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Spt5-mediated enhancer transcription directly couples enhancer activation with physical promoter interaction

Nature Genetics volume 52, pages 505–515 (2020)Cite this article

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Abstract

Active enhancers are frequently transcribed, yet the regulatory role of enhancer transcription remains debated. Here, we depleted the RNA polymerase II pausing and elongation factor Spt5 in activated mouse B cells and found that approximately 50% of enhancer–gene pairs showed co-regulated transcription, consistent with a potential functional requirement for enhancer transcription. In particular, Spt5 depletion led to loss of super-enhancer–promoter physical interaction and gene expression at the immunoglobulin heavy-chain locus (Igh), abrogating antibody class switch recombination. This defect correlated strictly with loss of enhancer transcription but did not affect acetylation of histone H3 at lysine 27, chromatin accessibility and occupancy of Mediator and cohesin at the enhancer. Strikingly, CRISPRa-mediated rescue of enhancer transcription in Spt5-depleted cells restored Igh gene expression. Our work suggests that Spt5-mediated enhancer transcription underlies the physical and functional interaction between a subset of active enhancers and their target promoters.

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Fig. 1: Spt5 depletion leads to loss of transcription at the Igh locus.
Fig. 2: Igh 3′RR super-enhancer function is dependent on Spt5.
Fig. 3: The Igh 3′RR super-enhancer chromatin is in an accessible and acetylated state in Spt5dep cells.
Fig. 4: CRISPRa-mediated transcriptional activation of the 3′RR partially restores Ighg1 transcription in Spt5dep cells.
Fig. 5: The maintenance of pre-established 3′RR interactions is independent of transcription, Pol II pausing and Spt5.
Fig. 6: Model for the role of Spt5-dependent enhancer transcription in Igh 3′RR–promoter interactions during antibody maturation.

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Data availability

All raw next-generation sequencing data (GRO-Seq, ChIP-Seq, ATAC-Seq, PRO-cap and mRNA-Seq) have been deposited in the Gene Expression Omnibus under accession number GSE132029. Source data for Figs. 1 and 2 are presented with the paper.

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Acknowledgements

We are grateful to the Vienna Biocenter Core Facilities for the next-generation sequencing, and to the IMP/IMBA core facilities, especially Comparitive Medicine, BioOptics, Molecular Biology Service and Protein Chemistry. We thank K. Uzunova for the Tn5 enzyme preparation, K. Mochizuki for the GRO-Seq reagents, and C. Umkehrer and A. Obenauf for the dual sgRNA vector. We thank A. Stark, M. Busslinger, C. Bücker and A. Andersen (Life Science Editors) for critical reading of the manuscript. This work was funded by Boehringer Ingelheim and the Austrian Research Promotion Agency (headquarter grant FFG-834223). U.E.S. is supported by the L’Oréal For Women in Science Austria fellowship (2015) and Austrian Science Fund (FWF T 795-B30).

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Authors and Affiliations

  1. Research Institute of Molecular Pathology (IMP), Vienna, Austria

    Johanna Fitz, Tobias Neumann, Monika Steininger, Eva-Maria Wiedemann, Adriana Cantoran Garcia, Alexander Athanasiadis, Ursula E. Schoeberl & Rushad Pavri

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  1. Johanna Fitz
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Contributions

J.F. designed and performed the experiments and co-wrote the manuscript. T.N. performed all of the bioinformatic analyses. M.S., E.-M.W., A.C.G., A.A. and U.E.S. designed and performed the experiments. R.P. conceived of the project, designed and performed the experiments and wrote the manuscript.

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Correspondence to Rushad Pavri.

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Extended data

Extended Data Fig. 1 Targeting strategy for generating Spt5-depleted (Spt5dep) mice and characterization of Spt5dep primary B cells.

a, Scheme for obtaining Spt5dep B cells (described in detail in Fitz et al. 23). Briefly, the targeting vector (Sanger Consortium) was used to generate knock-in mice, which were bred to Flp-expressing followed by EIIA-Cre-expressing mice to generate Supt5hF/+ and Supt5h-/+ genotypes, respectively. Supt5hF/F mice were crossed with Supt5h-/+Rosa26 Cre-ERT2/Cre-ERT2 mice to obtain the final genotype, Supt5hF/-Rosa26Cre-ERT2/+. b, Generation of WT (Rosa26Cre-ERT2/+) and Spt5dep (Supt5h-/-Rosa26Cre-ERT2/+) B cells. Primary B cells were isolated from spleens and cultured for 60 h with IL4 and LPS. 32 h prior to harvesting, 2 μM 4-hydroxytamoxifen (4-HT) was added to induce deletion of the floxed Supt5h exons. c, Multiplex PCR-based genotyping from genomic DNA from B cells of three Spt5dep mice at indicated times after 4-HT addition. d, Spt5 protein analysis from 10 μg nuclear extracts of WT and Spt5dep primary B cells (two mice each) treated with 4-HT for 0 h, 24 h and 32 h. Relative quantification was performed by normalization to histone H3 by setting the WT condition of each timepoint to 100%. e, Assessment of cell proliferation. Primary WT and Spt5dep B cells were stained with Cell Trace Violet upon isolation and analyzed by flow cytometry 32 h post 4-HT treatment. f, Viability curve using Trypan Blue exclusion for WT and Spt5dep primary B cells treated with 4-HT at the indicated time-points. The data represent the mean ± s.d. from eight replicates per genotype. g, Analysis of Pol II, Spt5 and histone H3 protein levels from nuclear extracts of WT and Spt5dep primary B cells treated with 4-HT for 32 h. Two replicate mice were used per genotype and a two-fold titration (3 μg and 10 μg) of nuclear extract was performed.

Extended Data Fig. 2 Replicate correlation analysis for all next-generation sequencing data used in this study.

a, Replicate correlation plots for total Pol II, Ser5P-Pol II, Ser2P-Pol II and Spt5 ChIP-seq datasets, as well as for GRO-seq, ATAC-seq and PRO-cap datasets from WT and Spt5dep primary B cells. Spearmann’s correlation coefficient (r) is indicated within the plots. b, Replicate correlation plots for mRNA-seq done in triplicate with Spearmann’s correlation coefficient indicated.

Extended Data Fig. 3 Identification of differentially expressed enhancer (DEE) groups.

a, Heatmaps in WT activated B cells centered on extragenic DNase hypersensitive site (DHS) peaks and ordered by decreasing DHS read density. Read densities of the following datasets are shown as reads per million (RPM): DHS-seq, ChIP-seq of total Pol II, serine 5-phosphorylated Pol II (Ser5P-Pol II), serine 2-phosphorylated Pol II (Ser2P-Pol II) and Spt5 and GRO-seq (sense and anti-sense strand reads are separated). DHS-seq data is from Kieffer-Kwon et al., 2013. b, Generating differentially expressed enhancer (DEE) groups. GRO-seq fold-changes (Spt5dep/WT) at extragenic DHSs (considered as putative enhancers) were clustered into three DEE groups: up-regulated, moderately down-regulated and strongly down-regulated (shown in Extended Data Fig. 5a). DEEs were linked to genes using Pol II ChIA-PET (Kieffer-Kwon et al., 2013). DEEs with (linked) or without (unlinked) PET links were divided into H3K27ac-positive (H3K27ac-pos) enhancers, H3K27ac-negative (H3K27ac-neg) enhancers and super-enhancer (SE) classes based on histone H3 lysine 27 acetylation (H3K27ac) (Whyte et al., 79). Finally, these enhancer classes were assigned to differentially expressed genes (DEGs) based on direct PET linkages (as in Extended Data Fig. 5c, d). A detailed explanation of this workflow is provided in the Methods under the Bioinformatics section. c, Upper panel: Diagram showing the calculation of the 5’ pausing index (5’PI). Note that we do not employ the conventional PI where the whole gene body is considered because of the processivity defects within gene bodies in Spt5dep cells seen in Extended Data Fig. 5a and described previously in Spt5dep MEFs (Fitz et al., 23). Lower panel: 5’ PI of DEGs assigned to DEEs. Each bar represents DEGs linked to the indicated DEE group. Each linked DEE group is split into the three enhancer classes as described in b. Statistical significance is calculated using the Student’s t test. Asterisks indicate P < 0.05 and ns = not significant.

Extended Data Fig. 4 Spt5 depletion results in elongation defects at long genes but does not lead to a global down-regulation of mRNA synthesis.

a, GRO-seq Spt5dep /WT ratio heatmap of all expressed minus-strand genes <100 kb in length (n=2943) generated using 1025 static bins and ordered by increasing gene length. Read densities are in reads per million (RPM). The transcription start site (TSS), and transcription termination site (TTS) are indicated. b, UCSC browser snapshots at one short and one long gene in WT and Spt5dep B cells. The tracks are overlaid with blue and red representing the WT and Spt5dep samples, respectively, and the overlapping region in black. The Y axis shows normalized read counts. c, UCSC browser snapshots of a representative up-regulated DEG (top) and down-regulated DEG (bottom). The WT and Spt5dep tracks are overlaid as described in b. d, Volcano plot of mRNA-seq data from WT and Spt5dep primary B cells performed in triplicate and calculated with the DEseq2 software. Genes having an expression fold-change >2 and an adjusted P value of < 0.1 are highlighted in red and considered as up- or down-regulated genes in this study. e, Box plots showing the distribution of mRNA fold-changes (Spt5dep/WT) within the corresponding GRO-seq-based DEG classes shown in Extended Data Fig. 5c. f, RT-qPCR analysis for mRNA abundance in WT and Spt5dep cells of five up-regulated and five down-regulated genes. Drosophila S2 cells were spiked in to normalize WT and Spt5dep qPCR values to the transcripts of the Drosophila-specific housekeeping gene, Act5c. The plotted data represents the mean of eight independent WT and Spt5dep samples ± s.d. Statistical significance in e and f were calculated using the two-tailed Student’s t test. Asterisks indicate P < 0.01.

Extended Data Fig. 5 A subset of enhancer-promoter pairs shows correlated transcriptional changes upon Spt5 depletion.

a, Left panel: WT GRO-seq read densities within the DEE groups (Extended Data Fig. 3b). DEEs linked to gene promoters using Pol II ChIA-PET, as well as DEEs with no PET links (unlinked DEEs), were further divided into three enhancer classes based on H3K27ac signal. DEEs with no PET links were termed unlinked enhancers. The number of DHSs in each row is indicated below the plot. Right panel: Same as before but now showing the fold-change in GRO-seq signal upon Spt5 depletion (Spt5dep/WT). Statistical significance for both plots is calculated using the two-tailed Student’s t test. Asterisks indicate P < 0.05 and ns = not significant. b, Mediator subunit, Med12, and histone H3 lysine 4 monomethylation (H3K4me1) levels within the DEE groups displayed exactly as in a. c, Left panel: Whole gene GRO-seq composite metagene analysis of WT and Spt5dep tracks representing the three differentially expressed gene (DEG) classes (Methods). Right panel: A magnified view of the gene body region (boxed in the left panel plots). d, Each bar represents DEGs linked to the indicated DEE group shown in a. Each linked DEE group is split into the three enhancer classes as described in b. The Y axis is set to 100% and the number of DEGs per bar is indicated above the bar. The numbers in white within the bars indicate the percentage of that DEG class relative to the total DEGs in that bar. Note the relatively equal distribution of DEGs showing up-, down- or intermediate regulation within each DEE group irrespective of linkage to an H3K27ac-pos enhancer, H3K27ac-neg enhancer or SE.

Extended Data Fig. 6 Effect of Spt5 depletion on B cell activation, expression of major B cells transcription factors and IgH GLTs under various stimulation conditions.

a-b, Flow cytometry analysis of a panel of B cell activation markers in WT and Spt5dep primary B cells after 32h of 4-HT treatment. Representative histogram plots for six activation markers is shown in (A) and bar graphs of mean fluorescence intensity for all tested activation markers is shown in b. Data in b represent the mean of eight independent WT and Spt5dep samples ± s.d. No statistically significant differences were detected between WT and Spt5dep activated B cells. c, RT-qPCR analysis for Igh germline transcript abundance in WT and Spt5dep cells activated with different stimulation conditions, as mentioned above each graph. For each condition, the expressed and expected Igh transcripts were analyzed, as indicated. The data represents the mean of three independent WT and Spt5dep samples ± s.d. Statistical significance was calculated using the two tailed Student’s t test (asterisks indicate P < 0.01 and ns = not significant). d, Scatter plot of mRNA-seq data for selected B cell transcription factors and cofactors as well as general transcription factors, Pol II, Mediator and cohesin (see Supplementary Table 1 for a complete list). The data plotted represent the mean reads per kilobase per million mapped reads (RPKM) of three independent WT and Spt5dep primary B cells samples. Genes showing 2-fold significant differential expression are marked in green (up-regulated) and red (down-regulated).

Extended Data Fig. 7 dCas9-VPR cannot stimulate Igh transcription in the absence of transcription.

a, Snapshot of the Igh locus showing the location of the uIghε sgRNA (~3 kb upstream of the Igh-ε promoter) relative to the IghH-γ1 and Igh-ε genes, and the 3’RR. b, Anti-Cas9 ChIP-qPCR at the hs4 and uIghε in cells infected with the indicated dCas9-VPR/sgRNA viruses, as described in Fig. 4a. c, RT-qPCR at hs4 and uIghε from the experiment in (B). For the uIghε, two sets of primers were used probing the region upstream and downstream of the sgRNA binding site. d, RT-qPCR for Igh-γ1 and Igh-μ transcripts in WT and Spt5dep cells infected with dCas9-VPR and uIghε sgRNA, as in Fig. 4a. In all cases, four independent experiments were performed and the two-tailed Student’s t test was used for statistical significance. Asterisks indicate P < 0.05 and ns, not significant.

Supplementary information

Supplementary Information

Supplementary Tables 1 and 2, and Discussion

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Source Data Fig. 1

Unprocessed western blots.

Source Data Fig. 2

Gating strategy for flow cytometry.

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Fitz, J., Neumann, T., Steininger, M. et al. Spt5-mediated enhancer transcription directly couples enhancer activation with physical promoter interaction. Nat Genet 52, 505–515 (2020). https://doi.org/10.1038/s41588-020-0605-6

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