ChIP-nexus Enables Improved Detection of in Vivo Transcription Factor Binding Footprints

Overview

Journal Nat Biotechnol

Specialty Biotechnology

Date 2015 Mar 10

PMID 25751057

Citations 130

Authors

Qiye He

Jeff Johnston

Julia Zeitlinger

Affiliations

Soon will be listed here.

Abstract

Understanding how eukaryotic enhancers are bound and regulated by specific combinations of transcription factors is still a major challenge. To better map transcription factor binding genome-wide at nucleotide resolution in vivo, we have developed a robust ChIP-exo protocol called ChIP-nexus (chromatin immunoprecipitation experiments with nucleotide resolution through exonuclease, unique barcode and single ligation), which utilizes an efficient DNA self-circularization step during library preparation. Application of ChIP-nexus to four proteins--human TBP and Drosophila NFkB, Twist and Max--shows that it outperforms existing ChIP protocols in resolution and specificity, pinpoints relevant binding sites within enhancers containing multiple binding motifs, and allows for the analysis of in vivo binding specificities. Notably, we show that Max frequently interacts with DNA sequences next to its motif, and that this binding pattern correlates with local DNA-sequence features such as DNA shape. ChIP-nexus will be broadly applicable to the study of in vivo transcription factor binding specificity and its relationship to cis-regulatory changes in humans and model organisms.

Citing Articles

ZBTB16/PLZF regulates juvenile spermatogonial stem cell development through an extensive transcription factor poising network.

Yi C, Kitamura Y, Maezawa S, Namekawa S, Cairns B Nat Struct Mol Biol. 2025; .

PMID: 40033150 DOI: 10.1038/s41594-025-01509-5.

Promoter-proximal RNA polymerase II termination regulates transcription during human cell type transition.

Lysakovskaia K, Devadas A, Schwalb B, Lidschreiber M, Cramer P Nat Struct Mol Biol. 2025; .

PMID: 39934431 DOI: 10.1038/s41594-025-01486-9.

ChromBPNet: bias factorized, base-resolution deep learning models of chromatin accessibility reveal cis-regulatory sequence syntax, transcription factor footprints and regulatory variants.

Pampari A, Shcherbina A, Kvon E, Kosicki M, Nair S, Kundu S bioRxiv. 2025; .

PMID: 39829783 PMC: 11741299. DOI: 10.1101/2024.12.25.630221.

Guidelines to Analyze ChIP-Seq Data: Journey Through QC and Analysis Considerations.

De Kumar B, Krishnan J Methods Mol Biol. 2025; 2889:193-206.

PMID: 39745614 DOI: 10.1007/978-1-0716-4322-8_14.

Cbf11 and Mga2 function together to activate transcription of lipid metabolism genes and promote mitotic fidelity in fission yeast.

Maresova A, Grulyova M, Hradilova M, Zemlianski V, Princova J, Prevorovsky M PLoS Genet. 2024; 20(12):e1011509.

PMID: 39652606 PMC: 11658701. DOI: 10.1371/journal.pgen.1011509.

References

Rhee H, Pugh B . Comprehensive genome-wide protein-DNA interactions detected at single-nucleotide resolution. Cell. 2011; 147(6):1408-19. PMC: 3243364. DOI: 10.1016/j.cell.2011.11.013. View

Ip Y, Park R, Kosman D, Bier E, Levine M . The dorsal gradient morphogen regulates stripes of rhomboid expression in the presumptive neuroectoderm of the Drosophila embryo. Genes Dev. 1992; 6(9):1728-39. DOI: 10.1101/gad.6.9.1728. View

Rhee H, Pugh B . ChIP-exo method for identifying genomic location of DNA-binding proteins with near-single-nucleotide accuracy. Curr Protoc Mol Biol. 2012; Chapter 21:Unit 21.24. PMC: 3813302. DOI: 10.1002/0471142727.mb2124s100. View

Ferre-DAmare A, Prendergast G, Ziff E, Burley S . Recognition by Max of its cognate DNA through a dimeric b/HLH/Z domain. Nature. 1993; 363(6424):38-45. DOI: 10.1038/363038a0. View

Feng J, Liu T, Qin B, Zhang Y, Liu X . Identifying ChIP-seq enrichment using MACS. Nat Protoc. 2012; 7(9):1728-40. PMC: 3868217. DOI: 10.1038/nprot.2012.101. View

Huang J, Schwyter D, Shirokawa J, Courey A . The interplay between multiple enhancer and silencer elements defines the pattern of decapentaplegic expression. Genes Dev. 1993; 7(4):694-704. DOI: 10.1101/gad.7.4.694. View

Casbon J, Osborne R, Brenner S, Lichtenstein C . A method for counting PCR template molecules with application to next-generation sequencing. Nucleic Acids Res. 2011; 39(12):e81. PMC: 3130290. DOI: 10.1093/nar/gkr217. View

Spitz F, Furlong E . Transcription factors: from enhancer binding to developmental control. Nat Rev Genet. 2012; 13(9):613-26. DOI: 10.1038/nrg3207. View

Yen K, Vinayachandran V, Batta K, Koerber R, Pugh B . Genome-wide nucleosome specificity and directionality of chromatin remodelers. Cell. 2012; 149(7):1461-73. PMC: 3397793. DOI: 10.1016/j.cell.2012.04.036. View

10.

Rhee H, Pugh B . Genome-wide structure and organization of eukaryotic pre-initiation complexes. Nature. 2012; 483(7389):295-301. PMC: 3306527. DOI: 10.1038/nature10799. View

11.

Kivioja T, Vaharautio A, Karlsson K, Bonke M, Enge M, Linnarsson S . Counting absolute numbers of molecules using unique molecular identifiers. Nat Methods. 2011; 9(1):72-4. DOI: 10.1038/nmeth.1778. View

12.

Serandour A, Brown G, Cohen J, Carroll J . Development of an Illumina-based ChIP-exonuclease method provides insight into FoxA1-DNA binding properties. Genome Biol. 2013; 14(12):R147. PMC: 4053927. DOI: 10.1186/gb-2013-14-12-r147. View

13.

Yang L, Zhou T, Dror I, Mathelier A, Wasserman W, Gordan R . TFBSshape: a motif database for DNA shape features of transcription factor binding sites. Nucleic Acids Res. 2013; 42(Database issue):D148-55. PMC: 3964943. DOI: 10.1093/nar/gkt1087. View

14.

Bardet A, Steinmann J, Bafna S, Knoblich J, Zeitlinger J, Stark A . Identification of transcription factor binding sites from ChIP-seq data at high resolution. Bioinformatics. 2013; 29(21):2705-13. PMC: 3799470. DOI: 10.1093/bioinformatics/btt470. View

15.

Venters B, Pugh B . Genomic organization of human transcription initiation complexes. Nature. 2013; 502(7469):53-8. PMC: 4018585. DOI: 10.1038/nature12535. View

16.

White M, Myers C, Corbo J, Cohen B . Massively parallel in vivo enhancer assay reveals that highly local features determine the cis-regulatory function of ChIP-seq peaks. Proc Natl Acad Sci U S A. 2013; 110(29):11952-7. PMC: 3718143. DOI: 10.1073/pnas.1307449110. View

17.

Zhou T, Yang L, Lu Y, Dror I, Dantas Machado A, Ghane T . DNAshape: a method for the high-throughput prediction of DNA structural features on a genomic scale. Nucleic Acids Res. 2013; 41(Web Server issue):W56-62. PMC: 3692085. DOI: 10.1093/nar/gkt437. View

18.

Gordan R, Shen N, Dror I, Zhou T, Horton J, Rohs R . Genomic regions flanking E-box binding sites influence DNA binding specificity of bHLH transcription factors through DNA shape. Cell Rep. 2013; 3(4):1093-104. PMC: 3640701. DOI: 10.1016/j.celrep.2013.03.014. View

19.

Wechsler D, Papoulas O, Dang C, Kingston R . Differential binding of c-Myc and Max to nucleosomal DNA. Mol Cell Biol. 1994; 14(6):4097-107. PMC: 358775. DOI: 10.1128/mcb.14.6.4097-4107.1994. View

20.

Szymanski P, Levine M . Multiple modes of dorsal-bHLH transcriptional synergy in the Drosophila embryo. EMBO J. 1995; 14(10):2229-38. PMC: 398329. DOI: 10.1002/j.1460-2075.1995.tb07217.x. View