Massively parallel decoding of mammalian regulatory sequences supports a flexible organizational model

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.

[1]  N. Rosenthal,et al.  A muscle-specific enhancer is located at the 3' end of the myosin light-chain 1/3 gene locus. , 1988, Genes & development.

[2]  J. Darnell,et al.  Liver-enriched transcription factor HNF-4 is a novel member of the steroid hormone receptor superfamily. , 1990, Genes & development.

[3]  R. Tjian,et al.  Characterization of a dimerization motif in AP-2 and its function in heterologous DNA-binding proteins. , 1991, Science.

[4]  I. Issemann,et al.  The peroxisome proliferator-activated receptor:retinoid X receptor heterodimer is activated by fatty acids and fibrate hypolipidaemic drugs. , 1993, Journal of molecular endocrinology.

[5]  T. Maniatis,et al.  Virus induction of human IFNβ gene expression requires the assembly of an enhanceosome , 1995, Cell.

[6]  Y. Benjamini,et al.  Controlling the false discovery rate: a practical and powerful approach to multiple testing , 1995 .

[7]  Michael Gribskov,et al.  Combining evidence using p-values: application to sequence homology searches , 1998, Bioinform..

[8]  J. Wolff,et al.  High levels of foreign gene expression in hepatocytes after tail vein injections of naked plasmid DNA. , 1999, Human gene therapy.

[9]  R. Dennis Cook,et al.  Detection of Influential Observation in Linear Regression , 2000, Technometrics.

[10]  Stephen S. Gisselbrecht,et al.  Ras Pathway Specificity Is Determined by the Integration of Multiple Signal-Activated and Tissue-Restricted Transcription Factors , 2000, Cell.

[11]  W. Wasserman,et al.  A predictive model for regulatory sequences directing liver-specific transcription. , 2001, Genome research.

[12]  D. Arnosti,et al.  Information display by transcriptional enhancers , 2003, Development.

[13]  J. D. Engel,et al.  Hepatic Erythropoietin Gene Regulation by GATA-4* , 2004, Journal of Biological Chemistry.

[14]  W. Miller,et al.  Mulan: multiple-sequence local alignment and visualization for studying function and evolution. , 2005, Genome research.

[15]  William Stafford Noble,et al.  Quantifying similarity between motifs , 2007, Genome Biology.

[16]  Alexander E. Kel,et al.  TRANSFAC® and its module TRANSCompel®: transcriptional gene regulation in eukaryotes , 2005, Nucleic Acids Res..

[17]  Christina Chaivorapol,et al.  Systematic Identification of cis-Regulatory Sequences Active in Mouse and Human Embryonic Stem Cells , 2007, PLoS genetics.

[18]  Christopher D. Brown,et al.  Supporting Online Material Materials and Methods Figs. S1 to S6 Table S1 References Functional Architecture and Evolution of Transcriptional Elements That Drive Gene Coexpression , 2022 .

[19]  Inna Dubchak,et al.  VISTA Enhancer Browser—a database of tissue-specific human enhancers , 2006, Nucleic Acids Res..

[20]  S. Duncan,et al.  Development of the mammalian liver and ventral pancreas is dependent on GATA4 , 2007, BMC Developmental Biology.

[21]  Edward J. Oakeley,et al.  Position dependencies in transcription factor binding sites , 2007, Bioinform..

[22]  A. Mortazavi,et al.  Genome-Wide Mapping of in Vivo Protein-DNA Interactions , 2007, Science.

[23]  A. Visel,et al.  Combinatorial Regulation of Endothelial Gene Expression by Ets and Forkhead Transcription Factors , 2008, Cell.

[24]  Nicholas G Martin,et al.  A single SNP in an evolutionary conserved region within intron 86 of the HERC2 gene determines human blue-brown eye color. , 2008, American journal of human genetics.

[25]  Xia Li,et al.  Regulation of a remote Shh forebrain enhancer by the Six3 homeoprotein , 2008, Nature Genetics.

[26]  T. Dickmeis,et al.  The words of the regulatory code are arranged in a variable manner in highly conserved enhancers. , 2008, Developmental biology.

[27]  Ole Winther,et al.  JASPAR, the open access database of transcription factor-binding profiles: new content and tools in the 2008 update , 2007, Nucleic Acids Res..

[28]  Clifford A. Meyer,et al.  FoxA1 Translates Epigenetic Signatures into Enhancer-Driven Lineage-Specific Transcription , 2008, Cell.

[29]  A. Visel,et al.  ChIP-seq accurately predicts tissue-specific activity of enhancers , 2009, Nature.

[30]  E. Siggia,et al.  Analysis of Combinatorial cis-Regulation in Synthetic and Genomic Promoters , 2008, Nature.

[31]  Martha L. Bulyk,et al.  UniPROBE: an online database of protein binding microarray data on protein–DNA interactions , 2008, Nucleic Acids Res..

[32]  Richard Durbin,et al.  Sequence analysis Fast and accurate short read alignment with Burrows – Wheeler transform , 2009 .

[33]  Daniel E. Newburger,et al.  Diversity and Complexity in DNA Recognition by Transcription Factors , 2009, Science.

[34]  A. Munnich,et al.  Highly conserved non-coding elements on either side of SOX9 associated with Pierre Robin sequence , 2009, Nature Genetics.

[35]  A. Visel,et al.  Homotypic clusters of transcription factor binding sites are a key component of human promoters and enhancers. , 2010, Genome research.

[36]  John M Westlund,et al.  Genome-wide discovery of human heart enhancers. , 2010, Genome research.

[37]  A. Visel,et al.  ChIP-Seq identification of weakly conserved heart enhancers , 2010, Nature Genetics.

[38]  Nathaniel D. Heintzman,et al.  9p21 DNA variants associated with Coronary Artery Disease impair IFNγ signaling response , 2011, Nature.

[39]  Nathan C. Sheffield,et al.  Open chromatin defined by DNaseI and FAIRE identifies regulatory elements that shape cell-type identity. , 2011, Genome research.

[40]  P. Kwok,et al.  Functional Characterization of Liver Enhancers That Regulate Drug‐Associated Transporters , 2011, Clinical pharmacology and therapeutics.

[41]  Michael A. Beer,et al.  Discriminative prediction of mammalian enhancers from DNA sequence. , 2011, Genome research.

[42]  Jay Shendure,et al.  Accurate gene synthesis with tag-directed retrieval of sequence-verified DNA molecules , 2012, Nature Methods.

[43]  Joseph B Hiatt,et al.  Massively parallel functional dissection of mammalian enhancers in vivo , 2012, Nature Biotechnology.

[44]  W. Edelmann,et al.  SLiCE: a novel bacterial cell extract-based DNA cloning method , 2012, Nucleic acids research.

[45]  E. Furlong,et al.  Transcription factors: from enhancer binding to developmental control , 2012, Nature Reviews Genetics.

[46]  Lee E. Edsall,et al.  A map of the cis-regulatory sequences in the mouse genome , 2012, Nature.

[47]  Abdelkader Essafi,et al.  Opposing Functions of the ETS Factor Family Define Shh Spatial Expression in Limb Buds and Underlie Polydactyly , 2012, Developmental cell.

[48]  Z. Yakhini,et al.  Inferring gene regulatory logic from high-throughput measurements of thousands of systematically designed promoters , 2012, Nature Biotechnology.

[49]  Juan M. Vaquerizas,et al.  DNA-Binding Specificities of Human Transcription Factors , 2013, Cell.

[50]  Beau Dabbs,et al.  Summary and discussion of : “ Controlling the False Discovery Rate : A Practical and Powerful Approach to Multiple Testing , 2014 .