Skip to main content

BRD2 interconnects with BRD3 to facilitate Pol II transcription initiation and elongation to prime promoters for cell differentiation

Abstract

The bromodomain and extraterminal motif (BET) proteins are critical drug targets for diseases. The precise functions and relationship of BRD2 with other BET proteins remain elusive mechanistically. Here, we used acute protein degradation and quantitative genomic and proteomic approaches to investigate the primary functions of BRD2 in transcription. We report that BRD2 is required for TAF3-mediated Pol II initiation at promoters with low levels of H3K4me3 and for R-loop suppression during Pol II elongation. Single and double depletion revealed that BRD2 and BRD3 function additively, independently, or perhaps antagonistically in Pol II transcription at different promoters. Furthermore, we found that BRD2 regulates the expression of different genes during embryonic body differentiation processes by promoter priming in embryonic stem cells. Therefore, our results suggest complex interconnections between BRD2 and BRD3 at promoters to fine-tune Pol II initiation and elongation for control of cell state.

This is a preview of subscription content, access via your institution.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7

Data availability

All sequencing datasets generated in this study, including ChIA-PET, ChIP-Seq, RNA-Seq, and 4C-Seq data, have been deposited in GEO with accession GSE160557. Mass spectrometry data can be found in the PRIDE database: PXD030774.

References

  1. Shi J, Vakoc CR (2014) The mechanisms behind the therapeutic activity of BET bromodomain inhibition. Mol Cell 54:728–736

    CAS  PubMed  Article  Google Scholar 

  2. Filippakopoulos P, Qi J, Picaud S, Shen Y, Smith WB, Fedorov O, Morse EM, Keates T, Hickman TT, Felletar I et al (2010) Selective inhibition of BET bromodomains. Nature 468:1067–1073

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  3. Zuber J, Shi J, Wang E, Rappaport AR, Herrmann H, Sison EA, Magoon D, Qi J, Blatt K, Wunderlich M et al (2011) RNAi screen identifies Brd4 as a therapeutic target in acute myeloid leukaemia. Nature 478:524–528

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  4. Nicodeme E, Jeffrey KL, Schaefer U, Beinke S, Dewell S, Chung CW, Chandwani R, Marazzi I, Wilson P, Coste H et al (2010) Suppression of inflammation by a synthetic histone mimic. Nature 468:1119–1123

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  5. Winter GE, Mayer A, Buckley DL, Erb MA, Roderick JE, Vittori S, Reyes JM, di Iulio J, Souza A, Ott CJ et al (2017) BET bromodomain proteins function as master transcription elongation factors independent of CDK9 recruitment. Mol Cell 67(5–18):e19

    Google Scholar 

  6. Yang Z, Yik JH, Chen R, He N, Jang MK, Ozato K, Zhou Q (2005) Recruitment of P-TEFb for stimulation of transcriptional elongation by the bromodomain protein Brd4. Mol Cell 19:535–545

    CAS  PubMed  Article  Google Scholar 

  7. Jang MK, Mochizuki K, Zhou M, Jeong HS, Brady JN, Ozato K (2005) The bromodomain protein Brd4 is a positive regulatory component of P-TEFb and stimulates RNA polymerase II-dependent transcription. Mol Cell 19:523–534

    CAS  PubMed  Article  Google Scholar 

  8. Zheng B, Aoi Y, Shah AP, Iwanaszko M, Das S, Rendleman EJ, Zha D, Khan N, Smith ER, Shilatifard A (2021) Acute perturbation strategies in interrogating RNA polymerase II elongation factor function in gene expression. Genes Dev 35:273–285

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  9. Stonestrom AJ, Hsu SC, Jahn KS, Huang P, Keller CA, Giardine BM, Kadauke S, Campbell AE, Evans P, Hardison RC, Blobel GA (2015) Functions of BET proteins in erythroid gene expression. Blood 125:2825–2834

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  10. Cheung KL, Zhang F, Jaganathan A, Sharma R, Zhang Q, Konuma T, Shen T, Lee JY, Ren C, Chen CH et al (2017) Distinct roles of Brd2 and Brd4 in potentiating the transcriptional program for Th17 Cell differentiation. Mol Cell 65:1068-1080.e1065

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  11. Surface LE, Fields PA, Subramanian V, Behmer R, Udeshi N, Peach SE, Carr SA, Jaffe JD, Boyer LA (2016) H2A.Z1 monoubiquitylation antagonizes BRD2 to maintain poised chromatin in ESCs. Cell Rep 14:1142–1155

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  12. Vardabasso C, Gaspar-Maia A, Hasson D, Punzeler S, Valle-Garcia D, Straub T, Keilhauer EC, Strub T, Dong J, Panda T et al (2015) Histone Variant H2A.Z.2 mediates proliferation and drug sensitivity of malignant melanoma. Mol Cell 59:75–88

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  13. Peng J, Dong W, Chen L, Zou T, Qi Y, Liu Y (2007) Brd2 is a TBP-associated protein and recruits TBP into E2F–1 transcriptional complex in response to serum stimulation. Mol Cell Biochem 294:45–54

    CAS  PubMed  Article  Google Scholar 

  14. Bagchi RA, Ferguson BS, Stratton MS, Hu T, Cavasin MA, Sun L, Lin YH, Liu D, Londono P, Song K et al (2018) HDAC11 suppresses the thermogenic program of adipose tissue via BRD2. JCI Insight. https://doi.org/10.1172/jci.insight.120159

    Article  PubMed  PubMed Central  Google Scholar 

  15. Izumikawa K, Ishikawa H, Yoshikawa H, Fujiyama S, Watanabe A, Aburatani H, Tachikawa H, Hayano T, Miura Y, Isobe T et al (2019) LYAR potentiates rRNA synthesis by recruiting BRD2/4 and the MYST-type acetyltransferase KAT7 to rDNA. Nucleic Acids Res 47:10357–10372

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  16. Denis GV, McComb ME, Faller DV, Sinha A, Romesser PB, Costello CE (2006) Identification of transcription complexes that contain the double bromodomain protein Brd2 and chromatin remodeling machines. J Proteome Res 5:502–511

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  17. Hsu SC, Gilgenast TG, Bartman CR, Edwards CR, Stonestrom AJ, Huang P, Emerson DJ, Evans P, Werner MT, Keller CA et al (2017) The BET protein BRD2 cooperates with CTCF to enforce transcriptional and architectural boundaries. Mol Cell 66(102–116):e107

    Google Scholar 

  18. Kim JJ, Lee SY, Gong F, Battenhouse AM, Boutz DR, Bashyal A, Refvik ST, Chiang CM, Xhemalce B, Paull TT et al (2019) Systematic bromodomain protein screens identify homologous recombination and R-loop suppression pathways involved in genome integrity. Genes Dev 33:1751–1774

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  19. Liu Z, Scannell DR, Eisen MB, Tjian R (2011) Control of embryonic stem cell lineage commitment by core promoter factor, TAF3. Cell 146:720–731

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  20. Wang C, Lee JE, Lai B, Macfarlan TS, Xu S, Zhuang L, Liu C, Peng W, Ge K (2016) Enhancer priming by H3K4 methyltransferase MLL4 controls cell fate transition. Proc Natl Acad Sci USA 113:11871–11876

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  21. Whyte WA, Bilodeau S, Orlando DA, Hoke HA, Frampton GM, Foster CT, Cowley SM, Young RA (2012) Enhancer decommissioning by LSD1 during embryonic stem cell differentiation. Nature 482:221–225

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  22. Lin C, Garruss AS, Luo Z, Guo F, Shilatifard A (2013) The RNA Pol II elongation factor Ell3 marks enhancers in ES cells and primes future gene activation. Cell 152:144–156

    CAS  PubMed  Article  Google Scholar 

  23. Pal DK, Evgrafov OV, Tabares P, Zhang F, Durner M, Greenberg DA (2003) BRD2 (RING3) is a probable major susceptibility gene for common juvenile myoclonic epilepsy. Am J Hum Genet 73:261–270

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  24. Velíšek L, Shang E, Velíšková J, Chachua T, Macchiarulo S, Maglakelidze G, Wolgemuth DJ, Greenberg DA (2011) GABAergic neuron deficit as an idiopathic generalized epilepsy mechanism: the role of BRD2 haploinsufficiency in juvenile myoclonic epilepsy. PLoS ONE 6:e23656

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  25. Shang E, Wang X, Wen D, Greenberg DA, Wolgemuth DJ (2009) Double bromodomain-containing gene Brd2 is essential for embryonic development in mouse. Dev Dyn 238:908–917

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  26. Gyuris A, Donovan DJ, Seymour KA, Lovasco LA, Smilowitz NR, Halperin AL, Klysik JE, Freiman RN (2009) The chromatin-targeting protein Brd2 is required for neural tube closure and embryogenesis. Biochim Biophys Acta 1789:413–421

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  27. Wang F, Liu H, Blanton WP, Belkina A, Lebrasseur NK, Denis GV (2009) Brd2 disruption in mice causes severe obesity without Type 2 diabetes. Biochem J 425:71–83

    PubMed  Article  CAS  Google Scholar 

  28. Jiang Y, Huang J, Lun K, Li B, Zheng H, Li Y, Zhou R, Duan W, Wang C, Feng Y et al (2020) Genome-wide analyses of chromatin interactions after the loss of Pol I, Pol II, and Pol III. Genome Biol 21:158

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  29. Zhang J, Liu Z, Jia J (2021) Mechanisms of smoothened regulation in hedgehog signaling. Cells 10:2138

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  30. Cheng ZY, He TT, Gao XM, Zhao Y, Wang J (2021) ZBTB transcription factors: key regulators of the development, differentiation and effector function of T cells. Front Immunol 12:713294

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  31. Edwards DS, Maganti R, Tanksley JP, Luo J, Park JJH, Balkanska-Sinclair E, Ling J, Floyd SR (2020) BRD4 prevents R-loop formation and transcription-replication conflicts by ensuring efficient transcription elongation. Cell Rep 32:108166

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  32. Chakraborty P, Huang JTJ, Hiom K (2018) DHX9 helicase promotes R-loop formation in cells with impaired RNA splicing. Nat Commun 9:4346

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  33. Brannan K, Kim H, Erickson B, Glover-Cutter K, Kim S, Fong N, Kiemele L, Hansen K, Davis R, Lykke-Andersen J, Bentley DL (2012) mRNA decapping factors and the exonuclease Xrn2 function in widespread premature termination of RNA polymerase II transcription. Mol Cell 46:311–324

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  34. Lauberth SM, Nakayama T, Wu X, Ferris AL, Tang Z, Hughes SH, Roeder RG (2013) H3K4me3 interactions with TAF3 regulate preinitiation complex assembly and selective gene activation. Cell 152:1021–1036

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  35. Vermeulen M, Mulder KW, Denissov S, Pijnappel WW, van Schaik FM, Varier RA, Baltissen MP, Stunnenberg HG, Mann M, Timmers HT (2007) Selective anchoring of TFIID to nucleosomes by trimethylation of histone H3 lysine 4. Cell 131:58–69

    CAS  PubMed  Article  Google Scholar 

  36. Lambert JP, Picaud S, Fujisawa T, Hou H, Savitsky P, Uusküla-Reimand L, Gupta GD, Abdouni H, Lin ZY, Tucholska M et al (2019) Interactome rewiring following pharmacological targeting of BET bromodomains. Mol Cell 73:621-638.e617

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  37. LeRoy G, Rickards B, Flint SJ (2008) The double bromodomain proteins Brd2 and Brd3 couple histone acetylation to transcription. Mol Cell 30:51–60

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  38. Donczew R, Hahn S (2021) BET family members Bdf1/2 modulate global transcription initiation and elongation in Saccharomyces cerevisiae. Elife. https://doi.org/10.7554/eLife.69619

    Article  PubMed  PubMed Central  Google Scholar 

  39. Boija A, Klein IA, Young RA (2021) Biomolecular condensates and cancer. Cancer Cell 39:174–192

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  40. Sabari BR, Dall’Agnese A, Boija A, Klein IA, Coffey EL, Shrinivas K, Abraham BJ, Hannett NM, Zamudio AV, Manteiga JC et al (2018) Coactivator condensation at super-enhancers links phase separation and gene control. Science. https://doi.org/10.1126/science.aar3958

    Article  PubMed  PubMed Central  Google Scholar 

  41. Daneshvar K, Ardehali MB, Klein IA, Hsieh FK, Kratkiewicz AJ, Mahpour A, Cancelliere SOL, Zhou C, Cook BM, Li W et al (2020) lncRNA DIGIT and BRD3 protein form phase-separated condensates to regulate endoderm differentiation. Nat Cell Biol 22:1211–1222

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  42. Weintraub AS, Li CH, Zamudio AV, Sigova AA, Hannett NM, Day DS, Abraham BJ, Cohen MA, Nabet B, Buckley DL et al (2017) YY1 is a structural regulator of enhancer-promoter loops. Cell 171(1573–1588):e1528

    Google Scholar 

  43. Kieffer-Kwon KR, Tang Z, Mathe E, Qian J, Sung MH, Li G, Resch W, Baek S, Pruett N, Grontved L et al (2013) Interactome maps of mouse gene regulatory domains reveal basic principles of transcriptional regulation. Cell 155:1507–1520

    CAS  PubMed  Article  Google Scholar 

  44. Dowen JM, Fan ZP, Hnisz D, Ren G, Abraham BJ, Zhang LN, Weintraub AS, Schujiers J, Lee TI, Zhao K, Young RA (2014) Control of cell identity genes occurs in insulated neighborhoods in mammalian chromosomes. Cell 159:374–387

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  45. Wang C, Lee JE, Cho YW, Xiao Y, Jin Q, Liu C, Ge K (2012) UTX regulates mesoderm differentiation of embryonic stem cells independent of H3K27 demethylase activity. Proc Natl Acad Sci USA 109:15324–15329

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  46. Berwick DC, Harvey K (2012) LRRK2 functions as a Wnt signaling scaffold, bridging cytosolic proteins and membrane-localized LRP6. Hum Mol Genet 21:4966–4979

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  47. Tokue M, Ikami K, Mizuno S, Takagi C, Miyagi A, Takada R, Noda C, Kitadate Y, Hara K, Mizuguchi H et al (2017) SHISA6 confers resistance to differentiation-promoting Wnt/β-catenin signaling in mouse spermatogenic stem cells. Stem Cell Reports 8:561–575

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  48. Andrieu GP, Denis GV (2018) BET proteins exhibit transcriptional and functional opposition in the epithelial-to-mesenchymal transition. Mol Cancer Res 16:580–586

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  49. Branigan GL, Olsen KS, Burda I, Haemmerle MW, Ho J, Venuto A, D’Antonio ND, Briggs IE, DiBenedetto AJ (2021) Zebrafish paralogs brd2a and brd2b are needed for proper circulatory, excretory and central nervous system formation and act as genetic antagonists during development. J Dev Biol. https://doi.org/10.3390/jdb9040046

    Article  PubMed  PubMed Central  Google Scholar 

  50. Wu SY, Chiang CM (2007) The double bromodomain-containing chromatin adaptor Brd4 and transcriptional regulation. J Biol Chem 282:13141–13145

    CAS  PubMed  Article  Google Scholar 

  51. Slaughter MJ, Shanle EK, Khan A, Chua KF, Hong T, Boxer LD, Allis CD, Josefowicz SZ, Garcia BA, Rothbart SB et al (2021) HDAC inhibition results in widespread alteration of the histone acetylation landscape and BRD4 targeting to gene bodies. Cell Rep 34:108638

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  52. Asangani IA, Dommeti VL, Wang X, Malik R, Cieslik M, Yang R, Escara-Wilke J, Wilder-Romans K, Dhanireddy S, Engelke C et al (2014) Therapeutic targeting of BET bromodomain proteins in castration-resistant prostate cancer. Nature 510:278–282

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  53. LeRoy G, Chepelev I, DiMaggio PA, Blanco MA, Zee BM, Zhao K, Garcia BA (2012) Proteogenomic characterization and mapping of nucleosomes decoded by Brd and HP1 proteins. Genome Biol 13:R68

    PubMed  PubMed Central  Article  Google Scholar 

  54. Anders L, Guenther MG, Qi J, Fan ZP, Marineau JJ, Rahl PB, Loven J, Sigova AA, Smith WB, Lee TI et al (2014) Genome-wide localization of small molecules. Nat Biotechnol 32:92–96

    CAS  PubMed  Article  Google Scholar 

  55. Warfield L, Ramachandran S, Baptista T, Devys D, Tora L, Hahn S (2017) Transcription of nearly all yeast RNA polymerase II-transcribed genes is dependent on transcription factor TFIID. Mol Cell 68(118–129):e115

    Google Scholar 

  56. Mylonas C, Lee C, Auld AL, Cisse II, Boyer LA (2021) 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

    CAS  PubMed  Article  Google Scholar 

  57. Wen Z, Zhang L, Ruan H, Li G (2020) Histone variant H2A.Z regulates nucleosome unwrapping and CTCF binding in mouse ES cells. Nucleic Acids Res 48:5939–5952

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  58. Bi M, Zhang Z, Jiang YZ, Xue P, Wang H, Lai Z, Fu X, De Angelis C, Gong Y, Gao Z et al (2020) Enhancer reprogramming driven by high-order assemblies of transcription factors promotes phenotypic plasticity and breast cancer endocrine resistance. Nat Cell Biol 22:701–715

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  59. Zhu C, Li L, Zhang Z, Bi M, Wang H, Su W, Hernandez K, Liu P, Chen J, Chen M et al (2019) A non-canonical role of YAP/TEAD is required for activation of estrogen-regulated enhancers in breast cancer. Mol Cell 75(791–806):e798

    Google Scholar 

  60. Jang Y, Park YK, Lee JE, Wan D, Tran N, Gavrilova O, Ge K (2021) MED1 is a lipogenesis coactivator required for postnatal adipose expansion. Genes Dev 35:713–728

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  61. Yang Y, Zhang L, Xiong C, Chen J, Wang L, Wen Z, Yu J, Chen P, Xu Y, Jin J et al (2021) HIRA complex presets transcriptional potential through coordinating depositions of the histone variants H3.3 and H2A.Z on the poised genes in mESCs. Nucleic Acids Res. https://doi.org/10.1093/nar/gkab1221

    Article  PubMed  PubMed Central  Google Scholar 

  62. Park YK, Lee JE, Yan Z, McKernan K, O’Haren T, Wang W, Peng W, Ge K (2021) Interplay of BAF and MLL4 promotes cell type-specific enhancer activation. Nat Commun 12:1630

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  63. Lai B, Lee JE, Jang Y, Wang L, Peng W, Ge K (2017) MLL3/MLL4 are required for CBP/p300 binding on enhancers and super-enhancer formation in brown adipogenesis. Nucleic Acids Res 45:6388–6403

    CAS  PubMed  PubMed Central  Article  Google Scholar 

Download references

Acknowledgements

We thank the members of the Ji laboratory for engaging in helpful discussions. We thank Dr. Richard Young for assistance during the beginning of the studies. We thank Dr. Mario Garcia-Dominguez for the BRD2 wild-type and mutant constructs. We thank the National Center for Protein Sciences at Peking University in Beijing, China, for assistance with high-resolution fluorescence imaging and LYD for help with flow cytometry sorting, DL for help with mass spectrometry detection, and CYS and HXL for help with cell imaging. We also thank Eric Spooner at the Whitehead Proteomics Core for mass spectrometry and Tom Volkert at the Whitehead Genome Technology Core for sequencing.

Funding

This work was supported by funds from the Ministry of Science and Technology of China and the National Natural Science Foundation of China (Grants 2017YFA0506600, 31871309 and 32170569, respectively), the Qidong-SLS Innovation Fund, and grants from the Peking-Tsinghua Center for Life Sciences and the Key Laboratory of Cell Proliferation and Differentiation of the Ministry of Education at Peking University School of Life Sciences to X. J. In addition, J.H. is supported in part by the China Postdoctoral Science Foundation (2017M610700). L.C. is supported by Ministry of Science and Technology of China and the National Natural Science Foundation of China (Grants 2021YFA1100500, 32171289).

Author information

Authors and Affiliations

Authors

Contributions

XJ conceived and supervised the project. CLW generated the degron mES cells and performed BRD2 degradation-related Western blots, 4C-Seq, ChIP-Seq/qPCR, RNA-Seq, differentiation-related analyses, and mass spectrometry analyses. QQX performed most of the analyses, including ChIP-Seq, RNA-Seq-related analyses and 4C-Seq, R-Loop, and conservation analyses. JH initially performed BRD2 ChIP-Seq and expression analyses during the embryonic body differentiation process. XHZ and LC generated the R-loop sequencing data. BA and DD helped with the bioinformatic analyses at the initial stage of the project. XJ performed BRD2 ChIA-PET experiments. All authors contributed to the data analyses and data interpretation. XJ wrote the manuscript with input from JH, CLW, QQX and help from the other authors.

Corresponding authors

Correspondence to Jie Huang or Xiong Ji.

Ethics declarations

Conflict of interest

The authors declare no conflicts of interest.

Additional information

Publisher's Note

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

Supplementary Information

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Wang, C., Xu, Q., Zhang, X. et al. BRD2 interconnects with BRD3 to facilitate Pol II transcription initiation and elongation to prime promoters for cell differentiation. Cell. Mol. Life Sci. 79, 338 (2022). https://doi.org/10.1007/s00018-022-04349-4

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1007/s00018-022-04349-4

Keywords

  • BET proteins
  • RNA Pol II
  • Cross-regulation
  • Embryonic body