Evaluation of the RET regulatory landscape reveals the biological relevance of a HSCR-implicated enhancer.

Evolutionary sequence conservation is now a relatively common approach for the prediction of functional DNA sequences. However, the fraction of conserved non-coding sequences with regulatory potential is still unknown. In this study, we focus on elucidating the regulatory landscape of RET, a crucial developmental gene within which we have recently identified a regulatory Hirschsprung disease (HSCR) susceptibility variant. We report a systematic examination of conserved non-coding sequences (n=45) identified in a 220 kb interval encompassing RET. We demonstrate that most of these conserved elements are capable of enhancer or suppressor activity in vitro, and the majority of the elements exert cell type-dependent control. We show that discrete sequences within regulatory elements can bind nuclear protein in a cell type-dependent manner that is consistent with their identified in vitro regulatory control. Finally, we focused our attention on the enhancer implicated in HSCR to demonstrate that this element drives reporter expression in cell populations of the excretory system and central nervous system (CNS) and peripheral nervous system (PNS), consistent with expression of the endogenous RET protein. Importantly, this sequence also modulates expression in the enteric nervous system consistent with its proposed role in HSCR.

[1]  D. Haussler,et al.  Ultraconserved Elements in the Human Genome , 2004, Science.

[2]  A. Reymond,et al.  Conserved non-genic sequences — an unexpected feature of mammalian genomes , 2005, Nature Reviews Genetics.

[3]  Nicholas L. Bray,et al.  AVID: A global alignment program. , 2003, Genome research.

[4]  Chaya Kalcheim,et al.  The Neural Crest: General Introduction , 1999 .

[5]  P. Leder,et al.  A conserved sequence in the immunoglobulin J kappa-C kappa intron: possible enhancer element. , 1983, Nature.

[6]  J. Osinga,et al.  Identifying candidate Hirschsprung disease-associated RET variants. , 2005, American journal of human genetics.

[7]  Lior Pachter,et al.  VISTA: computational tools for comparative genomics , 2004, Nucleic Acids Res..

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

[9]  A. McCallion,et al.  Genomic variation in multigenic traits: Hirschsprung disease. , 2003, Cold Spring Harbor symposia on quantitative biology.

[10]  M. Picciotto,et al.  Mouse genetics and transgenics: a practical approach , 2000, Heredity.

[11]  Jonathan C. Cohen,et al.  An Apolipoprotein Influencing Triglycerides in Humans and Mice Revealed by Comparative Sequencing , 2001, Science.

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

[13]  Nancy F. Hansen,et al.  Comparative analyses of multi-species sequences from targeted genomic regions , 2003, Nature.

[14]  Mouse Genome Sequencing Consortium.  Initial sequencing and comparative analysis of the mouse genome , 2002, Nature.

[15]  K. Correia,et al.  Whole-mount in situ hybridization to mouse embryos. , 2001, Methods.

[16]  B. Birren,et al.  Sequencing and comparison of yeast species to identify genes and regulatory elements , 2003, Nature.

[17]  Thomas J. Hudson,et al.  Cis-Acting Regulatory Variation in the Human Genome , 2004, Science.

[18]  Klaudia Walter,et al.  Open access, freely available online PLoS BIOLOGY Highly Conserved Non-Coding Sequences Are Associated with Vertebrate Development , 2022 .

[19]  F. Costantini,et al.  Common origin and developmental dependence on c-ret of subsets of enteric and sympathetic neuroblasts. , 1996, Development.

[20]  D. Haussler,et al.  Evolutionarily conserved elements in vertebrate, insect, worm, and yeast genomes. , 2005, Genome research.

[21]  D. Cox,et al.  Noncoding sequences conserved in a limited number of mammals in the SIM2 interval are frequently functional. , 2004, Genome research.

[22]  A. Munnich,et al.  Homozygosity for a frequent and weakly penetrant predisposing allele at the RET locus in sporadic Hirschsprung disease , 2005, Journal of Medical Genetics.

[23]  R. Gibbs,et al.  Large-scale comparative sequence analysis of the human and murine Bruton's tyrosine kinase loci reveals conserved regulatory domains. , 1997, Genome research.

[24]  F. Costantini,et al.  Expression of the c-ret proto-oncogene during mouse embryogenesis. , 1993, Development.

[25]  Minerva M. Carrasquillo,et al.  Genome-wide association study and mouse model identify interaction between RET and EDNRB pathways in Hirschsprung disease , 2002, Nature Genetics.

[26]  S. Batzoglou,et al.  Quantitative estimates of sequence divergence for comparative analyses of mammalian genomes. , 2003, Genome research.

[27]  D. Pribnow.  Nucleotide sequence of an RNA polymerase binding site at an early T7 promoter. , 1975, Proceedings of the National Academy of Sciences of the United States of America.

[28]  P. Stenson,et al.  Human Gene Mutation Database (HGMD , 2003 .

[29]  J. Milbrandt,et al.  Expression of Neurturin, GDNF, and GDNF Family-Receptor mRNA in the Developing and Mature Mouse , 1999, Experimental Neurology.

[30]  P. Stenson,et al.  Human Gene Mutation Database (HGMD®): 2003 update , 2003, Human mutation.

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

[32]  Lior Pachter,et al.  VISTA : visualizing global DNA sequence alignments of arbitrary length , 2000, Bioinform..

[33]  W. Miller,et al.  Identification of a coordinate regulator of interleukins 4, 13, and 5 by cross-species sequence comparisons. , 2000, Science.

[34]  R. Ravazzolo,et al.  A common haplotype at the 5′ end of the RET proto‐oncogene, overrepresented in Hirschsprung patients, is associated with reduced gene expression , 2005, Human mutation.

[35]  T. Ohta,et al.  On the rate of molecular evolution , 2005, Journal of Molecular Evolution.

[36]  E. Grice,et al.  A common sex-dependent mutation in a RET enhancer underlies Hirschsprung disease risk , 2005, Nature.

[37]  D. Haussler,et al.  Article Identification and Characterization of Multi-Species Conserved Sequences , 2022 .

[38]  J. Segre,et al.  Long-range comparison of human and mouse Sprr loci to identify conserved noncoding sequences involved in coordinate regulation. , 2004, Genome research.