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BRD4 orchestrates genome folding to promote neural crest differentiation

Nature Genetics volume 53, pages 1480–1492 (2021)Cite this article

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A Publisher Correction to this article was published on 01 November 2021

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Abstract

Higher-order chromatin structure regulates gene expression, and mutations in proteins mediating genome folding underlie developmental disorders known as cohesinopathies. However, the relationship between three-dimensional genome organization and embryonic development remains unclear. Here we define a role for bromodomain-containing protein 4 (BRD4) in genome folding, and leverage it to understand the importance of genome folding in neural crest progenitor differentiation. Brd4 deletion in neural crest results in cohesinopathy-like phenotypes. BRD4 interacts with NIPBL, a cohesin agonist, and BRD4 depletion or loss of the BRD4–NIPBL interaction reduces NIPBL occupancy, suggesting that BRD4 stabilizes NIPBL on chromatin. Chromatin interaction mapping and imaging experiments demonstrate that BRD4 depletion results in compromised genome folding and loop extrusion. Finally, mutation of individual BRD4 amino acids that mediate an interaction with NIPBL impedes neural crest differentiation into smooth muscle. Remarkably, loss of WAPL, a cohesin antagonist, rescues attenuated smooth muscle differentiation resulting from BRD4 loss. Collectively, our data reveal that BRD4 choreographs genome folding and illustrates the relevance of balancing cohesin activity for progenitor differentiation.

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Fig. 1: Loss of Brd4 in neural crest cells results in cohesinopathy features.
Fig. 2: BRD4 ET domain mediates a physical interaction with NIPBL.
Fig. 3: Acute degradation of BRD4 reduces NIPBL occupancy on chromatin.
Fig. 4: BRD4 maintains genome folding.
Fig. 5: A BRD4–NIPBL interaction is necessary for genome organization.
Fig. 6: Changes in transcription dynamics on BRD4 depletion.
Fig. 7: A BRD4–NIPBL interaction promotes smooth muscle cell differentiation of neural crest cells.
Fig. 8: Model depicting proposed mechanism of action of BRD4 to stabilize NIPBL on chromatin to maintain chromatin interactions and loop extrusion.

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Data availability

All data generated for this paper have been deposited at the National Center for Biotechnology Information Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo) and are available under accession no. GSE169516.

Mouse genome sequence mm10:

http://ftp.ebi.ac.uk/pub/databases/gencode/Gencode_mouse/release_M10/GRCm38.primary_assembly.genome.fa.gz

Saccharomyces cerevisiae genome sequence sacCer3: https://hgdownload.soe.ucsc.edu/goldenPath/sacCer3/bigZips/sacCer3.fa.gz

BRD4 ChIP–seq: http://dc2.cistrome.org/api/hgtext/34084/?db=mm10

NIPBL ChIP–seq: http://dc2.cistrome.org/api/hgtext/2864/?db=mm10

H3K27ac ChIP–seq: http://dc2.cistrome.org/api/hgtext/67169/?db=mm10

RAD21 ChIP–seq: http://dc2.cistrome.org/api/hgtext/39300/?db=mm10.Source data are provided with this paper.

Code availability

OligoMiner is available at https://github.com/beliveau-lab/OligoMiner and cLoops at https://github.com/YaqiangCao/cLoops. TADtool is available at https://github.com/vaquerizaslab/tadtool and Juicer Tools at https://github.com/aidenlab/juicer. The other tools used are indicated in Methods. We utilized existing published analysis tools as indicated above. No new analysis tools were generated, and scripts employed to analyze the data using the published tools are available upon request.

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Acknowledgements

We thank the Jain and Joyce laboratories for critical discussion. We thank M. Capelson, J. A. Epstein, D. Gilbert, M. Lakadamyali, K. Musunuru and E. Nora for critical reading of the manuscript, J. E. Phillips-Cremins and M. Mir for advice, A. Silva for help with illustrations, K. Ozato for sharing the Brd4 floxed mice, A. Ciulli for sharing the MZ3 compound, A. Tsirigos and R. Sitharaman at the NYU Technology Center for Hi-C library preparation, sequencing and early informatic advice, J. Rhoades for early informatic help, B. Nabet and N. Gray for providing the dTAG compounds, D. Janssens and S. Henikoff for CUT&RUN reagents and advice, C. Petucci for metabolomic RNA labeling help, and T. Finkes, J. Haglmueller and A. Tuerk at Lexogen for SLAM-seq and analysis. This work was supported by the National Institutes of Health (NIH) (grant nos. R01 HL139783 to R.J., F31 HL147416 to R.L.-S., T32 HL007843 to B.A.K.-B., T32 GM008216 to A.K., R35 GM128903 to E.F.J., F30 HD104360 to D.S.P., T32 GM008216 to J.M.L., F31 HD102084 to J.M.L., U01 DA052715 to G.V., and K08 HL157700 to A. Padmanabhan); support from grant no. R35 HL140018 funded A. Poleshko and C.L.S.; the following also provided support: Burroughs Wellcome Foundation Career Award for Medical Scientists (R.J.), Allen Foundation (R.J.) and American Heart Association (W.K. and R.J.), the National Science Foundation (15-48571 to R.L.-S. and R.J.), Michael Antonov Charitable Foundation (A. Padmanabhan), Tobacco-Related Disease Research Foundation (A. Padmanabhan), A.P. Giannini Foundation (A. Padmanabhan) and Sarnoff Foundation (A. Padmanabhan). G.V., G.A.B., E.F.J., R.J. and their laboratories are members of the NIH 4DN consortium and thank members for critical input.

Author information

Author notes

  1. These authors contributed equally: Ricardo Linares-Saldana, Wonho Kim, Nikhita A. Bolar.

Authors and Affiliations

  1. Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA

    Ricardo Linares-Saldana, Wonho Kim, Nikhita A. Bolar, Bailey A. Koch-Bojalad, Sora Yoon, Parisha P. Shah, Ashley Karnay, Daniel S. Park, Jennifer M. Luppino, Son C. Nguyen, Cheryl L. Smith, Andrey Poleshko, Qiaohong Wang, Li Li, Golnaz Vahedi, Gwang Hyeon Eom, Gerd A. Blobel, Eric F. Joyce & Rajan Jain

  2. Department of Cell and Developmental Biology, Department of Medicine, Institute of Regenerative Medicine, Penn Cardiovascular Institute, University of Pennsylvania, Philadelphia, PA, USA

    Ricardo Linares-Saldana, Wonho Kim, Nikhita A. Bolar, Bailey A. Koch-Bojalad, Parisha P. Shah, Ashley Karnay, Cheryl L. Smith, Andrey Poleshko, Qiaohong Wang, Li Li, Gwang Hyeon Eom & Rajan Jain

  3. Penn Epigenetics Institute, University of Pennsylvania, Philadelphia, PA, USA

    Ricardo Linares-Saldana, Wonho Kim, Nikhita A. Bolar, Bailey A. Koch-Bojalad, Sora Yoon, Parisha P. Shah, Ashley Karnay, Daniel S. Park, Jennifer M. Luppino, Son C. Nguyen, Cheryl L. Smith, Andrey Poleshko, Qiaohong Wang, Li Li, Golnaz Vahedi, Gwang Hyeon Eom, Gerd A. Blobel, Eric F. Joyce & Rajan Jain

  4. Institute of Molecular Physiology, Shenzhen Bay Laboratory, Shenzhen, China

    Haoyue Zhang

  5. Division of Hematology, The Children’s Hospital of Philadelphia, Philadelphia, PA, USA

    Haoyue Zhang & Gerd A. Blobel

  6. Department of Genetics, University of Pennsylvania, Philadelphia, PA, USA

    Sora Yoon, Daniel S. Park, Jennifer M. Luppino, Son C. Nguyen, Golnaz Vahedi & Eric F. Joyce

  7. Institute for Immunology, Epigenetics Institute, Institute for Diabetes, Obesity and Metabolism, University of Pennsylvania, Philadelphia, PA, USA

    Sora Yoon & Golnaz Vahedi

  8. Gladstone Institute of Cardiovascular Disease, San Francisco, CA, USA

    Arun Padmanabhan & Deepak Srivastava

  9. Division of Cardiology, Department of Medicine, University of California, San Francisco, CA, USA

    Arun Padmanabhan

  10. Roddenberry Stem Cell Center at the Gladstone Institutes, Departments of Pediatrics and Biochemistry & Biophysics, University of California San Francisco, San Francisco, CA, USA

    Deepak Srivastava

  11. Abramson Family Cancer Center, University of Pennsylvania, Philadelphia, PA, USA

    Golnaz Vahedi

  12. Department of Pharmacology, Chonnam National University Medical School, Hwasun, Republic of Korea

    Gwang Hyeon Eom

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  1. Ricardo Linares-Saldana
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Contributions

R.L.-S., W.K., N.A.B. and R.J. conceived and designed the study. R.L.-S., W.K., N.A.B., B.A.K.-B., A.K. and G.H.E. performed all the experiments except for generation of the dTAG-BRD4 mESC line (Q.W. and A. Padmanabhan) and immunohistochemistry of the mouse embryo (L.L.). R.L.-S., W.K., N.A.B., H.Z., S.Y., G.H.E. and R.J. analyzed the data. R.L.-S., W.K., N.A.B., P.P.S., G.H.E. and R.J. wrote the manuscript. D.S.P., J.M.L., S.C.N., C.L.S., A. Poleshko, D.S., G.V., G.A.B. and E.F.J. assisted with data interpretation. R.J. supervised the project. All authors edited and approved the manuscript.

Corresponding author

Correspondence to Rajan Jain.

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Competing interests

D.S. is a scientific cofounder, shareholder and director of Tenaya Therapeutics. The remaining authors declare no competing interests.

Additional information

Peer review information Nature Genetics thanks the anonymous reviewers for their contribution to the peer review of this work.

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

Extended data

Extended Data Fig. 1 Characterization of BRD4 in neural crest and the interaction with NIPBL.

(a) GFP epifluorescence and immunohistochemistry (coronal section) of E11.5 Brd4f/f, Wnt1Cre+, R26mt-mg/+ and Brd4f/+, Wnt1Cre+, R26mt-mg/+ embryos. Scale bar, epifluorescence: 500 mm; immunohistochemistry: 500 µm. (b) Multiple sections of E16.5 Brd4f/f, Wnt1Cre+ and Brd4f/+, Wnt1Cre+ embryo showing outflow tract and heart. Mutant demonstrates right-sided aortic arch and truncus arteriosus. (Ao, Aorta; RV, right ventricle; LV, left ventricle; TA, truncus arteriosus). Second image duplicated from Fig. 1. (* denotes sectioning artifact). Scale bar, 1 mm. (c) Percentage of phospho-Histone H3 + cells in neural crest-derived craniofacial mesenchyme (each point represents one field of view, 35 (Brd4f/+, Wnt1Cre+, R26mt-mg/+) and 37 (Brd4f/f, Wnt1Cre+, R26mt-mg/+) fields of view total from two embryos, mean and SD shown, student’s T-test). (d) Expression of indicated genes in E15.0 secondary palate tissue (Unpaired T-test; ****p < 0.0001. Error bars denote SEM). Gene expression normalized to the expression of Hprt1. (e) Immunoprecipitation of endogenous BRD4 blotted for BRD4 and NIPBL (murine neural crest cells, O9-1 cells). (f) Immunoprecipitation (30 min) of endogenous BRD4 from and blotted for BRD4 and NIPBL (HEK293T cells). (g) Immunoprecipitation of endogenous BRD4 and blotted for indicated proteins (HEK293T cells). (h) Immunoprecipitation of endogenous BRD4 and blotted for indicated proteins (mESCs). (i) Indicated FLAG-tagged versions of BRD4 immunoprecipitated (HEK293T cells). Immunoblots probed with indicated antibodies. The NIPBL and FLAG immunoblots are reproduced from Fig. 2e as the proteins were all blotted for from the same blot. (j) Alignment of BRD4-interacting peptides of NSD3, LANA, JMJD6 and MLV-γ-integrase and putative BRD4-interacting peptide of NIPBL. Residues highlighted by gray are conserved across other peptides. Consensus motif indicated at the bottom (Φ represents a hydrophobic amino acid and X any amino acid). Predicted α-helix, β-strand and coil structures are marked by pink, yellow and gray color, respectively. Predictions based on PSPIRED71. The consensus motif is highlighted in the gray box.

Source data

Extended Data Fig. 2 Generation of dTAG-BRD4 mouse embryonic stem cell line.

(a) Venn diagram showing the comparison of BRD4 and NIPBL peaks from publicly available datasets (see Methods). (b) PCR of genomic DNA from clones of indicated genotype using oligos annealing outside of area of homologous recombination. “Unsuccessfully targeted” is a line which was targeted by sgRNA and Cas9, but dTAG was not inserted. Of clones shown, only Brd4dtag/dtag was confirmed by Sanger sequencing. (Ladder: Thermo, #10787-018). (c) Immunoblot of chromatin fraction from parental and dTAG-BRD4 mESCs. (d) Confirmation of fractionation protocol. Cytosol, membrane, nucleoplasm, and chromatin fractions from dTAG-BRD4 mESCs in control and the degron conditions were blotted for indicated proteins. (e) OCT4 immunofluorescence from dTAG-BRD4 mESCs in control and the degron conditions. Scale bar, 20 µm. (f) Flow cytometry-based analysis of proliferation/doubling time using CellTrace CFSE at 4.5 h post treatment with DMSO or 500 nM dTAG-13. Rectangle and circle in left and middle graphs indicates gate in which cells analyzed using Cell Proliferation tool on ModFIT LT v5.0 (n = 23823 and 23859 cells in DMSO and degron, respectively).

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Extended Data Fig. 3 BRD4 regulates NIPBL occupancy.

(a) Co-occupancy analysis of BRD4 (green), NIPBL (blue) and H3K27ac (purple) at BRD4, NIPBL, or BRD4/NIPBL peaks as defined in Extended Data Fig. 2a. (b) NIPBL occupancy [log2 normalized counts (Degron/DMSO)] across top 10 K peaks. (c) Immunoblot of chromatin fractionations from DMSO, dTAG-13 (500 nM), dTAGv-1 (500 nM), and MZ3 (1 µM)-treated cells for 4.5 h for indicated proteins. The samples were derived from the same experiment and blots were processed in parallel. (d) Expression of indicated BRD4-mutants in dTAG-BRD4 mESCs upon degradation of endogenous BRD4. HDAC2 is used as a loading control. Relative band intensity ratio (BRD4:HDAC2) is indicated. (e) ChIP-qPCR of H3K27ac at regions indicated in Fig. 3e. Two-tailed T-test was used for comparison. Error bars denote the mean + SEM. (f) ChIP-qPCR of BRD4 or IgG at the same two regions assayed in Fig. 3f. One-way ANOVA Dunnett’s test was used to compare conditions to empty vector transfection with DMSO treatment. *p < 0.05. Error bars denote the mean + SD.

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Extended Data Fig. 4 BRD4 maintains higher order chromatin structure.

(a) Hi-C matrix comparing c-loop identified loops in DMSO, dTAG-BRD4 mESCs (blue) to mESCs profiled in Mumbach et al., 2016 (black) at two different regions. (b) Mean counts/bin of gained interactions in DMSO and the degron conditions. T-test used for comparison. (c) NIPBL occupancy at BRD4-sensitive and -resistant loop anchors in DMSO and the degron conditions. (d) Stratification of BRD4-resistant and -sensitive loops. Structural loops refer to loops with CTCF and RAD21 peaks at both anchors and 0-1 anchors have H3K27ac peak. CTCF-Cohesin enhancer-promoter (EP) loops refer to loops with H3K27ac, CTCF and RAD21 peaks at both anchors. CTCF-Cohesin independent loops refer to loops with CTCF and RAD21 peaks at 0-1 anchors and both anchors have H3K27ac peak. Other refers to all loops which do not fall into these categories. Anchor regions were extended by 15 kb upstream and downstream for peak intersections. (e) Change in interaction frequency at BRD4, NIPBL, RAD21, and CTCF peaks. (f) Change in interaction frequency at NIPBL peaks (stratified into deciles of peak strength) upon depletion of BRD4 (left) and BRD4 occupancy (normalized counts) at NIPBL peaks (right) (stratified into deciles of peak strength). (g) Hi-C matrix comparing TADs in DMSO, dTAG-BRD4 mESCs (blue) to mESCs profiled in Dixon et al.51 (black) at two different regions. Adjacent bar plot compares the percentage of basepairs uniquely found in a TAD in DMSO, dTAG-BRD4 mESCs (light blue), Dixon et al.51 mESCs (black) or shared between the datasets (navy). Jaccard index between datasets 0.82. (h) Correlation of insulation score specifically at TAD boundaries between DMSO and the degron conditions.

Extended Data Fig. 5 BRD4 loss does not affect compartmentalization.

(a) Eigenvalues in 250 kb bins across the entire genome in DMSO (blue) and the degron (red) conditions, per chromosome. (b) Genome wide comparison of eigenvector values (250 kb bins) in DMSO (blue) and the degron (red) conditions. (c) Correlation of eigenvector values between DMSO and the degron conditions. T-test used for comparison.

Extended Data Fig. 6 Loss of BRD4 abrogates genome folding.

(a) Hi-C matrix (10 kb resolution) of region painted with Oligopaints (as indicated by the green and magenta bars below matrix, from Fig. 5a). Below matrix is insulation score and NIPBL occupancy in DMSO and degron conditions. (b) Immunoblot of NIPBL in shScramble and shNipbl treatment in mESCs. HDAC2 was blotted as a loading control. (c) FISH signal compactness (see methods) of upstream and downstream probes used in Fig. 5. (d) Hi-C matrix (10 kb resolution) with areas painted with Oligopaints, insulation score, BRD4 and NIPBL occupancy (in wildtype mESCs), NIPBL occupancy in DMSO and degron samples shown below. Area highlighted by red dotted line indicates sub-TAD and arrows point to a domain affected upon BRD4 depletion. On right is a representative image from DMSO and the degron conditions with regions painted indicated below matrix. Below the representative FISH images is the plot of center-to-center signal distances and compactness of FISH signals. For visualization purposes only, only probe center-to-center distances up to 0.6 μm shown. Wilcoxon-rank sum test. Scale bar, 1 µm. (e) Hi-C matrix (10 kb resolution) with areas painted with Oligopaints, insulation score, BRD4 and NIPBL occupancy (in wildtype mESCs), and NIPBL occupancy in DMSO and degron samples shown below. In middle is representative image from DMSO and the degron conditions with regions indicated on matrix painted. On right is the plot of center-to-center distances. Wilcoxon-rank sum test used for comparison. Scale bar, 1 µm. All images are maximum-projections. (f) Representative GFP immunofluorescence from conditions used in Fig. 5c. Scale bar, 100 µm. (g) ChIP-qPCR of BRD4 or IgG at the two regions indicated in Fig. 5a by red bars. One-way ANOVA Dunnett’s test was used to compare conditions to empty vector transfection with DMSO treatment. *p < 0.05. Error bars denote the mean + SD.

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Extended Data Fig. 7 BRD4 mediates loop extrusion.

(a) Chromatin fractionation immunoblot of dTAG-BRD4 mESCs treated with DMSO, dTAG-13 (500 nM), dTAGv-1 (500 nM), or MZ3 (1 µM) for 4.5 h. The NIPBL, BRD4 and HDAC2 immunoblots are reproduced from Extended Data Fig. 2f as the proteins were all blotted from the same blot. The samples were derived from the same experiment and blots were processed in parallel. (b) Occupancy from public datasets (see methods) of indicated factors (black) and random shuffle (gray) across stripe domains in DMSO-treated dTAG-BRD4 mESCs. Note the enrichment of factor occupancy at stripe anchors. (c) Hi-C matrix (top DMSO, bottom Degron) showing an example of a stripe domains (10 kb resolution), which is magnified on the right. Black arrows indicate a stripe domain which is weaker in degron compared to control. Color bar refers normalized count. (d) Hi-C matrix (top DMSO, bottom Degron) showing another example of stripe (indicated by arrow) which is weaker in degron compared to DMSO (10 kb resolution). (e) Change in stripe score and median intensity of stripe domains in degron compared to DMSO. Stripe domains defined in DMSO condition and same region compared in the degron condition. T-test used for comparison. (f–i) Occupancy of NIPBL (F and G) or RAD21 (H and I) at RAD21-NIPBL peaks (F and H) or in stripe domains (G-NIPBL peaks and I-RAD21 peaks) in DMSO and the degron conditions. (j) The percentage of differentially expressed genes per TAD is plotted and TADs are broken into descending deciles of change in interaction frequency. Individual data points shown as well as superimposed box and whisker plot (median and interquartile range indicated by box). (k) Cumulative density of differentially expressed genes (DEG, FDR < 0.05, |log2FC | >1, red) or all genes (black) in relation to proximity to BRD4-sensitive (left) and -resistant (right) loop anchors.

Source data

Extended Data Fig. 8 BRD4 mediates differentiation of neural crest progenitor cells.

(a) Immunoblot showing levels of BRD4 upon depletion of BRD4 and introduction of wildtype BRD4 or various mutants unable to bind NIPBL. EV: empty vector. Undif.: Undifferentiated condition. (b) Representative image of undifferentiated O9-1 cells infected with shRenilla and stained with SMA antibody. Scale bar, 100 μm. (c) Acta2 and Cnn1 expression in indicated conditions. One-way ANOVA Tukey’s multiple comparison test is used to compare across conditions (*p < 0.05, ***p < 0.001). Error bars denote the mean ± SD. Undif.: Undifferentiated condition. (d) Nipbl expression in Brd4-depleted O9-1 cells. Unpaired t-test was used for comparison. All error bars denote the mean ± SEM. (e) Immunoblots of BRD4, WAPL and NIPBL in various knockdown conditions. Histone H3 and GAPDH are loading controls. Undif.: Undifferentiated condition. (f) Representative image of undifferentiated O9-1 cells infected with shRenilla and transfected with empty vector and stained with SMA antibody. Scale bar, 100 μm. (g, h) Acta2 and Cnn1 expression in indicated conditions. One-way ANOVA Tukey’s multiple comparison test is used to compare across conditions (*p < 0.05, **p < 0.01, ***p < 0.001). Error bars denote the mean ± SD. Undif.: Undifferentiated condition.

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Linares-Saldana, R., Kim, W., Bolar, N.A. et al. BRD4 orchestrates genome folding to promote neural crest differentiation. Nat Genet 53, 1480–1492 (2021). https://doi.org/10.1038/s41588-021-00934-8

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