title: Massively parallel decoding of mammalian regulatory sequences supports a flexible organizational model.
abstract: Despite continual progress in the cataloging of vertebrate regulatory elements, little is known about their organization and regulatory architecture. Here we describe a massively parallel experiment to systematically test the impact of copy number, spacing, combination and order of transcription factor binding sites on gene expression. A complex library of ∼5,000 synthetic regulatory elements containing patterns from 12 liver-specific transcription factor binding sites was assayed in mice and in HepG2 cells. We find that certain transcription factors act as direct drivers of gene expression in homotypic clusters of binding sites, independent of spacing between sites, whereas others function only synergistically. Heterotypic enhancers are stronger than their homotypic analogs and favor specific transcription factor binding site combinations, mimicking putative native enhancers. Exhaustive testing of binding site permutations suggests that there is flexibility in binding site order. Our findings provide quantitative support for a flexible model of regulatory element activity and suggest a framework for the design of synthetic tissue-specific enhancers.
authors.affiliation: Department of Bioengineering and Therapeutic Sciences, University of California, San Francisco, San Francisco, California, USA.|Institute for Human Genetics, University of California, San Francisco, San Francisco, California, USA.
authors.forename: Robin P
authors.initials: RP
authors.lastname: Smith
authors.affiliation: Computational Biology Branch, National Center for Biotechnology Information, National Library of Medicine, US National Institutes of Health, Bethesda, Maryland, USA.|Institute for Biostatistics and Informatics in Medicine and Ageing Research, University of Rostock, Rostock, Germany.
authors.forename: Leila
authors.initials: L
authors.lastname: Taher
authors.affiliation: Department of Genome Sciences, University of Washington, Seattle, Washington, USA.
authors.forename: Rupali P
authors.initials: RP
authors.lastname: Patwardhan
authors.affiliation: Department of Bioengineering and Therapeutic Sciences, University of California, San Francisco, San Francisco, California, USA.|Institute for Human Genetics, University of California, San Francisco, San Francisco, California, USA.
authors.forename: Mee J
authors.initials: MJ
authors.lastname: Kim
authors.affiliation: Department of Bioengineering and Therapeutic Sciences, University of California, San Francisco, San Francisco, California, USA.|Institute for Human Genetics, University of California, San Francisco, San Francisco, California, USA.
authors.forename: Fumitaka
authors.initials: F
authors.lastname: Inoue
authors.affiliation: Department of Genome Sciences, University of Washington, Seattle, Washington, USA.
authors.forename: Jay
authors.initials: J
authors.lastname: Shendure
authors.affiliation: Computational Biology Branch, National Center for Biotechnology Information, National Library of Medicine, US National Institutes of Health, Bethesda, Maryland, USA.
authors.forename: Ivan
authors.initials: I
authors.lastname: Ovcharenko
authors.affiliation: Department of Bioengineering and Therapeutic Sciences, University of California, San Francisco, San Francisco, California, USA.|Institute for Human Genetics, University of California, San Francisco, San Francisco, California, USA.
authors.forename: Nadav
authors.initials: N
authors.lastname: Ahituv
mesh_terms: D000818:Animals; D001665:Binding Sites; D002460:Cell Line; D016000:Cluster Analysis; D004742:Enhancer Elements, Genetic; D005784:Gene Amplification; D018628:Gene Dosage; D015870:Gene Expression; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D006801:Humans; D008099:Liver / Q000378:metabolism; D008297:Male; D051379:Mice; D008954:Models, Biological; D059372:Nucleotide Motifs; D009928:Organ Specificity / Q000235:genetics; D011485:Protein Binding; D012045:Regulatory Sequences, Nucleic Acid; D014157:Transcription Factors / Q000378:metabolism*
chemical_list: D014157:Transcription Factors
references.citation: Cell. 2008 Mar 21;132(6):958-70
references.pmid: 18358809
references.citation: Science. 1991 Mar 1;251(4997):1067-71
references.pmid: 1998122
references.citation: Curr Opin Genet Dev. 2001 Apr;11(2):205-8
references.pmid: 11250145
references.citation: Am J Hum Genet. 2008 Feb;82(2):424-31
references.pmid: 18252222
references.citation: Nat Genet. 2009 Mar;41(3):359-64
references.pmid: 19234473
references.citation: J Biol Chem. 2004 Jan 23;279(4):2955-61
references.pmid: 14583613
references.citation: Science. 2009 Jun 26;324(5935):1720-3
references.pmid: 19443739
references.citation: Nucleic Acids Res. 2012 Apr;40(8):e55
references.pmid: 22241772
references.citation: Cell. 1995 Dec 29;83(7):1091-100
references.pmid: 8548797
references.citation: Nature. 2011 Feb 10;470(7333):264-8
references.pmid: 21307941
references.citation: Genes Dev. 1990 Dec;4(12B):2353-65
references.pmid: 2279702
references.citation: Genome Res. 2001 Sep;11(9):1559-66
references.pmid: 11544200
references.citation: Bioinformatics. 1998;14(1):48-54
references.pmid: 9520501
references.citation: Bioinformatics. 2009 Jul 15;25(14):1754-60
references.pmid: 19451168
references.citation: Dev Cell. 2012 Feb 14;22(2):459-67
references.pmid: 22340503
references.citation: Clin Pharmacol Ther. 2011 Apr;89(4):571-8
references.pmid: 21368754
references.citation: Genome Res. 2005 Jan;15(1):184-94
references.pmid: 15590941
references.citation: Hum Gene Ther. 1999 Jul 1;10(10):1735-7
references.pmid: 10428218
references.citation: BMC Dev Biol. 2007 Apr 23;7:37
references.pmid: 17451603
references.citation: Genome Res. 2011 Dec;21(12):2167-80
references.pmid: 21875935
references.citation: Dev Biol. 2008 Jun 15;318(2):366-77
references.pmid: 18455719
references.citation: Nat Genet. 2008 Nov;40(11):1348-53
references.pmid: 18836447
references.citation: Genome Res. 2010 Mar;20(3):381-92
references.pmid: 20075146
references.citation: Genome Res. 2011 Oct;21(10):1757-67
references.pmid: 21750106
references.citation: Nat Biotechnol. 2012 Feb 26;30(3):271-7
references.pmid: 22371084
references.citation: Nat Genet. 2010 Sep;42(9):806-10
references.pmid: 20729851
references.citation: Nat Biotechnol. 2012 Feb 26;30(3):265-70
references.pmid: 22371081
references.citation: Science. 2007 Sep 14;317(5844):1557-60
references.pmid: 17872446
references.citation: Nucleic Acids Res. 2007 Jan;35(Database issue):D88-92
references.pmid: 17130149
references.citation: Nature. 2012 Aug 2;488(7409):116-20
references.pmid: 22763441
references.citation: Nat Rev Genet. 2012 Sep;13(9):613-26
references.pmid: 22868264
references.citation: Genome Res. 2010 May;20(5):565-77
references.pmid: 20363979
references.citation: Development. 2003 Dec;130(26):6569-75
references.pmid: 14660545
references.citation: Nat Biotechnol. 2012 May 20;30(6):521-30
references.pmid: 22609971
references.citation: Nature. 2009 Jan 8;457(7226):215-8
references.pmid: 19029883
references.citation: Cell. 2008 Dec 12;135(6):1053-64
references.pmid: 19070576
references.citation: J Mol Endocrinol. 1993 Aug;11(1):37-47
references.pmid: 8240670
references.citation: Cell. 2000 Sep 29;103(1):63-74
references.pmid: 11051548
references.citation: Science. 2007 Jun 8;316(5830):1497-502
references.pmid: 17540862
references.citation: PLoS Genet. 2007 Aug;3(8):e145
references.pmid: 17784790
references.citation: Genes Dev. 1988 Dec;2(12B):1779-90
references.pmid: 3240859
references.citation: Nature. 2009 Feb 12;457(7231):854-8
references.pmid: 19212405
references.citation: Bioinformatics. 2007 Apr 15;23(8):933-41
references.pmid: 17308339
references.citation: Nat Methods. 2012 Sep;9(9):913-5
references.pmid: 22886093