Genome-wide investigation of transcription factor footprints and dynamics using cFOOT-seq

Abstract

Gene regulation relies on the precise binding of transcription factors (TFs) at regulatory elements, but simultaneously detecting hundreds of TFs on chromatin is challenging. We developed cFOOT-seq, a cytosine deaminase-based TF footprinting assay, for high-resolution, quantitative genome-wide assessment of TF binding in both open and closed chromatin regions, even with small cell numbers. By utilizing the dsDNA deaminase SsdA_tox, cFOOT-seq converts accessible cytosines to uracil while preserving genomic integrity, making it compatible with techniques like ATAC-seq for sensitive and cost-effective detection of TF occupancy at the single-molecule and single-cell level. Our approach enables the delineation of TF footprints, quantification of occupancy, and examination of chromatin influences on TF binding. Notably, cFOOT-seq, combined with FootTrack analysis, enables de novo prediction of TF binding sites and tracking of TF occupancy dynamics. We demonstrate its application in capturing cell type-specific TFs, analyzing TF dynamics during reprogramming, and revealing TF dependencies on chromatin remodelers. Overall, cFOOT-seq represents a robust approach for investigating the genome-wide dynamics of TF occupancy and elucidating the cis-regulatory architecture underlying gene regulation.

Keywords

gene regulation / transcription factor / TF footprint / chromatin landscape / chromatin accessibility / nucleosome position / chromatin remodeling

References

[1] Ahmad K , Brahma S , Henikoff S . Epigenetic pioneering by SWI/SNF family remodelers. Mol Cell 2024; 84: 194- 201.

[2] Ai S , Xiong H , Li CC et al. Profiling chromatin states using single-cell itChIP-seq. Nat Cell Biol 2019; 21: 1164- 1172.

[3] Altemose N , Maslan A , Smith OK et al. DiMeLo-seq: a long-read, single-molecule method for mapping protein-DNA interactions genome wide. Nat Methods 2022; 19: 711- 723.

[4] Alver BH , Kim KH , Lu P et al. The SWI/SNF chromatin remodelling complex is required for maintenance of lineage specific enhancers. Nat Commun 2017; 8: 14648.

[5] Barisic D , Stadler MB , Iurlaro M et al. Mammalian ISWI and SWI/SNF selectively mediate binding of distinct transcription factors. Nature 2019; 569: 136- 140.

[6] Bartosovic M , Castelo-Branco G . Multimodal chromatin profiling using nanobody-based single-cell CUT&Tag. Nat Biotechnol 2023; 41: 794- 805.

[7] Bartosovic M , Kabbe M , Castelo-Branco G . Single-cell CUT&Tag profiles histone modifications and transcription factors in complex tissues. Nat Biotechnol 2021; 39: 825- 835.

[8] Basurto-Cayuela L , Guerrero-Martinez JA , Gomez-Marin E et al. SWI/SNF-dependent genes are defined by their chromatin landscape. Cell Rep 2024; 43: 113855.

[9] Becker PB , Workman JL . Nucleosome remodeling and epigenetics. Cold Spring Harb Perspect Biol 2013; 5: a017905.

[10] Bentsen M , Goymann P , Schultheis H et al. ATAC-seq footprinting unravels kinetics of transcription factor binding during zygotic genome activation. Nat Commun 2020; 11: 4267.

[11] Bentsen M , Heger V , Schultheis H et al. TF-COMB-discovering grammar of transcription factor binding sites. Comput Struct Biotechnol J 2022; 20: 4040- 4051.

[12] Bomber ML , Wang J , Liu Q et al. Human SMARCA5 is continuously required to maintain nucleosome spacing. Mol Cell 2023; 83: 507- 522.e6.

[13] Bracken AP , Brien GL , Verrijzer CP . Dangerous liaisons: interplay between SWI/SNF, NuRD, and Polycomb in chromatin regulation and cancer. Genes Dev 2019; 33: 936- 959.

[14] Brahma S , Henikoff S . The BAF chromatin remodeler synergizes with RNA polymerase II and transcription factors to evict nucleosomes. Nat Genet 2024; 56: 100- 111.

[15] Buenrostro JD , Giresi PG , Zaba LC et al. Transposition of native chromatin for fast and sensitive epigenomic profiling of open chromatin, DNA-binding proteins and nucleosome position. Nat Methods 2013; 10: 1213- 1218.

[16] Cai Y , Jin J , Yao T et al. YY1 functions with INO80 to activate transcription. Nat Struct Mol Biol 2007; 14: 872- 874.

[17] Carter B , Ku WL , Kang JY et al. Mapping histone modifications in low cell number and single cells using antibody-guided chromatin tagmentation (ACT-seq). Nat Commun 2019; 10: 3747.

[18] Cenik BK , Shilatifard A . COMPASS and SWI/SNF complexes in development and disease. Nat Rev Genet 2021; 22: 38- 58.

[19] Chronis C , Fiziev P , Papp B et al. Cooperative binding of transcription factors orchestrates reprogramming. Cell 2017; 168: 442- 459.e20.

[20] Clapier CR , Iwasa J , Cairns BR et al. Mechanisms of action and regulation of ATP-dependent chromatin-remodelling complexes. Nat Rev Mol Cell Biol 2017; 18: 407- 422.

[21] De Boer CG , Taipale J . Hold out the genome: a roadmap to solving the cis-regulatory code. Nature 2024; 625: 41- 50.

[22] De Moraes MH , Hsu F , Huang D et al. An interbacterial DNA deaminase toxin directly mutagenizes surviving target populations. eLife 2021; 10: e62967.

[23] Eraslan G , Avsec Z , Gagneur J et al. Deep learning: new computational modelling techniques for genomics. Nat Rev Genet 2019; 20: 389- 403.

[24] Ernst J , Kellis M . ChromHMM: automating chromatinstate discovery and characterization. Nat Methods 2012; 9: 215- 216.

[25] Eustermann S , Patel AB , Hopfner KP et al. Energy-driven genome regulation by ATP-dependent chromatin remodellers. Nat Rev Mol Cell Biol 2024; 25: 309- 332.

[26] Gerstein MB , Kundaje A , Hariharan M et al. Architecture of the human regulatory network derived from ENCODE data. Nature 2012; 489: 91- 100.

[27] Gilmour DS , Lis JT . Detecting protein-DNA interactions in vivo: distribution of RNA polymerase on specific bacterial genes. Proc Natl Acad Sci U S A 1984; 81: 4275- 4279.

[28] Gopalan S , Wang Y , Harper NW et al. Simultaneous profiling of multiple chromatin proteins in the same cells. Mol Cell 2021; 81: 4736- 4746.e5.

[29] Grosselin K , Durand A , Marsolier J et al. High-throughput single-cell ChIP-seq identifies heterogeneity of chromatin states in breast cancer. Nat Genet 2019; 51: 1060- 1066.

[30] Harada A , Maehara K , Handa T et al. A chromatin integration labelling method enables epigenomic profiling with lower input. Nat Cell Biol 2019; 21: 287- 296.

[31] He HH , Meyer CA , Hu SS et al. Refined DNase-seq protocol and data analysis reveals intrinsic bias in transcription factor footprint identification. Nat Methods 2014; 11: 73- 78.

[32] He Q , Johnston J , Zeitlinger J . ChIP-nexus enables improved detection of in vivo transcription factor binding footprints. Nat Biotechnol 2015; 33: 395- 401.

[33] He R , Dong W , Wang Z et al. Genome-wide single-cell and single-molecule footprinting of transcription factors with deaminase. Proc Natl Acad Sci U S A 2024; 121: e2423270121.

[34] Hesselberth JR , Chen X , Zhang Z et al. Global mapping of protein-DNA interactions in vivo by digital genomic footprinting. Nat Methods 2009; 6: 283- 289.

[35] Hu Y , Horlbeck MA , Zhang R et al. Multiscale footprints reveal the organization of cis-regulatory elements. Nature 2025; 638: 779- 786.

[36] Iurlaro M , Stadler MB , Masoni F et al. Mammalian SWI/SNF continuously restores local accessibility to chromatin. Nat Genet 2021; 53: 279- 287.

[37] Iurlaro M , Masoni F , Flyamer IM et al. Systematic assessment of ISWI subunits shows that NURF creates local accessibility for CTCF. Nat Genet 2024; 56: 1203- 1212.

[38] Johnson DS , Mortazavi A , Myers RM et al. Genome-wide mapping of in vivo protein-DNA interactions. Science 2007; 316: 1497- 1502.

[39] Jolma A , Kivioja T , Toivonen J et al. Multiplexed massively parallel SELEX for characterization of human transcription factor binding specificities. Genome Res 2010; 20: 861- 873.

[40] Jolma A , Yan J , Whitington T et al. DNA-binding specificities of human transcription factors. Cell 2013; 152: 327- 339.

[41] Jolma A , Yin Y , Nitta KR et al. DNA-dependent formation of transcription factor pairs alters their binding specificity. Nature 2015; 527: 384- 388.

[42] Kadoch C , Hargreaves DC , Hodges C et al. Proteomic and bioinformatic analysis of mammalian SWI/SNF complexes identifies extensive roles in human malignancy. Nat Genet 2013; 45: 592- 601.

[43] Kaya-Okur HS , Wu SJ , Codomo CA et al. CUT&Tag for efficient epigenomic profiling of small samples and single cells. Nat Commun 2019; 10: 1930.

[44] Kim S , Wysocka J . Deciphering the multi-scale, quantitative cis-regulatory code. Mol Cell 2023; 83: 373- 392.

[45] Klemm SL , Shipony Z , Greenleaf WJ . Chromatin accessibility and the regulatory epigenome. Nat Rev Genet 2019; 20: 207- 220.

[46] Krebs AR . Studying transcription factor function in the genome at molecular resolution. Trends Genet 2021; 37: 798- 806.

[47] Krebs AR , Imanci D , Hoerner L et al. Genome-wide single-molecule footprinting reveals high RNA polymerase II turnover at paused promoters. Mol Cell 2017; 67: 422.e4.

[48] Lambert SA , Jolma A , Campitelli LF et al. The human transcription factors. Cell 2018; 172: 650- 665.

[49] Li D , Liu J , Yang X et al. Chromatin accessibility dynamics during iPSC reprogramming. Cell Stem Cell 2017; 21: 819- 833.e6.

[50] Li Z , Schulz MH , Look T et al. Identification of transcription factor binding sites using ATAC-seq. Genome Biol 2019; 20: 45.

[51] Liu Y , He J , Chen R et al. AP-1 activity is a major barrier of human somatic cell reprogramming. Cell Mol Life Sci 2021; 78: 5847- 5863.

[52] Lochs SJA , Van Der Weide RH , De Luca KL et al. Combinatorial single-cell profiling of major chromatin types with MAbID. Nat Methods 2024; 21: 72- 82.

[53] Martin BJE , Ablondi EF , Goglia C et al. Global identification of SWI/SNF targets reveals compensation by EP400. Cell 2023; 186: 5290- 5307.e26.

[54] Meers MP , Llagas G , Janssens DH et al. Multifactorial profiling of epigenetic landscapes at single-cell resolution using MulTI-Tag. Nat Biotechnol 2023; 41: 708- 716.

[55] Mi L , Shi M , Li YX et al. DddA homolog search and engineering expand sequence compatibility of mitochondrial base editing. Nat Commun 2023; 14: 874.

[56] Mok BY , De Moraes MH , Zeng J et al. A bacterial cytidine deaminase toxin enables CRISPR-free mitochondrial base editing. Nature 2020; 583: 631- 637.

[57] Moyers BA , Partridge EC , Mackiewicz M et al. Characterization of human transcription factor function and patterns of gene regulation in HepG2 cells. Genome Res 2023; 33: 1879- 1892.

[58] Neph S , Stergachis AB , Reynolds A et al. Circuitry and dynamics of human transcription factor regulatory networks. Cell 2012a; 150: 1274- 1286.

[59] Neph S , Vierstra J , Stergachis AB et al. An expansive human regulatory lexicon encoded in transcription factor footprints. Nature 2012b; 489: 83- 90.

[60] Papillon JPN , Nakajima K , Adair CD et al. Discovery of orally active inhibitors of Brahma homolog (BRM)/SMARCA2 ATPase activity for the treatment of Brahma related gene 1 (BRG1)/SMARCA4-mutant cancers. J Med Chem 2018; 61: 10155- 10172.

[61] Partridge EC , Chhetri SB , Prokop JW et al. Occupancy maps of 208 chromatin-associated proteins in one human cell type. Nature 2020; 583: 720- 728.

[62] Preissl S , Gaulton KJ , Ren B . Characterizing cis-regulatory elements using single-cell epigenomics. Nat Rev Genet 2022; 24: 21- 43.

[63] Rauluseviciute I , Riudavets-Puig R , Blanc-Mathieu R et al. JASPAR 2024: 20th anniversary of the open-access database of transcription factor binding profiles. Nucleic Acids Res 2024; 52: D174- D182.

[64] Rhee HS , Pugh BF . Comprehensive genome-wide proteinDNA interactions detected at single-nucleotide resolution. Cell 2011; 147: 1408- 1419.

[65] Schick S , Grosche S , Kohl KE et al. Acute BAF perturbation causes immediate changes in chromatin accessibility. Nat Genet 2021; 53: 269- 278.

[66] Shain AH , Pollack JR . The spectrum of SWI/SNF mutations, ubiquitous in human cancers. PLoS One 2013; 8: e55119.

[67] Shipony Z , Marinov GK , Swaffer MP et al. Long-range single-molecule mapping of chromatin accessibility in eukaryotes. Nat Methods 2020; 17: 319- 327.

[68] Skene PJ , Henikoff S . An efficient targeted nuclease strategy for high-resolution mapping of DNA binding sites. eLife 2017; 6: e21856.

[69] Sonmezer C , Kleinendorst R , Imanci D et al. Molecular cooccupancy identifies transcription factor binding cooperativity in vivo. Mol Cell 2021; 81: 267.e6.

[70] Spitz F , Furlong EE . Transcription factors: from enhancer binding to developmental control. Nat Rev Genet 2012; 13: 613- 626.

[71] Stergachis AB , Debo BM , Haugen E et al. Single-molecule regulatory architectures captured by chromatin fiber sequencing. Science 2020; 368: 1449- 1454.

[72] Sung MH , Baek S , Hager GL . Genome-wide footprinting: ready for prime time? Nat Methods 2016; 13: 222- 228.

[73] Vaquerizas JM , Kummerfeld SK , Teichmann SA et al. A census of human transcription factors: function, expression and evolution. Nat Rev Genet 2009; 10: 252- 263.

[74] Vierstra J , Lazar J , Sandstrom R et al. Global reference mapping of human transcription factor footprints. Nature 2020; 583: 729- 736.

[75] Vinckevicius A , Parker JB , Chakravarti D . Genomic determinants of THAP11/ZNF143/HCFC1 complex recruitment to chromatin. Mol Cell Biol 2015; 35: 4135- 4146.

[76] Wang Q , Xiong H , Ai S et al. CoBATCH for high-throughput single-cell epigenomic profiling. Mol Cell 2019; 76: 206- 216.e7.

[77] Weng Z , Ruan F , Chen W et al. BIND&MODIFY: a long-range method for single-molecule mapping of chromatin modifications in eukaryotes. Genome Biol 2023; 24: 61.

[78] Wiechens N , Singh V , Gkikopoulos T et al. The chromatin remodelling enzymes SNF2H and SNF2L position nucleosomes adjacent to CTCF and other transcription factors. PLoS Genet 2016; 12: e1005940.

[79] Xie Z , Sokolov I , Osmala M et al. DNA-guided transcription factor interactions extend human gene regulatory code. Nature 2025; 641: 1329- 1338.

[80] Xiong H , Wang Q , Li CC et al. Single-cell joint profiling of multiple epigenetic proteins and gene transcription. Sci Adv 2024; 10: eadi3664.

[81] Yue X , Xie Z , Li M et al. Simultaneous profiling of histone modifications and DNA methylation via nanopore sequencing. Nat Commun 2022; 13: 7939.

[82] Zheng R , Wan C , Mei S et al. Cistrome data browser: expanded datasets and new tools for gene regulatory analysis. Nucleic Acids Res 2019; 47: D729- D735.

RIGHTS & PERMISSIONS

The Author(s). Published by Oxford University Press on behalf of Higher Education Press.