during unzipping. (D) Representative unzipping traces of tetrasome (cyan), WT (red), H2A.Z (blue), and uH2B (green) nucleosomes. For clarity, only the region after entering the second NPS (corresponding to the boxed region in (C)) is shown, with the unzipped bp normalized to the beginning of the second NPS. The three dashed lines are entry, dyad, and exit of the second NPS, respectively. Rezipping traces, identical to those of B and C, are not shown. (E) Topography maps are plotted as force-weighted residence time (RT) histograms of the unzipping fork along bare NPS DNA, tetrasome and different types of nucleosomes during unzipping at constant trap separation speed of 20 nm/s. The gray histograms with colored stripes (excluding Bare NPS DNA and WT Nucleosome) are residual plots found by subtracting the WT nucleosome RTs. Unzipped bp are normalized to the beginning of the second NPS core. Major peaks are highlighted with gray dashed lines, with the peak positions (in bp) labeled above the peaks. (Left to right: 17, 22, 26, 31, 35, 41, 52, 61, 69, 109, 112, 122 bp). n = 34, 41, 34, 39, 35, 10, respectively for NPS DNA, hWT, H2A.Z, M3_M7, uH2B nucleosome and tetrasome. See also Figure 1—figure supplement 1 for representative unzipping traces and analysis.

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Video 8. Pol II transcription through hWT nucleosome, NPS zoom.

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Figure 3. Observation of Multiple Nucleosomal States at the Proximal Dimer Region. ; (A) Time traces of number of base pairs unzipped (relative to beginning of the second NPS) with hWT nucleosome for fixed trap separations of 1031 nm, 1045 nm, and 1060 nm (top to bottom). Color indicates increasing trap separation (purple to red), corresponding to clusters in Figure 3—figure supplement 1F. Gray dashed lines indicate 17, 23, and 28 base pairs unzipped. (B) Probability distributions for the number of DNA bps unzipped, computed from force-extension data in Figure 3—figure supplement 1F. Each curve is from a different trap separation, matching colors in A and Figure 3—figure supplement 1F. Distributions are shown for both bare DNA (top) and WT nucleosome (bottom). Vertical black dotted line indicates the start of the second NPS. Vertical gray dashed lines indicate peak positions for bare DNA (with position in bp labeled), showing that WT nucleosome shifts the first peak within the NPS, and gives rise to an additional peak at 28 bp. See Figure 3—figure supplement 1F for force-extension data. (C) Zoomed-in view of the black dashed box in (B). Peak positions are labeled in bp. (D) DNA unzipping energy computed by assuming the unzipped bp distributions from data in Figure 3—figure supplement 1F (including distributions in B) correspond to equilibrium Boltzmann statistics. Inset ∆E shows the DNA-octamer interaction energy, computed as the difference between unzipping energies in the presence of WT (red), H2A.Z (blue), and uH2B (green) nucleosomes and unzipping energies for bare DNA only (black). Vertical black dashed lines and * indicate peak positions (labeled in bp). See also Figure 3—figure supplement 1 on hopping traces and analysis of energy landscape from equilibrium data.

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Video 2. Unzipping-rezipping of hWT nucleosome.

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Video 7. Pol II transcription through hWT nucleosome.

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Figure 5—figure supplement 1. Long-lived Pauses of Pol II in the Nucleosome are Associated with Backtracking and Hopping Dynamics. ; (A, B) Representative traces of backtracking (A) and hopping (B) dynamics of Pol II during transcription through an hWT nucleosome. The trace is the same as the red trace in Figure 5A and B. The black arrowheads in (A) highlight backtracking events right before long-lived pauses. The triple-stars highlight the region where Pol II has hopping dynamics, the zoomed in view of which is shown in (B). The hopping trace is fitted as two states with a classic hidden Markov model (red is raw data, black is fitted trace). The fitted parameters and histogram of Pol II position counts are shown to the right side. d is the distance transcribed, p is the probability in that state, and kout is the rate of transitioning to the other state. (C, D) Representative traces of backtracking (C) and hopping (D) dynamics of Pol II during transcription through an H2A.Z nucleosome. The trace is the same as the orange trace in Figure 5C and D. The data is analyzed and shown as in (A, B). (E, F) Representative traces of backtracking (E) and hopping (F) dynamics of Pol II during transcription through an uH2B nucleosome. The trace is the same as the gray trace in Figure 5E and F. The data is analyzed and shown as in (A, B) except that the trace in (F) is fitted as three-states.

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Figure 2. Transcriptome dystregulation in H3K36R mutants is correlated with H3K36me3 ChIP-seq. ; (A) Metagene plot describing the density of H3K36me3 (top), H3K36me2 (middle), and H3K36me1 (bottom) ChIP-seq across genes that are upregulated (purple), unchanged (blue), or downregulated (yellow) in H3K36R mutants as compared with HWT controls. (B) Boxplot of differential expression of gene cohorts stratified by density of H3K36me3 signal in the 3’ UTR (1=lowest density decile, 10=highest decile). (C) MA plot with accompanying LOESS regression line plotting log2 fold change (y-axis) vs. HWT FPKM (x-axis) interpreted from poly-A RNA-seq data.

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Figure 6—figure supplement 1. RT-PCR controls, alternative polyadenylation analysis and schematic of assay for gene-specific poly A tail length assay (LM-PAT) showing relative positions of primers. ; (A) RT-PCR for select genes from cytoplasmic (lanes 1 and 2) or nuclear (lanes 3 and 4) RNA from HWT (lanes 1 and 3) or K36R (lanes 2 and 4) animals. 7SK RNA is a control for nuclear enrichment. (B) RT-qPCR for select genes from Figure 6B measuring normalized Log2 fold change (HWT/K36R) in no RNAi or Dcp2 RNAi background. P-value obtained via t-test. (C) Schematic of modified LM-PAT assay, in which an adenylated oligonucleotide anchor is ligated to the 3’ end of total RNA, cDNA is generated using an anchor-specific RT primer, and genes of interest are amplified using a gene-specific forward primer and an anchor specific reverse primer that contains either an oligo-T sequence at its 3’ end (tail-anchored) so as to extend from the ends of poly-A tails, or an oligo-T-N sequence (UTR-anchored) in order to extend from the terminus of the 3’ UTR. (D) RT-qPCR for YFP measuring normalized Log2 fold change (HWT/K36R) in no RNAi, pcm RNAi, or twin RNAi backgrounds. P-value obtained via t-test. (E) Genome wide analysis of alternative polyadenylation using DaPars (Xia et al., 2014), with percentage of distal poly-A site usage (PDUI) for each gene in HWT and K36R plotted on the x- and y-axes, respectively.

2017

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Figure 4. H3K36 modification does not suppress cryptic transcription initiation in coding regions. ; (A) Representative browser shot of gene containing novel unannotated transcription start sites (nuTSSs, highlighted in red). Direction of transcription denoted by arrow, and read counts denoted on Y-axis. (B) Boxplot describing the fold change in Start-seq signal for nuTSSs classified by their genomic localization and strand of origin relative to the resident gene if applicable. Lower boxplot describes H3K36me3 ChIP-seq signal (ChIP/input) for the same gene cohorts. (C) Scatterplot of normalized nuclear RNA-seq reads mapping antisense to genes in the dm3 reference gene model in HWT (x-axis) or K36R (y-axis). Genes containing or within 1 kb of a local H3K36me3-ChIP-seq peak are denoted by red dots. (D) Hex-plot heatmap plotting nuTSSs by their location relative to the gene boundaries of the nearest gene, and the absolute change in their Start-seq signal (K36R – HWT).

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Figure 6. A class of highly expressed genes is subject to exonuclease degradation and inefficient post-transcriptional processing in H3K36R mutants. ; (A) LOESS regression lines generated from MA plots of either nuclear or poly-A RNA-seq, plotting gene log2 fold change (y-axis) vs. normalized read counts in HWT (x-axis). (B) Log2 fold change values between K36R and HWT in nuclear (left) and poly-A (right) RNA-seq, plotted for genes selected for further RT-PCR analysis. (C) RT-qPCR quantification of differential expression between HWT and K36R for select genes in a no RNAi, pacman RNAi, twin RNAi, or Pop2 RNAi background, using the -∆∆Ct method. (D) LM-PAT assay results for the YFP transcript in HWT and K36R, in a no RNAi, pcm RNAi, twin RNAi, or Pop2 RNAi background. Sanger sequencing trace confirming the poly-A site (leftmost panel) and differential tail lengths (right two panels) is shown below.

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Figure 4—figure supplement 1. Metagene analysis of Start-seq reads at novel, unannotated (nu)TSSs in comparison to open chromatin, nucleosome positioning and H3K36 trimethylation. ; (A) Metaplot of ATAC-seq signal mapping in a 1 kb window around nuTSSs, classified as in Figure 4B. (B) Metaplots of H3 (left) and H3K36me3 (right) ChIP-seq signal mapping to a 4 kb window around nuTSSs, separated by quartiles of absolute Start-seq signal change between K36R and HWT. (C) Metaplots of HWT ATAC-seq signal mapping to a 4 kb window around nuTSSs, separated by log2 fold change in Start-seq signal. ‘Up’ denoted increased by more than two fold in K36R, ‘Down’ denotes decreased by more than two fold in K36R, and ‘Unchanged’ denotes all other nuTSSs. (D) Heatmaps displaying H3 (left) and H3K36me3 (right) ChIP-seq signal mapping to a 4 kb window around nuTSSs representing the catergories listed in B. (E) Heatmaps displaying H3 (left) and H3K36me3 (right) ChIP-seq signal mapping to a 4 kb window around nuTSSs representing the catergories listed in C.

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Figure 5. H3K36 modification does not regulate alternative splicing. ; (A) Density plots reflecting the distributions of change in percent spliced in (∆PSI) values for skipped exon (red) or retained intron (blue) alternative splicing events manually classified as significant based on MISO parameters (see Materials and methods). (B) Volcano plots for skipped exon (left) and retained intron (right) events, with a local regression line (blue line) reflecting the skew in ∆PSI values (x-axis) based on Bayes factor (y-axis). (C) Global analysis of splice junction usage, where R denotes the ‘retention ratio’ in one condition, and ∆R denotes the difference in R between K36R and HWT.

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Supplementary file 1. Excel file summarizing DESeq2 (Love et al., 2014) results from comparing K36R to HWT from total nuclear or poly-A RNA-seq. ; For each gene, and each experiment (nuclear and poly-A), there are listed values (from left to right) for mean counts, log2 fold change (K36R/HWT), log2 fold change standard error, test statistic, p-value, and adjusted p-value.

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Figure 3—figure supplement 2. Metagene analysis of Start-seq reads at previously annotated (observed) transcription start sites, obsTSSs. ; (A) Metaplot of Start-seq signal aligned in a 100 nt window around all annotated TSSs in the dm3 reference gene model. (B) Metaplot of HWT and K36R nuclear RNA-seq signal aligned in a 1 kb window around all obsTSSs identified in Start-seq data. (C) Representative browser shot of Start-seq signal pileup at annotated gene promoter (obsTSS). Direction of transcription denoted by arrow. (D) Metaplot of ATAC-seq signal in a 4 kb window surrounding obsTSSs identified by Start-seq. obsTSSs are binned by the average normalized signal across the window (denoted in the legend).

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Figure 3. H4 acetylation enrichment in mutants does not result in open-chromatin-dependent changes in gene expression. ; (A) Western blots measuring enrichment of histone H3, H3K36me3, H3K27me3, and pan H4 acetylation (H4ac) in H3K36R mutants and HWT controls. Signal relative to first lane is denoted below each band. (B) Scatterplot of ATAC-seq signal mapping in a 200 nt window (as denoted at top) around obsTSSs, with R2 value indicated. (C) Scatterplot of log2 fold change of poly-A RNA-seq (x-axis) vs. that of ATAC-seq (y-axis) signal in a window around the corresponding gene’s transcription start site (as identified by start-seq). Genes with codirectional, statistically significant changes in both RNA-seq and ATAC-seq are indicated in red. Example browser shot of a gene differentially expressed in mutants in the absence of changes in chromatin accessibility at its start site is shown at right.

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Figure 4—figure supplement 1. Supplemental data on the HLE’s recruitment of dynein and motile properties. ; (A) Distributions and means (± SEM) of number of GFP decay steps for GFP::Dlic associated with HLE RNPs, compared to data for hWT RNPs that were acquired in experiments performed in parallel. ***p800 nm or >1000 nm·s−1 were binned together in these plots. Data were acquired at 4.2 fps for these and other experiments involving the HLE, which alters the measured run lengths and velocities for hWT RNPs compared to those obtained from 15 fps imaging (see Figure 4 legends for further details).

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