A systematic approach to identify functional motifs within vertebrate developmental enhancers.

Uncovering the cis-regulatory logic of developmental enhancers is critical to understanding the role of non-coding DNA in development. However, it is cumbersome to identify functional motifs within enhancers, and thus few vertebrate enhancers have their core functional motifs revealed. Here we report a combined experimental and computational approach for discovering regulatory motifs in developmental enhancers. Making use of the zebrafish gene expression database, we computationally identified conserved non-coding elements (CNEs) likely to have a desired tissue-specificity based on the expression of nearby genes. Through a high throughput and robust enhancer assay, we tested the activity of approximately 100 such CNEs and efficiently uncovered developmental enhancers with desired spatial and temporal expression patterns in the zebrafish brain. Application of de novo motif prediction algorithms on a group of forebrain enhancers identified five top-ranked motifs, all of which were experimentally validated as critical for forebrain enhancer activity. These results demonstrate a systematic approach to discover important regulatory motifs in vertebrate developmental enhancers. Moreover, this dataset provides a useful resource for further dissection of vertebrate brain development and function.

[1]  Venky N. Iyer,et al.  Sepsid even-skipped Enhancers Are Functionally Conserved in Drosophila Despite Lack of Sequence Conservation , 2008, PLoS genetics.

[2]  Klaudia Walter,et al.  Highly Conserved Non-Coding Sequences Are Associated with Vertebrate Development , 2004, PLoS biology.

[3]  Charles Elkan,et al.  Fitting a Mixture Model By Expectation Maximization To Discover Motifs In Biopolymer , 1994, ISMB.

[4]  T. Hirano,et al.  Expression of the zinc ®nger gene fez-like in zebra®sh forebrain , 2000 .

[5]  J. B. Jaynes,et al.  Transcription of bxd Noncoding RNAs Promoted by Trithorax Represses Ubx in cis by Transcriptional Interference , 2006, Cell.

[6]  Qiang Li,et al.  cneViewer: a database of conserved non-coding elements for studies of tissue-specific gene regulation , 2008, Bioinform..

[7]  H Okamoto,et al.  Visualization of Cranial Motor Neurons in Live Transgenic Zebrafish Expressing Green Fluorescent Protein Under the Control of the Islet-1 Promoter/Enhancer , 2000, The Journal of Neuroscience.

[8]  T. Hirano,et al.  Zinc finger gene fez‐like functions in the formation of subplate neurons and thalamocortical axons , 2004, Developmental dynamics : an official publication of the American Association of Anatomists.

[9]  Alan M. Moses,et al.  In vivo enhancer analysis of human conserved non-coding sequences , 2006, Nature.

[10]  B. Goossens,et al.  Aerial Surveys Give New Estimates for Orangutans in Sabah, Malaysia , 2004, PLoS biology.

[11]  A. Visel,et al.  Ultraconservation identifies a small subset of extremely constrained developmental enhancers , 2008, Nature Genetics.

[12]  C. Kimmel,et al.  Stages of embryonic development of the zebrafish , 1995, Developmental dynamics : an official publication of the American Association of Anatomists.

[13]  W. Driever,et al.  Mutations in the zebrafish unmask shared regulatory pathways controlling the development of catecholaminergic neurons. , 1999, Developmental biology.

[14]  V. Korzh,et al.  Zebrafish primary neurons initiate expression of the LIM homeodomain protein Isl-1 at the end of gastrulation. , 1993, Development.

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

[16]  M. Gulisano,et al.  Nested expression domains of four homeobox genes in developing rostral brain , 1992, Nature.

[17]  E. Boncinelli,et al.  Transcription factors and head formation in vertebrates , 1997, BioEssays : news and reviews in molecular, cellular and developmental biology.

[18]  A. Shima,et al.  Identification of the Tol2 transposase of the medaka fish Oryzias latipes that catalyzes excision of a nonautonomous Tol2 element in zebrafish Danio rerio. , 1999, Gene.

[19]  Ivan Ovcharenko,et al.  ECR Browser: a tool for visualizing and accessing data from comparisons of multiple vertebrate genomes , 2004, Nucleic Acids Res..

[20]  Xin Chen,et al.  TRANSFAC: an integrated system for gene expression regulation , 2000, Nucleic Acids Res..

[21]  S. Aizawa,et al.  Acetylated YY1 regulates Otx2 expression in anterior neuroectoderm at two cis‐sites 90 kb apart , 2007, The EMBO journal.

[22]  Andrew Lumsden,et al.  Patterning the Vertebrate Neuraxis , 1996, Science.

[23]  L. Pennacchio,et al.  Genomic strategies to identify mammalian regulatory sequences , 2001, Nature Reviews Genetics.

[24]  Ran Kafri,et al.  The regulatory utilization of genetic redundancy through responsive backup circuits. , 2006, Proceedings of the National Academy of Sciences of the United States of America.

[25]  P. Gruss,et al.  Enhancer elements , 1983, Cell.

[26]  H. Bussemaker,et al.  Building a dictionary for genomes: identification of presumptive regulatory sites by statistical analysis. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[27]  H. Okamoto,et al.  Molecular heterogeneity among primary motoneurons and within myotomes revealed by the differential mRNA expression of novel islet-1 homologs in embryonic zebrafish. , 1995, Developmental biology.

[28]  J. T. Kadonaga,et al.  *To whom correspondence should be addressed. E- , 2022 .

[29]  Colin N. Dewey,et al.  Initial sequencing and comparative analysis of the mouse genome. , 2002 .

[30]  I. Ovcharenko,et al.  Human-zebrafish non-coding conserved elements act in vivo to regulate transcription , 2005, Nucleic acids research.

[31]  W. Talbot,et al.  Zinc finger protein too few controls the development of monoaminergic neurons , 2003, Nature Neuroscience.

[32]  Boris Lenhard,et al.  Systematic human/zebrafish comparative identification of cis-regulatory activity around vertebrate developmental transcription factor genes. , 2009, Developmental biology.

[33]  P. Arlotta,et al.  Fezl Is Required for the Birth and Specification of Corticospinal Motor Neurons , 2005, Neuron.

[34]  Panayiotis V. Benos,et al.  STAMP: a web tool for exploring DNA-binding motif similarities , 2007, Nucleic Acids Res..

[35]  Tom H. Pringle,et al.  The human genome browser at UCSC. , 2002, Genome research.

[36]  L. Wolpert.  Developmental Biology , 1968, Nature.

[37]  Stephen W. Wilson,et al.  Neurogenin1 is a determinant of zebrafish basal forebrain dopaminergic neurons and is regulated by the conserved zinc finger protein Tof/Fezl. , 2006, Proceedings of the National Academy of Sciences of the United States of America.

[38]  Tilman Sanchez-Elsner,et al.  Noncoding RNAs of Trithorax Response Elements Recruit Drosophila Ash1 to Ultrabithorax , 2006, Science.

[39]  M. Nóbrega,et al.  Scanning Human Gene Deserts for Long-Range Enhancers , 2003, Science.

[40]  S. Mcconnell,et al.  Fezl regulates the differentiation and axon targeting of layer 5 subcortical projection neurons in cerebral cortex. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[41]  William Stafford Noble,et al.  Assessing computational tools for the discovery of transcription factor binding sites , 2005, Nature Biotechnology.

[42]  R. Place,et al.  MicroRNA-373 induces expression of genes with complementary promoter sequences , 2008, Proceedings of the National Academy of Sciences.

[43]  Yang Liu,et al.  Mouse Brain Organization Revealed Through Direct Genome-Scale TF Expression Analysis , 2004, Science.

[44]  W. J. Kent,et al.  Environmentally Induced Foregut Remodeling by PHA-4/FoxA and DAF-12/NHR , 2004, Science.

[45]  A. Lumsden,et al.  Compartments and their boundaries in vertebrate brain development , 2005, Nature Reviews Neuroscience.

[46]  C. Redies,et al.  Modularity in vertebrate brain development and evolution , 2001, BioEssays : news and reviews in molecular, cellular and developmental biology.

[47]  A. Orth,et al.  Large-scale analysis of the human and mouse transcriptomes , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[48]  E. Davidson.  Genomic Regulatory Systems: Development and Evolution , 2005 .

[49]  J. Bieker.  Krüppel-like Factors: Three Fingers in Many Pies* , 2001, The Journal of Biological Chemistry.

[50]  R. Tjian,et al.  Transcription regulation and animal diversity , 2003, Nature.

[51]  C. Stern.  Initial patterning of the central nervous system: How many organizers? , 2001, Nature Reviews Neuroscience.

[52]  Stephen W. Wilson,et al.  Early steps in the development of the forebrain. , 2004, Developmental cell.

[53]  J. Mattick,et al.  Raising the estimate of functional human sequences. , 2007, Genome research.

[54]  William Stafford Noble,et al.  Identification and analysis of functional elements in 1% of the human genome by the ENCODE pilot project , 2007, Nature.

[55]  T. Curiel.  Regulatory T-cell development: is Foxp3 the decider? , 2007, Nature Medicine.

[56]  J. Rubenstein,et al.  Regionalization of the prosencephalic neural plate. , 1998, Annual review of neuroscience.

[57]  J. Eisen,et al.  Motoneuron fate specification revealed by patterned LIM homeobox gene expression in embryonic zebrafish. , 1995, Development.

[58]  Debbie K Goode,et al.  BMC Developmental Biology BioMed Central Database , 2007 .

[59]  Y. Yoshihara,et al.  Zinc-finger gene Fez in the olfactory sensory neurons regulates development of the olfactory bulb non-cell-autonomously , 2006, Development.

[60]  Peter W. Markstein,et al.  A regulatory code for neurogenic gene expression in the Drosophila embryo , 2004, Development.

[61]  K. Kawakami,et al.  Patterning the zebrafish diencephalon by the conserved zinc-finger protein Fezl , 2007, Development.

[62]  H. Okamoto,et al.  Developmental regulation of Islet‐1 mRNA expression during neuronal differentiation in embryonic zebrafish , 1994, Developmental dynamics : an official publication of the American Association of Anatomists.

[63]  Monte Westerfield,et al.  The Zebrafish Information Network: the zebrafish model organism database , 2005, Nucleic Acids Res..

[64]  Michael Levine,et al.  Whole-Genome Analysis of Dorsal-Ventral Patterning in the Drosophila Embryo , 2002, Cell.

[65]  M. Levine,et al.  Regulation of even‐skipped stripe 2 in the Drosophila embryo. , 1992, The EMBO journal.

[66]  G. Lutfalla,et al.  Identification of the Zebrafish IFN Receptor: Implications for the Origin of the Vertebrate IFN System12 , 2007, The Journal of Immunology.

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