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lncRNA DIGIT and BRD3 protein form phase-separated condensates to regulate endoderm differentiation

Nature Cell Biology volume 22, pages 1211–1222 (2020)Cite this article

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Abstract

Cooperation between DNA, RNA and protein regulates gene expression and controls differentiation through interactions that connect regions of nucleic acids and protein domains and through the assembly of biomolecular condensates. Here, we report that endoderm differentiation is regulated by the interaction between the long non-coding RNA (lncRNA) DIGIT and the bromodomain and extraterminal domain protein BRD3. BRD3 forms phase-separated condensates of which the formation is promoted by DIGIT, occupies enhancers of endoderm transcription factors and is required for endoderm differentiation. BRD3 binds to histone H3 acetylated at lysine 18 (H3K18ac) in vitro and co-occupies the genome with H3K18ac. DIGIT is also enriched in regions of H3K18ac, and the depletion of DIGIT results in decreased recruitment of BRD3 to these regions. Our findings show that cooperation between DIGIT and BRD3 at regions of H3K18ac regulates the transcription factors that drive endoderm differentiation and suggest that protein–lncRNA phase-separated condensates have a broader role as regulators of transcription.

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Fig. 1: DIGIT interacts with BRD3.
Fig. 2: DIGIT interacts with recombinant BRD3 in vitro, and bromodomains mediate the interaction between BRD3 and DIGIT.
Fig. 3: BRD3 forms protein–RNA phase-separated condensates.
Fig. 4: DIGIT promotes the formation of BRD3 phase-separated condensates.
Fig. 5: BRD3 regulates endoderm genes.
Fig. 6: BRD3 and DIGIT interact with acetylated H3K18.
Fig. 7: BRD3 interacts with DIGIT-regulated genes on H3K18ac-marked chromatin.
Fig. 8: Loss of DIGIT inhibits the recruitment of BRD3.

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Data availability

ChIP–seq and RIP–seq data generated by this study have been deposited in the GEO under the accession codes GSE126661 (BRD3 ChIP–seq) and GSE149032 (BRD3 RIP–seq). Previously published RNA-seq data that were reanalysed can be found at GSE75297. Previously published ChIP–seq data that were reanalysed can be found at GSE16256. All other data supporting the findings of this study are available from the corresponding author on reasonable request. Source data are provided with this paper.

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Acknowledgements

We thank R. Tomaino and staff at the Taplin Mass Spectrometry Core at Harvard Medical School, the MGH nextGen Sequencing Core, the MGH PMB Microscopy Core Facility and the HSCI-CRM Flow Cytometry Core Facility at MGH; T. Kitao-Ando (Gifu University) for providing consultation in the optimization of the RNA pull-down protocol; and M. Beal, J. Ryu and R. Cook of Optical Biosystems for their continuous help and collaboration with RNA detection and high-throughput microscopy. A.C.M. was supported by NIH/NICHD grant R01HD09277302 and a Pew Biomedical Scholars Award. R.E.K. was supported by NIH grants R01GM043901, R37GM048405 and R35GM131743. R.A.Y. was supported by NIH grant R01GM123511.

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Authors and Affiliations

  1. Gastrointestinal Unit, Massachusetts General Hospital, Boston, MA, USA

    Kaveh Daneshvar, Arcadia J. Kratkiewicz, Amin Mahpour, Chan Zhou, Wenyang Li, Joshua V. Pondick, Sweta K. Gupta, Sean P. Moran & Alan C. Mullen

  2. Harvard Medical School, Boston, MA, USA

    Kaveh Daneshvar, Arcadia J. Kratkiewicz, Amin Mahpour, Sabrina O. L. Cancelliere, Chan Zhou, Wenyang Li, Joshua V. Pondick, Sweta K. Gupta, Sean P. Moran & Alan C. Mullen

  3. Department of Molecular Biology and MGH Research Institute, Massachusetts General Hospital, Boston, MA, USA

    M. Behfar Ardehali, Fu-Kai Hsieh & Robert E. Kingston

  4. Department of Genetics, Harvard Medical School, Boston, MA, USA

    M. Behfar Ardehali, Fu-Kai Hsieh & Robert E. Kingston

  5. Whitehead Institute for Biomedical Research, Cambridge, MA, USA

    Isaac A. Klein & Richard A. Young

  6. Department of Medical Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA, USA

    Isaac A. Klein

  7. Harvard Stem Cell Institute, Cambridge, MA, USA

    Sabrina O. L. Cancelliere & Alan C. Mullen

  8. Optical Biosystems, Inc, Santa Clara, CA, USA

    Brett M. Cook

  9. Department of Biology, Massachusetts Institute of Technology, Cambridge, MA, USA

    Richard A. Young

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Contributions

K.D. and A.C.M. conceived the study. K.D. and A.C.M. designed the experiments with M.B.A., I.A.K. and A.M.; M.B.A. made a conceptual contribution to the functional characterization of BRD3. Computational analyses were performed by C.Z. and A.M. with help from K.D., A.J.K. and S.P.M. Bench experiments were performed by K.D., M.B.A., I.A.K, F.-K.H., A.J.K., S.O.L.C., A.M., W.L., B.M.C., S.K.G. and J.V.P. The manuscript was written by K.D. and A.C.M. with input from R.A.Y., R.E.K. and all of the other authors.

Corresponding author

Correspondence to Alan C. Mullen.

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Competing interests

A.C.M. receives research funding from Boehringer Ingelheim, Bristol-Myers Squibb, Roche Pharmaceuticals and Takeda Pharmaceuticals for unrelated projects. R.A.Y. is a founder and shareholder of Syros Pharmaceuticals, Camp4 Therapeutics, Dewpoint Therapeutics and Omega Therapeutics. I.A.K. is a consultant and a shareholder of Dewpoint Therapeutics.

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Extended data

Extended Data Fig. 1 Optimization of RNA pull-down, endoderm differentiation, and evaluation of GATA6-AS in endoderm.

a, Recovery of DIGIT-4xS1m versus endogenous GAPDH mRNA with increasing concentrations of KCl. RNA recovered with 250 mM KCl is set to 100 for each RNA species. Error bars represent the standard deviation of n = 3 independent experiments. b, Enrichment of SCRM-4xS1m after streptavidin pull-down as quantified by RT-qPCR. The transcript level in input was set to 1.0. RNA levels are normalized to GAPDH. **** indicates p < 0.0001 (Student’s t-test, n = 3 independent experiments). Box plots show the first and third quartiles, median (horizontal line), and minimum and maximum values (whiskers). c, Immunostaining shows the differentiation of hESCs to definitive endoderm (DE). FOXA2 (red) and SOX17 (green) are used as markers of definitive endoderm. SOX2 (yellow) is used as a marker of pluripotency. Hoechst staining (blue) marks nuclei. Scale bar represents 100 μm. This experiment was performed twice with similar results. d, Flow cytometry plots show the population of cells that are CXCR4 and c-KIT double-positive in hESCs and DE cells generated using the high-efficiency DE differentiation protocol54. This experiment was performed three times with similar results. e, The diagram shows the gene encoding lncRNA GATA6-AS, which is divergently transcribed from the gene encoding GATA6. Annotated GATA6-AS transcripts are shown in red and the transcript cloned in DE cells is shown in black. f, qRT-PCR shows levels of GATA6-AS in hESCs and on the indicated days of endoderm differentiation. Box plots show the first and third quartiles, median (horizontal line), and minimum and maximum values (whiskers). **** indicates p < 0.0001 compared to day 0 (One-way ANOVA, n = 4 independent experiments). Statistical source data are provided in Source data extended data Fig. 1.

Source data

Extended Data Fig. 2 RIP-seq, expression of BET proteins in endoderm, and interactions with SMARCD1.

a, RIP shows the levels of DIGIT following SMARCD1 and IgG immunoprecipitation. *** indicates p < 0.001 n = 3 independent experiments. Box plots show the first and third quartiles, median (horizontal line), and minimum and maximum values (whiskers). b, Co-immunoprecipitation followed by immunoblotting shows the interaction of BRD3 and SMARCD1. This experiment was performed twice with similar results. c, Volcano plots show transcripts enriched with BRD3 RIP. Transcripts from BRD3 RIP were normalized to IgG RIP for endoderm cells (DE, Fig. 1g) and normalized to total nuclear RNA (input), left. BRD3 RIP normalized to IgG (center) and input (right) are shown for hESCs. The 10 most enriched transcripts in common between IgG and input for hESCs are labeled on the volcano plots. Points are colored to indicate different thresholds as labeled on the far right. RIP-seq was performed in two independent samples and pooled for analysis. d, Volcano plots show lncRNA transcripts enriched with BRD3 RIP when normalized to IgG or input in DE (left) and hESCs (right). lncRNA transcripts in common between IgG and input controls are labeled (purple). Points are shaded to indicate different thresholds as in (c). RIP-seq was performed in two independent samples and pooled for analysis. e, The table (left) shows normalized expression values (RPKM) in endoderm differentiation4. Immunoblot (right) shows the protein levels of BET proteins with differentiation. Beta-Actin (ACTB) is used as a loading control. This experiment was performed twice with similar results. f, Immunoprecipitation was performed using antibodies specific to each BET protein. IgG serves as a control. This experiment was performed twice with similar results. Unprocessed blots and statistical source data are provided in Source data extended data Fig. 2.

Source data

Extended Data Fig. 3 Generation of recombinant BRD3 and GFP, production of Cy-5 labeled RNAs, EMSA replicate, localization of BRD3 domains, and immunoprecipitation of BRD3 domains.

a, Immunoblot was performed to detect recombinant FLAG-mEGFP-BRD3 (long isoform) and FLAG-mEGFP using an anti-GFP antibody (left) and an anti-BRD3 antibody (right). b, Fluorescent detection of the Cy5-labeled DIGIT and SCRM on a 1% bleach-agarose gel. Experiments in A and B were performed twice with similar results. c, Replicate electrophoretic mobility shift assay (EMSA) of in vitro transcribed DIGIT (left, top) and SCRM (left, bottom), which are labeled with Cy5 and incubated with recombinant BRD3. Migration was visualized on the right by quantifying the signal for DIGIT (top) or >SCRM (bottom) across the gel. Migration of 0 on the x-axis was defined by the peak of DIGIT and SCRM with 0 nM BRD3 protein, and migration of 100 was defined by the peak of DIGIT and SCRM with 2048 nM BRD3 protein. This experiment shows a second replicate of Fig. 2c. d, The dissociation constants (Kd) were calculated for (c). e, Fluorescence microscopy shows the nuclear localization of ectopically expressed GFP, GFP fused to full-length BRD3, and GFP fused to the indicated bromodomains (BD) or extra terminal (ET) domains of BRD3 in HEK 293 cells. Scale bar represents 20 μm. This experiment was performed twice with similar results. f, Immunoprecipitation followed by immunoblotting using an antibody against GFP shows the precipitation of the proteins described in (e). This experiment was performed twice with similar results. Unprocessed blots are provided in Source data extended data Fig. 3.

Source data

Extended Data Fig. 4 Prediction of ordered/disordered protein structures, strategy for tagging endogenous BRD3 with mEGFP, formation of BRD3 puncta in hESCs, and in vitro droplet formation.

a, PONDR VSL2 plots showing the ordered and disordered regions of the long (left) and short (middle) isoforms of BRD3, and BRD2 (right). b, Creation of mEGFP-BRD3 fusion protein. The cDNA encoding mEGFP was inserted at the N-terminus of BRD3. This terminus is shared by the long and short isoforms of BRD3. The site targeted by gRNA is indicated with an arrow. The start of the coding sequence (CDS) is indicated, and the mEGFP cDNA is inserted in frame after the start codon. c, Design of the homology vector for the insertion of mEGFP. The genomic locations of homology arms are indicated. Arrow indicates that mEGFP was inserted after the start codon for BRD3. d, Immunoblot using anti-BRD3 antibody confirms the generation of mEGFP-BRD3 fused protein. hESCs were lysed and IP was performed using an IgG isotype control and an antibody recognizing GFP. Total cell lysates (input) and the IPs were then probed with an anti-BRD3 antibody. This experiment was performed twice with similar results. e, Immunofluorescence of an hESC showing BRD3 puncta (green) in the nucleus (blue). Scale bar represents 10 ÎĽm. This experiment was performed twice with similar results. f, Droplet assay shows the formation of mEGFP-BRD3 droplets in the presence of three different molecular crowding agents. Scale bar represents 2 ÎĽm. This experiment was performed three times with similar results. g, Fluorescent detection of the Cy3-labeled DIGIT on a 1% bleach-agarose gel. This experiment was performed twice with similar results.

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Extended Data Fig. 5 Homology construct for targeting BRD3, and analysis of long and short isoforms of BRD3.

a, Map of the homology construct for insertion of a puromycin resistance cassette and a stop codon cassette downstream of the sixth codon of the gene encoding BRD3. b, hESCs were differentiated towards DE for three days prior to qRT-PCR. Primers that uniquely recognize either the long (left) or short (right) isoforms of BRD3 were used for amplification. Cells were also transfected with plasmids expressing the long (left) and short (right) isoforms of BRD3 as positive (+) controls for the primers. The long isoform of BRD3 is detected with DE differentiation while the short isoform is not detected. ACTB was used as a control. This experiment was performed twice with similar results. c, Wildtype and BRD3-/- hESCs were transfected with plasmids expressing GFP (green), the short isoform of BRD3 (yellow), and the long isoform of BRD3 (orange). Expression of CXCR4 and SOX17 was quantified by qRT-PCR after three days of DE differentiation. Ectopic expression of both the long and short isoform of BRD3 rescued expression of CXCR4 and SOX17. * indicates p < 0.05 (n = 3 independent experiments). Box plots show the first and third quartiles, median (horizontal line), and minimum and maximum values (whiskers). Statistical source data are provided in Source data extended data Fig. 5.

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Extended Data Fig. 6 Binding of BRD3 to modified histones, effects of inhibition of P300 on the enrichment of BRD3, the activity of MNase in presence of Ba+2 and Sr+2 cations, and enrichment of lncRNAs in specific histone modifications.

a, Peptide arrays show the binding of recombinant BRD3 to histone modifications. b, Spot intensity of BRD3 (long and short isoforms, top and bottom respectively) binding to the top ten histone modifications as quantified by image processing software (see Materials and Methods). c, Immunoblots to detect the levels of acetylation on H3K18 and H3K27, as well as levels of BRD3 and total H3 in hESCs treated with DMSO, A485, or GNE049. This experiment was performed twice with similar results. d, CUT&RUN followed by qPCR shows the enrichment of BRD3 at the HEXIM2 and ZFP36L1 loci in hESCs. Box plots show the first and third quartiles, median (horizontal line), and minimum and maximum values (whiskers). *** indicates p < 0.001 (Student’s t-test, n = 3 independent experiments). e, RNase activity of MNase in the presence of Ba2+ and Sr2+ cations. This experiment was performed twice with similar results. f, DNase activity of MNase in the presence of Ba2+ and Sr2+ cations. This experiment was performed twice with similar results. g, CUT&RUNER followed by qPCR shows the enrichment of MALAT1 and MEG3 transcripts in IgG control, markers of gene activation (H3K4me3 and H3K18ac) and repression (H3K27me3). Box plots show the first and third quartiles, median (horizontal line), and minimum and maximum values (whiskers). * indicates p < 0.05, *** indicates p < 0.001, and **** indicates p < 0.0001 compared to IgG (Student’s t-test, n = 3 independent experiments). Statistical source data are provided in Source data extended data Fig. 6.

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Extended Data Fig. 7 BRD3 occupies endoderm genes and enhancers and interaction between BRD3 and DIGIT is not dependent on location of transcription.

a, The number of BRD3 regions (y-axis) that contain promoters and enhancers are shown for hESCs (white) and endoderm cells (black). BRD3 regions are defined as containing promoters if the region is located within 2 kb of a transcription start site (TSS). BRD3 regions are defined as containing enhancers if the region overlaps with enhancers defined by H3K27ac. Regions that contain both promoters and enhancers are counted in both categories. In Fig. 7a, BRD3 regions are first evaluated for association with promoters. Regions that are not associated with promoters are then evaluated for association with enhancers (See Supplementary Methods). Data from two independent BRD3 ChIPs were pooled for analysis of hESCs and DE. b, Box plots show the length of regions occupied by BRD3 for the categories described in (a). The bold horizontal line represents the median for each box plot. The lower edge of the box represents the first quartile (Q1) and the upper edge of the box represents the third quartile (Q3). The lower whiskers represent Q1–1.5*(Q3-Q1) and the upper whisker represents Q3 + 1.5*(Q3-Q1). The width of each box is proportional to the number of BRD3 regions in each category. Data from two independent BRD3 ChIPs were pooled for analysis of hESCs and DE. The width of each box is proportional to the number of BRD3 regions in each category. Data from two independent BRD3 ChIPs were pooled for analysis of hESCs and DE. c, Transcript enrichment from BRD3 RIP in DE vs IgG control (Fig. 1g, y-axis) is shown for each gene occupied by BRD3 in DE (x-axis). Endoderm genes are labeled in red. Data from two independent BRD3 ChIPs and two independent BRD3 RIPs were pooled for analysis.

Extended Data Fig. 8 BRD3 occupancy at genes not regulated by DIGIT.

ChIP-seq data shows H3K18ac (top, green) and BRD3 occupancy (bottom, black) at HEXIM2, PITX2/PANCR and ZAP70 in hESCs and endoderm/mesendoderm cells. BRD3 occupies HEXIM2 in hESCs and mesendoderm/endoderm. BRD3 occupies PITX2/PANCR and ZAP70 in mesendoderm/endoderm but not in hESCs. Data from two independent BRD3 ChIPs were pooled for analysis. H3K18ac was analyzed from GSE16256.

Supplementary information

Supplementary Information

Supplementary Fig. 1

Reporting Summary

Supplementary Table 1

MS and RIP–seq analysis, related to Fig. 1.

Supplementary Table 2

Histone array data, related to Fig. 6.

Supplementary Table 3

Regions occupied by BRD3, H3K18ac and H3K27ac, related to Figs. 6 and 7.

Supplementary Table 4

Genes, transcripts and regions analysed in Fig. 7.

Supplementary Table 5

Sequences of oligonucleotides, gRNAs and RNA probes.

Supplementary Table 6

Antibody catalogue numbers and dilutions.

Source data

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Unprocessed western blots.

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Daneshvar, K., Ardehali, M.B., Klein, I.A. et al. lncRNA DIGIT and BRD3 protein form phase-separated condensates to regulate endoderm differentiation. Nat Cell Biol 22, 1211–1222 (2020). https://doi.org/10.1038/s41556-020-0572-2

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