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Cited by 82Genomics and Chromatin DynamicsChromatin Remodeling and CancerEpigenetics and DNA Methylation

Cooperative binding between distant transcription factors is a hallmark of active enhancers

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Rao, Satyanarayan, Ahmad, Kami, Ramachandran, Srinivas·2021·Molecular Cell
DOI 10.1016/j.molcel.2021.02.014PMC PMC8052300

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Abstract

Enhancers harbor binding motifs that recruit transcription factors (TFs) for gene activation. While cooperative binding of TFs at enhancers is known to be critical for transcriptional activation of a handful of developmental enhancers, the extent of TF cooperativity genome-wide is unknown. Here, we couple high-resolution nuclease footprinting with single-molecule methylation profiling to characterize TF cooperativity at active enhancers in the Drosophila genome. Enrichment of short micrococcal nuclease (MNase)-protected DNA segments indicates that the majority of enhancers harbor two or more TF-binding sites, and we uncover protected fragments that correspond to co-bound sites in thousands of enhancers. From the analysis of co-binding, we find that cooperativity dominates TF binding in vivo at the majority of active enhancers. Cooperativity is highest between sites spaced 50 bp apart, indicating that cooperativity occurs without apparent protein-protein interactions. Our findings suggest nucleosomes promoting cooperativity because co-binding may effectively clear nucleosomes and promote enhancer function.

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  • Single molecule footprinting showed high transcription factor cooperativity at active enhancers and identified cases where transcription factors bind independently, sequentially or simultaneously.

    “SMF showed high TF cooperativity at active enhancers and identified cases where TFs bind independently, sequentially or simultaneously [118].”

    From Transcriptional enhancers and their communication with gene promoters (2021) · cites this paper as [118]

    The Discussion (page unavailable in our copy) reports substantial TF cooperativity at 64% of active fly enhancers and independent binding at some others. The Results section, “Cooperative binding is common at active enhancers” (page unavailable), identifies TFs co-bound on single molecules and potential initiating sites. These observations support co-occupancy and a possible binding hierarchy, but do not establish that TFs bind sequentially or simultaneously in time. (LLM, conf=0.86)

    Experimental — accuracy not yet measured

  • Co-occupied DNA-bound transcription factors in Drosophila were often spaced quite widely apart, more than 50 bp.

    “Interestingly, at non-dimeric sites, physical interactions between co-bound TFs were not necessary [119], consistent with evidence from Drosophila where co-occupied DNA-bound TFs were often spaced quite widely apart (> 50 bp) [118].”

    From Transcriptional enhancers and their communication with gene promoters (2021) · cites this paper as [118]

    The Results section, “Cooperative binding is common at active enhancers,” reports co-binding across spacings of 50–90 bp and significant cooperativity at distances up to 140 bp, supporting the point that Drosophila TFs can co-occupy widely separated sites. But “Many active enhancers contain multiple bound TFs” says ~50 bp was the most observed spacing; the copy does not establish that co-occupied factors were often more than 50 bp apart. (LLM, conf=0.83)

    Experimental — accuracy not yet measured

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Research works printed but not linked: 54

  1. Adams CC, and Workman JL (1995). Binding of disparate transcriptional activators to nucleosomal DNA is inherently cooperative. Mol Cell Biol 15, 1405– 1421. 7862134 PMC230365
  2. Ahmad K, and Spens AE (2019). Separate Polycomb Response Elements control chromatin state and activation of the vestigial gene. PLoS Genet 15, e1007877. 31425502 PMC6730940
  3. Andersson R, and Sandelin A (2020). Determinants of enhancer and promoter activities of regulatory elements. Nat Rev Genet 21, 71– 87. 31605096
  4. Arnosti DN, and Kulkarni MM (2005). Transcriptional enhancers: Intelligent enhanceosomes or flexible billboards? J Cell Biochem 94, 890– 898. 15696541
  5. Banerji J, Rusconi S, and Schaffner W (1981). Expression of a beta-globin gene is enhanced by remote SV40 DNA sequences. Cell 27, 299– 308. 6277502
  6. Bintu L, Buchler NE, Garcia HG, Gerland U, Hwa T, Kondev J, and Phillips R (2005). Transcriptional regulation by the numbers: models. Curr Opin Genet Dev 15, 116– 124. 15797194 PMC3482385
  7. Cherbas L, Willingham A, Zhang D, Yang L, Zou Y, Eads BD, Carlson JW, Landolin JM, Kapranov P, Dumais J, (2011). The transcriptional diversity of 25 Drosophila cell lines. Genome Res 21, 301– 314. 21177962 PMC3032933
  8. Dunipace L, Ozdemir A, and Stathopoulos A (2011). Complex interactions between cis-regulatory modules in native conformation are critical for Drosophila snail expression. Development 138, 4075– 4084. 21813571 PMC3160101
  9. Enuameh MS, Asriyan Y, Richards A, Christensen RG, Hall VL, Kazemian M, Zhu C, Pham H, Cheng Q, Blatti C, (2013). Global analysis of Drosophila Cys(2)-His(2) zinc finger proteins reveals a multitude of novel recognition motifs and binding determinants. Genome Res 23, 928– 940. 23471540 PMC3668361
  10. Fornes O, Castro-Mondragon JA, Khan A, van der Lee R, Zhang X, Richmond PA, Modi BP, Correard S, Gheorghe M, Baranasic D, (2020). JASPAR 2020: update of the open-access database of transcription factor binding profiles. Nucleic Acids Res 48, D87– D92. 31701148 PMC7145627
  11. Gordan R, Shen N, Dror I, Zhou T, Horton J, Rohs R, and Bulyk ML (2013). Genomic regions flanking E-box binding sites influence DNA binding specificity of bHLH transcription factors through DNA shape. Cell Rep 3, 1093– 1104. 23562153 PMC3640701
  12. Grant CE, Bailey TL, and Noble WS (2011). FIMO: scanning for occurrences of a given motif. Bioinformatics 27, 1017– 1018. 21330290 PMC3065696
  13. Gupta S, Stamatoyannopoulos JA, Bailey TL, and Noble WS (2007). Quantifying similarity between motifs. Genome Biol 8, R24. 17324271 PMC1852410
  14. Haruki H, Nishikawa J, and Laemmli UK (2008). The anchor-away technique: rapid, conditional establishment of yeast mutant phenotypes. Mol Cell 31, 925– 932. 18922474
  15. He Q, Johnston J, and Zeitlinger J (2015). ChIP-nexus enables improved detection of in vivo transcription factor binding footprints. Nat Biotechnol 33, 395– 401. 25751057 PMC4390430
  16. Henikoff JG, Belsky JA, Krassovsky K, MacAlpine DM, and Henikoff S (2011). Epigenome characterization at single base-pair resolution. Proc Natl Acad Sci U S A 108, 18318– 18323. 22025700 PMC3215028
  17. Henikoff S, Henikoff JG, Sakai A, Loeb GB, and Ahmad K (2009). Genome-wide profiling of salt fractions maps physical properties of chromatin. Genome Res 19, 460– 469. 19088306 PMC2661814
  18. Inukai S, Kock KH, and Bulyk ML (2017). Transcription factor-DNA binding: beyond binding site motifs. Curr Opin Genet Dev 43, 110– 119. 28359978 PMC5447501
  19. Iwafuchi-Doi M, and Zaret KS (2014). Pioneer transcription factors in cell reprogramming. Genes Dev 28, 2679– 2692. 25512556 PMC4265672
  20. Jessen WJ, Dhasarathy A, Hoose SA, Carvin CD, Risinger AL, and Kladde MP (2004). Mapping chromatin structure in vivo using DNA methyltransferases. Methods 33, 68– 80. 15039089
  21. Joshi R, Passner JM, Rohs R, Jain R, Sosinsky A, Crickmore MA, Jacob V, Aggarwal AK, Honig B, and Mann RS (2007). Functional specificity of a Hox protein mediated by the recognition of minor groove structure. Cell 131, 530– 543. 17981120 PMC2709780
  22. Kasinathan S, Orsi GA, Zentner GE, Ahmad K, and Henikoff S (2014). High-resolution mapping of transcription factor binding sites on native chromatin. Nat Methods 11, 203– 209. 24336359 PMC3929178
  23. Kelly TK, Liu Y, Lay FD, Liang G, Berman BP, and Jones PA (2012). Genome-wide mapping of nucleosome positioning and DNA methylation within individual DNA molecules. Genome Res 22, 2497– 2506. 22960375 PMC3514679
  24. Krassovsky K, Henikoff JG, and Henikoff S (2012). Tripartite organization of centromeric chromatin in budding yeast. Proc Natl Acad Sci U S A 109, 243– 248. 22184235 PMC3252899
  25. Krebs AR, Imanci D, Hoerner L, Gaidatzis D, Burger L, and Schübeler D (2017). Genome-wide Single-Molecule Footprinting Reveals High RNA Polymerase II Turnover at Paused Promoters. Mol Cell 67, 411– 422.e414. 28735898 PMC5548954
  26. Krueger F, and Andrews SR (2011). Bismark: a flexible aligner and methylation caller for Bisulfite-Seq applications. Bioinformatics 27, 1571– 1572. 21493656 PMC3102221
  27. Lagha M, Bothma JP, and Levine M (2012). Mechanisms of transcriptional precision in animal development. Trends Genet 28, 409– 416. 22513408 PMC4257495
  28. Levine M (2010). Transcriptional enhancers in animal development and evolution. Curr Biol 20, R754– 763. 20833320 PMC4280268
  29. Ludwig MZ, Manu, Kittler R, White KP, and Kreitman M (2011). Consequences of eukaryotic enhancer architecture for gene expression dynamics, development, and fitness. PLoS Genet 7, e1002364. 22102826 PMC3213169
  30. Mann RS, and Affolter M (1998). Hox proteins meet more partners. Curr Opin Genet Dev 8, 423– 429. 9729718
  31. Melnikova L, Juge F, Gruzdeva N, Mazur A, Cavalli G, and Georgiev P (2004). Interaction between the GAGA factor and Mod(mdg4) proteins promotes insulator bypass in Drosophila. Proc Natl Acad Sci U S A 101, 14806– 14811. 15465920 PMC522021
  32. Mirny LA (2010). Nucleosome-mediated cooperativity between transcription factors. Proc Natl Acad Sci U S A 107, 22534– 22539. 21149679 PMC3012490
  33. Morgunova E, and Taipale J (2017). Structural perspective of cooperative transcription factor binding. Curr Opin Struct Biol 47, 1– 8. 28349863
  34. Moyle-Heyrman G, Tims HS, and Widom J (2011). Structural constraints in collaborative competition of transcription factors against the nucleosome. J Mol Biol 412, 634– 646. 21821044 PMC3534743
  35. Mueller B, Mieczkowski J, Kundu S, Wang P, Sadreyev R, Tolstorukov MY, and Kingston RE (2017). Widespread changes in nucleosome accessibility without changes in nucleosome occupancy during a rapid transcriptional induction. Genes Dev 31, 451– 462. 28356342 PMC5393060
  36. Paakinaho V, Presman DM, Ball DA, Johnson TA, Schiltz RL, Levitt P, Mazza D, Morisaki T, Karpova TS, and Hager GL (2017). Single-molecule analysis of steroid receptor and cofactor action in living cells. Nat Commun 8, 15896. 28635963 PMC5482060
  37. Polach KJ, and Widom J (1996). A model for the cooperative binding of eukaryotic regulatory proteins to nucleosomal target sites. J Mol Biol 258, 800– 812. 8637011
  38. Ramachandran S, Ahmad K, and Henikoff S (2017). Transcription and Remodeling Produce Asymmetrically Unwrapped Nucleosomal Intermediates. Mol Cell 68, 1038– 1053 e1034. 29225036 PMC6421108
  39. Ramachandran S, and Henikoff S (2016). Transcriptional Regulators Compete with Nucleosomes Post-replication. Cell 165, 580– 592. 27062929 PMC4855302
  40. Rastogi C, Rube HT, Kribelbauer JF, Crocker J, Loker RE, Martini GD, Laptenko O, Freed-Pastor WA, Prives C, Stern DL, (2018). Accurate and sensitive quantification of protein-DNA binding affinity. Proc Natl Acad Sci U S A 115, E3692– e3701. 29610332 PMC5910815
  41. Ray-Gallet D, Woolfe A, Vassias I, Pellentz C, Lacoste N, Puri A, Schultz DC, Pchelintsev NA, Adams PD, Jansen LE, (2011). Dynamics of histone H3 deposition in vivo reveal a nucleosome gap-filling mechanism for H3.3 to maintain chromatin integrity. Mol Cell 44, 928– 941. 22195966
  42. Reiter F, Wienerroither S, and Stark A (2017). Combinatorial function of transcription factors and cofactors. Curr Opin Genet Dev 43, 73– 81. 28110180
  43. Rhee HS, and Pugh BF (2011). Comprehensive genome-wide protein-DNA interactions detected at single-nucleotide resolution. Cell 147, 1408– 1419. 22153082 PMC3243364
  44. Schones DE, Cui K, Cuddapah S, Roh TY, Barski A, Wang Z, Wei G, and Zhao K (2008). Dynamic regulation of nucleosome positioning in the human genome. Cell 132, 887– 898. 18329373 PMC10894452
  45. Skene PJ, and Henikoff S (2017). An efficient targeted nuclease strategy for high-resolution mapping of DNA binding sites. Elife 6. PMC5310842 28079019
  46. Slattery M, Riley T, Liu P, Abe N, Gomez-Alcala P, Dror I, Zhou T, Rohs R, Honig B, Bussemaker HJ, (2011). Cofactor binding evokes latent differences in DNA binding specificity between Hox proteins. Cell 147, 1270– 1282. 22153072 PMC3319069
  47. Sonmezer C, Kleinendorst R, Imanci D, Villacorta L, Schubeler D, Benes V, and Krebs AR (2020). Single molecule occupancy patterns of transcription factors reveal determinants of cooperative binding in vivo. bioRxiv.
  48. Spitz F, and Furlong EE (2012). Transcription factors: from enhancer binding to developmental control. Nat Rev Genet 13, 613– 626. 22868264
  49. Stergachis AB, Debo BM, Haugen E, Churchman LS, and Stamatoyannopoulos JA (2020). Single-molecule regulatory architectures captured by chromatin fiber sequencing. Science 368, 1449– 1454. 32587015
  50. Teves SS, and Henikoff S (2011). Heat shock reduces stalled RNA polymerase II and nucleosome turnover genome-wide. Genes Dev 25, 2387– 2397. 22085965 PMC3222904
  51. von Hippel PH, and Berg OG (1986). On the specificity of DNA-protein interactions. Proc Natl Acad Sci U S A 83, 1608– 1612. 3456604 PMC323132
  52. Wilczynski B, and Furlong EE (2010). Dynamic CRM occupancy reflects a temporal map of developmental progression. Mol Syst Biol 6, 383. 20571532 PMC2913398
  53. Yanez-Cuna JO, Arnold CD, Stampfel G, Boryn LM, Gerlach D, Rath M, and Stark A (2014). Dissection of thousands of cell type-specific enhancers identifies dinucleotide repeat motifs as general enhancer features. Genome Res 24, 1147– 1156. 24714811 PMC4079970
  54. Zentner GE, and Henikoff S (2013). Mot1 redistributes TBP from TATA-containing to TATA-less promoters. Mol Cell Biol 33, 4996– 5004. 24144978 PMC3889552

Cited by

Sorted by citations

Authors
Rao, S., Ahmad, K., & Ramachandran, S.
Affiliations
  • University of Colorado Anschutz (US)
  • Fred Hutch Cancer Center (US)
Corresponding author
Ramachandran, Srinivas
ORCID
Venue
Molecular Cell
Publisher
Elsevier BV
Published
2021-04
Volume
81
Issue
8
Pages
1651-1665.e4
ISSN
1097-2765 (ISSN-L)
DOI
10.1016/j.molcel.2021.02.014
PMC
PMC8052300
OpenAlex
W3135190230
Cited by
82
References
6
Citations by year
2026: 14 · 2025: 15 · 2024: 18 · 2023: 13 · 2022: 15 · 2021: 7
FWCI
4.71
Type
journal article
Language
en
Open access
Licence
Locations
  • Molecular Cell (publishedVersion)
  • bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion, cc-by-nc-nd)
  • SSRN Electronic Journal (acceptedVersion)
  • PubMed (publishedVersion)
  • PubMed Central (acceptedVersion)
Funders
NIGMS
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Genomics and Chromatin Dynamics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Chromatin Remodeling and Cancer
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Epigenetics and DNA Methylation
Keywords
transcription factor cooperativity, enhancers, DNA methylation profiling, Drosophila, transcriptional activation, enhancer function
MeSH terms
Animals, Binding Sites, Cell Line, Drosophila, Enhancer Elements, Genetic, Micrococcal Nuclease, Nucleosomes, Protein Binding, Transcription Factors, Transcriptional Activation, Genome, Protein Interaction Maps

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