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A dual role for H2A.Z.1 in modulating the dynamics of RNA polymerase II initiation and elongation

Abstract

RNA polymerase II (RNAPII) pausing immediately downstream of the transcription start site is a critical rate-limiting step for the expression of most metazoan genes. During pause release, RNAPII encounters a highly conserved +1 H2A.Z nucleosome, yet how this histone variant contributes to transcription is poorly understood. Here, using an inducible protein degron system combined with genomic approaches and live cell super-resolution microscopy, we show that H2A.Z.1 modulates RNAPII dynamics across most genes in murine embryonic stem cells. Our quantitative analysis shows that H2A.Z.1 slows the rate of RNAPII pause release and consequently impacts negative elongation factor dynamics as well as nascent transcription. Consequently, H2A.Z.1 also impacts re-loading of the pre-initiation complex components TFIIB and TBP. Altogether, this work provides a critical mechanistic link between H2A.Z.1 and the proper induction of mammalian gene expression programs through the regulation of RNAPII dynamics and pause release.

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Fig. 1: H2A.Z.1 attenuates the release of RNAPII towards progressive elongation.
Fig. 2: H2A.Z.1 controls NELF and RNAPII dynamics at a single-molecule level.
Fig. 3: H2A.Z.1 controls the RNAPII half-life of less stable promoters.
Fig. 4: H2A.Z.1 controls PIC recruitment at promoters.

Data availability

Raw and normalized sequencing data generated in this study have been deposited under GEO accession no. GSE143737. We also used publicly available ChIP-seq data for H3K4me3, H3K27ac and H3K27me3 (GSE47950)68, H2A.Z.1-GFP (GSE40063)13 and RING1B (GSE69955)69. Bona fide active (H3K4me3 only) and bivalent (H3K4me3 and H3K27me3) gene coordinates were downloaded from Mas et al. 2018 (GSE99530)70. Source data are provided with this paper.

Code availability

We have made use of publicly available scripts, software and tools. A more specific code used to analyze the data in this study is available from the corresponding author upon request.

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Acknowledgements

We thank the Boyer Lab, S. Vos, E. Calo and C. Peterson for helpful discussions and insightful comments on the manuscript. This work was supported by NIGMS R01-GM134734 to I.I.C., NHLBI R01-HL140471 to L.A.B. and the Koch Institute Core Grant P30-CA14051 from the National Cancer Institute.

Author information

Authors and Affiliations

Authors

Contributions

C.M. and L.A.B. designed the study. C.M. performed experiments and analyzed data. C.L. and I.I.C. assisted with tcPALM. A.L.A. assisted with FRAP image acquisition and analysis. C.M. and L.A.B. wrote the manuscript with input from all authors.

Corresponding author

Correspondence to Laurie A. Boyer.

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Competing interests

The authors declare no competing interests.

Additional information

Peer review information Nature Structural & Molecular Biology thanks the anonymous reviewers for their contribution to the peer review of this work. Peer reviewer reports are available. Beth Moorefield and Anke Sparmann were the primary editors on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Extended Data Fig. 1 Endogenous tagging of H2A.Z.1 with HA-FKBP-V in mESCs.

a, Schematic diagram of endogenous tagging of H2afz using CRISPR-Cas9 and homologous directed repair (HDR). b, Genotyping of WT and H2A.Z.1-FKBP-V mESCs cells using primers (Primer 1 & Primer 2) binding around the C-terminus of the H2afz gene. PCR amplified samples were run on a 1% agarose gel. c, Western blotting with Anti-HA and Anti-GAPDH antibodies in WT and H2A.Z.1-FKBP-(Control or dTAG-13 treated) mESCs. H2A.Z.1-FKBP-V cells were treated with dTAG-13 for 0,1,2,4,6, and 8 hours. Maximum H2A.Z.1 depletion was observed after 8h of treatment with the small molecule. d, Cumulative distribution plot of H2A.Z.1 ChIP-seq over active protein-coding promoters (n = 7,789), active distal enhancers (n = 6,856), and super-enhancers (n=231). e, Scatterplot of H2A.Z.1-HA counts (log2), H2A.Z.1-GFP counts (log2), and Total H2A.Z counts (log2) over the promoters of n=12,737 uniquely annotated protein-coding genes. Spearman correlation in indicated in red. f, Single gene plots of several ChIP-seq datasets (RNAPII, H3K4me3, H3K27Ac, H2A.Z.1- GFP, H2A.Z.1-HA (Control & dTAG-13), Input). g, ChIP-seq heatmaps over n=12,031 significant H2A.Z.1-HA peaks (FDR < 10−5) in Control and dTAG-13 treated cells. A heatmap of Input control is also presented.

Source data

Extended Data Fig. 2 H2A.Z.1 is a barrier to RNAPII progression.

a, ChIP-seq heatmaps over n=10,878 uniquely annotated genes for H2A.Z.1, RNAPII, H3K4me3, H3K27me3, and RING1B. Genes are sorted by H3K4me3 levels. Corresponding mRNA levels are highlighted in red. b, Western blotting with Anti-Pol II S2ph, Anti-Pol II S5ph, Anti-HA (H2A.Z.1), and Anti-GAPDH after DRB treatment (2h) and subsequent wash-off (1h). DMSO concentration is at 2%. c, Average RNAPII metaplot profiles of n=7,624 uniquely annotated genes for Control (DMSO), DRB, and wash-off over TSS, gene body, and TES. d, Boxplots quantifying either promoter-proximal or elongating RNAPII for n=7,624 uniquely annotated genes for Control (DMSO), DRB, and wash-off conditions. The median value for each condition is shown with a horizontal black line. Boxes show values between the first and the third quartiles. The lower and upper whiskers show the smallest and the highest values, respectively. Outliers are shown in black circles. e, Scatterplot of H2A.Z.1 ChIP-seq logFC (DRB / DMSO) versus RNAPII Pausing Index logFC (DRB / DMSO) over n=4,166 Active genes (H3K4me3 only). Spearman correlation in indicated in red (ρ = 0.52). f, ChIP-seq heatmaps over n=4,166 Active genes (H3K4me3 only) for RNAPII (DMSO, and DRB), H2A.Z.1 (DMSO, and DRB), H3K4me3, and H3K27me3. Genes are sorted by H3K4me3 levels. g, Scatterplot of H2A.Z.1 ChIP-seq logFC (DRB / DMSO) versus RNAPII Pausing Index logFC (DRB / DMSO) over n=2,240 Bivalent genes (H3K4me3 & H3K27me3). Spearman correlation in indicated in red (ρ = 0.12). h, ChIP-seq heatmaps over n=2,240 Bivalent genes (H3K4me3 & H3K27me3) for RNAPII (DMSO, and DRB), H2A.Z.1 (DMSO, and DRB), H3K4me3, and H3K27me3. Genes are sorted by H3K4me3 levels.

Source data

Extended Data Fig. 3 H2A.Z.1 acts as a transcriptional repressor.

a, Correlation plots for biological NET-seq replicates (Control – dTAG-13) at 4h and 8h of treatment. b, Metaplot profiles and heatmaps of RNAPII (NET-seq) over n=4,184 protein-coding RefSeq TSS or START-seq TSS. H2A.Z.1 (DMSO, and DRB) ChIP-seq heatmaps are also displayed over n=4,184 protein-coding START-seq TSS. c, Boxplots measuring promoter proximal (−30 to +250 bp of TSS) RNAPII density log2(dTAG-13 vs Control) at 4h and 8h of dTAG-13 treatment over n=4,184 protein-coding genes. Significance is calculated using a paired two-sided Wilcoxon rank test. d, Boxplots measuring elongating (+300 bp to TES) RNAPII density log2(dTAG-13 vs Control) at 4h and 8h of dTAG-13 treatment over n=4,184 protein-coding genes. Significance is calculated using a paired two-sided Wilcoxon rank test. For c,d, the median value for each condition is shown with a horizontal black line. Boxes show values between the first and the third quartiles. The lower and upper whiskers show the smallest and the highest values, respectively. Outliers are shown in black circles. e, MA plot of RNA-seq between dTAG-13 vs Control. Significant genes are highlighted in red and blue (logFC < −0.6 or logFC > 0.6 & adj.P.Value < 0.05). f, Scatterplot of gene body RNAPII (NET-seq) of log2(dTAG-13 vs Control) and mRNA (RNA-seq) of log2(dTAG-13 vs Control) over n=6,216 protein-coding genes with detectable gene body RNAPII signal (RPKM > 0.1). Regression coefficient and p-value are shown in red. g, Heatmaps of RNAPII, H2A.Z.1, and nucleosomes (chemical mapping Voong et al, 2016) over n=5,350 protein-coding genes. Genes are sorted by RNAPII levels. Chemical mapping nucleosome heatmaps are centred relative to the +1 dyad. h, Metaplots of RNAPII (NET-seq) between Control and dTAG-13 (8h) relative to the +1 nucleosome dyad of n=2,604 protein-coding genes with the highest RNAPII density (RPKM > 1). H2A.Z.1 ChIP-seq metaplot is shown in grey.

Extended Data Fig. 4 H2A.Z.1 depletion does not impact chromatin architecture.

a, Plot depicting frequency of paired-end fragment length distribution of ATAC-seq reads in Control (black) and dTAG-13 (red) treated cells. b, Correlation plots of ATAC-seq reads between biological replicates for Control and dTAG-13 conditions. c, ATAC-seq metaplots of open chromatin and mono-nucleosomes over the TSS of n=7,624 uniquely annotated genes for Control (black) and dTAG-13 (red) treated cells. Shaded area represents the 95% confidence interval. d, Genome browser snapshot displaying H2A.Z.1 (ChIP-seq) density, open chromatin (ATAC-seq), and mono-nucleosomes (ATAC-seq) between Control (black) and dTAG-13 (red) treated cells. e, H3 ChIP-seq metaplots over the TSS of n=7,624 uniquely annotated genes for Control (black) and dTAG-13 (red) treated cells. f, Genotyping of H2afzdTAG and H2afzdTAG + H2afv3xFLAG mESCs cells using primers (Primer 1 & Primer 2) binding around the C-terminus of the H2afv gene. PCR amplified samples were run on a 1% agarose gel. g, H3K4me3, H2A.Z.1 (Control & dTAG-13), and H2A.Z.2 (Control & dTAG-13) ChIP-seq heatmaps over the TSS of n=7,624 uniquely annotated genes. Genes are sorted by H3K4me3 levels. h, Genome browser snapshot displaying H2A.Z.1, and H2A.Z.2 ChIP-seq density between Control (black) and dTAG-13 (red) treated cells.

Source data

Extended Data Fig. 5 The genome-wide impact of H2A.Z.1 depletion on NELF-B and SPT5.

a, Western blotting with Anti-NELFB and Anti-GAPDH antibodies in H2A.Z.1dTAG and H2A.Z.1dTAG +NELF-B-GFP mESCs. b, Correlation plots of NELF-B-GFP and SPT5 ChIP-nexus between biological replicates for Control and dTAG-13 conditions. c, Boxplots measuring promoter proximal (±300 bp of TSS) NELF density between Control and dTAG-13 over n=4,184 protein-coding genes. Significance is calculated using a paired two-sided Wilcoxon rank test. d, Boxplots measuring either promoter proximal (±300 bp of TSS) or gene body (+300 bp of TSS until TES) SPT5 density between Control and dTAG-13 over n=4,184 protein-coding genes. Significance is calculated using a paired two-sided Wilcoxon rank test. For c,d, the median value for each condition is shown with a horizontal black line. Boxes show values between the first and the third quartiles. The lower and upper whiskers show the smallest and the highest values, respectively. Outliers are shown in black circles.

Source data

Extended Data Fig. 6 Rapid dynamics of endogenously tagged RNAPII.

a, Single gene plots of unphosphorylated RNAPII (ChIP-seq), Total RNAPII (Anti-Dendra2 ChIP-seq), and NET-seq. b, Heatmaps of Anti-Dendra2-RNAPII (ChIP-seq) and NET-seq over n=11,315 protein-coding genes. c, Correlation plot between ChIP-seq (Anti-Dendra2-RNAPII) and NET-seq over 11,315 protein-coding genes. Pearson correlation is indicated on the plot (R = 0.75). d, Histogram of total RNAPII lifetime (Transient and stable clusters) between Control (n=473 clusters from 32 cells examined over 3 independent experiments) and dTAG-13 (n=495 clusters from 35 cells examined over 3 independent experiments) measured by time correlated PALM (tcPALM). Median and average (±SEM) RNAPII lifetime values are indicated on the histogram. e, Live-cell direct image of pre-converted Dendra2-RNAPII (left), super-resolution image of post-converted Dendra2-RNAPII (middle), and DBSCAN analysis of post-converted Dendra2-RNAPII (right). Cells were treated either with DMSO, DRB or TRI for 45 min before photo conversion. Scale bar 5 µm. f, Boxplots measuring the number of RNAPII transient cluster per nucleus in Control (n=16 cells examined over 2 independent experiments), DRB (n=10 cells examined over 2 independent experiments), and TRI-treated (n=13 cells examined over 2 independent experiments) cells. Significance was calculated using a Student’s two-sided t-test (**p<10−9). The median value for each condition is shown with a horizontal black line. Boxes show values between the first and the third quartiles. The lower and upper whiskers show the smallest and the highest values, respectively. Outliers are shown in black circles.

Source data

Extended Data Fig. 7 Rapid dynamics of endogenously tagged NELF.

a, Western blotting with Anti-NELFB and Anti-GAPDH antibodies in H2A.Z.1dTAG and H2A.Z.1dTAG +NELF-B-Halo mESCs. b, Live-cell direct images of NELF-Halo in DMSO, DRB, and TRI-treated cells (45min). NELF-B-Halo-TMR (561nm). Left to right for each panel: 1. Maximum intensity projection of 2D stack. 2. Median-filtered image. 3. Raw image after background subtraction. 4. Smoothed with Gaussian kernel (1 pxl radius) for peak detection above background fluctuations (red crosses). Scale bar 5 µm. c, Dot plots of the number of NELF-B stable clusters per nucleus in Control (n=41), DRB (n=36), and TRI-treated (n=42) cells. Scale bar 5 µm. d, Density plots of the number of NELF-B transient clusters per nucleus in Control (n=21 cells examined over 2 independent experiments), DRB (n=21 cells examined over 2 independent experiments), and TRI-treated (n=28 cells examined over 2 independent experiments) cells. Significance is calculated using a two-sided Student’s t-test (* p = 0.001; ** p = 10−9). e, NELF-B ChIP-nexus heatmaps in Control, DRB, and TRI-treated cells over 4,184 protein-coding TSSs. f, Live-cell direct images of NELF-Halo in Control and dTAG-13 conditions. NELF-B-Halo-TMR (561nm). Left to right for each panel: 1. Maximum intensity projection of 2D stack. 2. Median-filtered image. 3. Raw image after background subtraction. 4. Smoothed with Gaussian kernel (1 pxl radius) for peak detection above background fluctuations (red crosses). Scale bar 5 µm. g, Normalized intensity of all NELF-Halo clusters displayed in (f) (n=4,899 clusters from 30 cells in Control; n=7,281 clusters from 29 cells examined over 2 independent experiments in dTAG-13). Significance is calculated using an unpaired two-sided Wilcoxon rank test (*** p = 6.9 x 10−216). Median value is shown with a horizontal black line. Boxes show values between the first and the third quartiles. The lower and upper whiskers show the smallest and the highest values, respectively. h, Density plot of the average number of total NELF clusters per nucleus in Control (n=40 cells) and dTAG-13 (n=34) cells examined over 2 independent experiments. A student’s two-sided t-test was used for significance. i, FRAP analysis of NELF clusters. Images of a Halo-NELF cell before (0 sec), and immediately after (1–10 sec) bleaching. The black box indicates an unbleached control locus. The golden box indicates the position of the cluster on which the FRAP beam was focused. j, The normalized recovery curve for NELF yielded a recovery fraction of ~80% during the 60 sec observation in Control (n = 22 cells) conditions that increased to ~90% in dTAG-13 (n=24 cells) treated cells. Data was gathered from 2 independent experiments. Dots and shaded areas represent mean and SEM values, respectively. An unpaired two-sided student’s t-test was used for significance. k, Boxplots measuring mobile fraction in Control (n=22 cells) and dTAG-13 (n=24 cells) examined over 2 independent experiments.

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Extended Data Fig. 8 RNAPII ChIP-nexus over the course of TRI treatment and stable pausing.

a, Pearson correlation heatmap of all RNAPII ChIP-nexus replicates (Control or dTAG-13 and no TRI, TRI 10 min, TRI 20 min, and TRI 40min). b, Metaplot profiles of RNAPII (ChIP-nexus) over n=4,558 protein-coding TSSs for Control and dTAG-13 cells over the course of triptolide (TRI) treatment. c, Cumulative distribution plot of RNAPII (ChIP-nexus) Pausing Index for Control and H2A.Z.1-depleted (dTAG-13) mESCs, over n=4,184 uniquely annotated protein-coding genes. Significance was calculated using a paired two-sided Wilcoxon rank test. d, Boxplots measuring mRNA abundance (FPKM) for the three different gene classes. Significance was calculated using an unpaired two-sided Wilcoxon rank test. e, Boxplots measuring GC content ± 250bp around the TSS for the three different gene classes. Significance was calculated using an unpaired two-sided Wilcoxon rank test. f, Metaplots of GC frequency for the three different gene classes ± 1000bp around the TSS. g, Boxplots measuring log2 FC (wash-off / DRB) of promoter-proximal RNAPII for the three different gene classes. Significance was calculated using an unpaired two-sided Wilcoxon rank test. h, Boxplots measuring log2 FC (RA 6h / DMSO) of promoter-proximal RNAPII (Lin et al 2011) for the three different gene classes. Significance was calculated using an unpaired two-sided Wilcoxon rank test. For d-h the number of genes for the different categories is as follows; short half-life (n=1,449), medium half-life (n=730), long half-life (n=728). For d,e,g,h, the median value for each condition is shown with a horizontal black line. Boxes show values between the first and the third quartiles. The lower and upper whiskers show the smallest and the highest values, respectively. Outliers are shown in black circles.

Extended Data Fig. 9 TRI treatment reveals RNAPII at the PIC area.

a, Western blotting with Anti-Pol IIS5ph, Anti-Pol II S2ph, and Anti-HA (H2A.Z.1) antibodies over the course of TRI treatment. DMSO concentration is at 2%. b, Metaplot profiles of RNAPII (ChIP-nexus) over 1,460 protein-coding genes displaying the highest RNAPII at the PIC region. First column (Control), second column (dTAG-13), third column (log2 dTAG-13/Control – Positive strand). c, Single gene plot (Tex19.1) of RNAPII ChIP-nexus profiles (Control, dTAG-13) over the course of Triptolide treatment (No TRI, 10 min, 20 min, and 40 min). RNAPII density is displayed both in the positive (blue) and negative (red) strand. Initiating RNAPII is highlighted in yellow. d, Correlation plots for biological TBP ChIP-nexus replicates. e, Correlation plots for biological TFIIB ChIP-nexus replicates.

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Extended Data Fig. 10 Rapid dynamics of endogenously tagged TFIIB and TBP.

a, Metaplots of TBP ChIP-nexus in Control conditions with (‘TRI 40min’) or without (‘No TRI’) triptolide. b, Metaplots of TBP ChIP-nexus in H2A.Z.1-depleted (dTAG-13) conditions with (‘TRI 40min’) or without (‘No TRI’) triptolide. c, Boxplots measuring log2 FC (TRI 40min vs No TRI) TBP density between Control and dTAG-13 in an area 250 bp upstream of the TSS (n = 2,143 genes). Significance was calculated using a Wilcoxon rank test. d, Boxplots measuring log2 FC (TRI 40min vs No TRI) of TFIIB and TBP at promoters of the three different gene classes between Control and dTAG-13. The number of genes for the different categories is as follows; short half-life (n=1,449), medium half-life (n=730), long half-life (n=728). Significance was calculated using an unpaired two-sided Wilcoxon rank test. For c,d, the median value for each condition is shown with a horizontal black line. Boxes show values between the first and the third quartiles. The lower and upper whiskers show the smallest and the highest values, respectively. Outliers are not shown. e, Western blotting with Anti-TFIIB and Anti-GAPDH antibodies in H2A.Z.1dTAG and H2A.Z.1dTAG +TFIIB-Halo mESCs. f, Live-cell direct images of TFIIB-Halo, NELF-GFP, and composite. TFIIB-Halo-JF646 (646nm). Scale bar 5 µm and 5 nm (zoomed in clusters). g, Western blotting with Anti-TBP and Anti-GAPDH antibodies in H2A.Z.1dTAG and H2A.Z.1dTAG +TBP-Halo mESCs. h, Live-cell super-resolution image of photoactivated Halo-TBP (PA-JF549) and corresponding time-correlated photoactivation localization microscopy (tcPALM) traces. Scale bar 5 µm and 5 nm (zoomed in clusters). i, Cumulative distribution and histogram of TBP transient cluster lifetime between Control (n=301 clusters from 16 cells examined over 2 independent experiments) and dTAG-13 (n=321 clusters from 17 cells examined over 2 independent experiments) measured by time correlated PALM (tcPALM). Significance is calculated using an unpaired two-sided Wilcoxon rank test.

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Supplementary information

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Supplementary Table 1

List of genes with short, medium and long RNAPII half-life.

Supplementary Table 2

List of guide RNA sequences.

Supplementary Table 3

Differential expression gene list from RNA-seq.

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Mylonas, C., Lee, C., Auld, A.L. et al. A dual role for H2A.Z.1 in modulating the dynamics of RNA polymerase II initiation and elongation. Nat Struct Mol Biol 28, 435–442 (2021). https://doi.org/10.1038/s41594-021-00589-3

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