HOT regions function as patterned developmental enhancers and have a distinct cis-regulatory signature.

HOT (highly occupied target) regions bound by many transcription factors are considered to be one of the most intriguing findings of the recent modENCODE reports, yet their functions have remained unclear. We tested 108 Drosophila melanogaster HOT regions in transgenic embryos with site-specifically integrated transcriptional reporters. In contrast to prior expectations, we found 102 (94%) to be active enhancers during embryogenesis and to display diverse spatial and temporal patterns, reminiscent of expression patterns for important developmental genes. Remarkably, HOT regions strongly activate nearby genes and are required for endogenous gene expression, as we show using bacterial artificial chromosome (BAC) transgenesis. HOT enhancers have a distinct cis-regulatory signature with enriched sequence motifs for the global activators Vielfaltig, also known as Zelda, and Trithorax-like, also known as GAGA. This signature allows the prediction of HOT versus control regions from the DNA sequence alone.

[1]  Colin N. Dewey,et al.  Discovery of functional elements in 12 Drosophila genomes using evolutionary signatures , 2007, Nature.

[2]  Michael B. Eisen,et al.  Zelda Binding in the Early Drosophila melanogaster Embryo Marks Regions Subsequently Activated at the Maternal-to-Zygotic Transition , 2011, PLoS genetics.

[3]  Raymond K. Auerbach,et al.  modENCODE Project Genome by the Caenorhabditis elegans Integrative Analysis of the , 2011 .

[4]  D. W. Knowles,et al.  Transcription Factors Bind Thousands of Active and Inactive Regions in the Drosophila Blastoderm , 2008, PLoS biology.

[5]  J. Bernués,et al.  The N-terminal POZ Domain of GAGA Mediates the Formation of Oligomers That Bind DNA with High Affinity and Specificity* , 1999, The Journal of Biological Chemistry.

[6]  E. Lécuyer,et al.  Fluorescent in situ hybridization protocols in Drosophila embryos and tissues. , 2008, Methods in molecular biology.

[7]  M. Blaxter.  Revealing the Dark Matter of the Genome , 2010, Science.

[8]  Stephen Butcher,et al.  Temporal Coordination of Gene Networks by Zelda in the Early Drosophila Embryo , 2011, PLoS genetics.

[9]  T. Shimojima,et al.  Drosophila GAGA factor directs histone H3.3 replacement that prevents the heterochromatin spreading. , 2007, Genes & development.

[10]  Thomas Lengauer,et al.  ROCR: visualizing classifier performance in R , 2005, Bioinform..

[11]  James B. Brown,et al.  Developmental roles of 21 Drosophila transcription factors are determined by quantitative differences in binding to an overlapping set of thousands of genomic regions , 2009, Genome Biology.

[12]  Ling V. Sun,et al.  Hotspots of transcription factor colocalization in the genome of Drosophila melanogaster , 2006, Proceedings of the National Academy of Sciences.

[13]  Hugo J. Bellen,et al.  P[acman]: A BAC Transgenic Platform for Targeted Insertion of Large DNA Fragments in D. melanogaster , 2006, Science.

[14]  B. Williams,et al.  Mapping and quantifying mammalian transcriptomes by RNA-Seq , 2008, Nature Methods.

[15]  M. Metzstein,et al.  The zinc-finger protein Zelda is a key activator of the early zygotic genome in Drosophila , 2008, Nature.

[16]  E. Furlong,et al.  Tissue-specific analysis of chromatin state identifies temporal signatures of enhancer activity during embryonic development , 2012, Nature Genetics.

[17]  R. Treisman,et al.  The POZ domain: a conserved protein-protein interaction motif. , 1994, Genes & development.

[18]  S. Elgin,et al.  (CT)n (GA)n repeats and heat shock elements have distinct roles in chromatin structure and transcriptional activation of the Drosophila hsp26 gene , 1993, Molecular and cellular biology.

[19]  Peter J. Bickel,et al.  The Developmental Transcriptome of Drosophila melanogaster , 2010, Nature.

[20]  G. Rubin,et al.  Tools for neuroanatomy and neurogenetics in Drosophila , 2008, Proceedings of the National Academy of Sciences.

[21]  F. Karch,et al.  The Trithorax-like gene encodes the Drosophila GAGA factor , 1994, Nature.

[22]  Gos Micklem,et al.  Supporting Online Material Materials and Methods Figs. S1 to S50 Tables S1 to S18 References Identification of Functional Elements and Regulatory Circuits by Drosophila Modencode , 2022 .

[23]  L. M. Lira,et al.  Sequence-specific antirepression of histone H1-mediated inhibition of basal RNA polymerase II transcription. , 1991, Science.

[24]  John R. ten Bosch,et al.  The TAGteam DNA motif controls the timing of Drosophila pre-blastoderm transcription , 2006, Development.

[25]  M. Ashburner,et al.  Systematic determination of patterns of gene expression during Drosophila embryogenesis , 2002, Genome Biology.

[26]  N. Perrimon,et al.  Exploiting position effects and the gypsy retrovirus insulator to engineer precisely expressed transgenes , 2008, Nature Genetics.

[27]  Robert L. Grossman,et al.  A cis-regulatory map of the Drosophila genome , 2011, Nature.

[28]  M. Bate,et al.  twist: A Myogenic Switch in Drosophila , 1996, Science.

[29]  Steven M. Gallo,et al.  REDfly v3.0: toward a comprehensive database of transcriptional regulatory elements in Drosophila , 2010, Nucleic Acids Res..

[30]  Rahul Satija,et al.  The TAGteam motif facilitates binding of 21 sequence-specific transcription factors in the Drosophila embryo. , 2012, Genome research.

[31]  E. Furlong.  Molecular biology: A fly in the face of genomics , 2011, Nature.