title: In vivo enhancer analysis of human conserved non-coding sequences.
abstract: Identifying the sequences that direct the spatial and temporal expression of genes and defining their function in vivo remains a significant challenge in the annotation of vertebrate genomes. One major obstacle is the lack of experimentally validated training sets. In this study, we made use of extreme evolutionary sequence conservation as a filter to identify putative gene regulatory elements, and characterized the in vivo enhancer activity of a large group of non-coding elements in the human genome that are conserved in human-pufferfish, Takifugu (Fugu) rubripes, or ultraconserved in human-mouse-rat. We tested 167 of these extremely conserved sequences in a transgenic mouse enhancer assay. Here we report that 45% of these sequences functioned reproducibly as tissue-specific enhancers of gene expression at embryonic day 11.5. While directing expression in a broad range of anatomical structures in the embryo, the majority of the 75 enhancers directed expression to various regions of the developing nervous system. We identified sequence signatures enriched in a subset of these elements that targeted forebrain expression, and used these features to rank all approximately 3,100 non-coding elements in the human genome that are conserved between human and Fugu. The testing of the top predictions in transgenic mice resulted in a threefold enrichment for sequences with forebrain enhancer activity. These data dramatically expand the catalogue of human gene enhancers that have been characterized in vivo, and illustrate the utility of such training sets for a variety of biological applications, including decoding the regulatory vocabulary of the human genome.
authors.affiliation: US Department of Energy Joint Genome Institute, Walnut Creek, California 94598, USA. LAPennacchio@lbl.gov
authors.forename: Len A
authors.initials: LA
authors.lastname: Pennacchio
authors.forename: Nadav
authors.initials: N
authors.lastname: Ahituv
authors.forename: Alan M
authors.initials: AM
authors.lastname: Moses
authors.forename: Shyam
authors.initials: S
authors.lastname: Prabhakar
authors.forename: Marcelo A
authors.initials: MA
authors.lastname: Nobrega
authors.forename: Malak
authors.initials: M
authors.lastname: Shoukry
authors.forename: Simon
authors.initials: S
authors.lastname: Minovitsky
authors.forename: Inna
authors.initials: I
authors.lastname: Dubchak
authors.forename: Amy
authors.initials: A
authors.lastname: Holt
authors.forename: Keith D
authors.initials: KD
authors.lastname: Lewis
authors.forename: Ingrid
authors.initials: I
authors.lastname: Plajzer-Frick
authors.forename: Jennifer
authors.initials: J
authors.lastname: Akiyama
authors.forename: Sarah
authors.initials: S
authors.lastname: De Val
authors.forename: Veena
authors.initials: V
authors.lastname: Afzal
authors.forename: Brian L
authors.initials: BL
authors.lastname: Black
authors.forename: Olivier
authors.initials: O
authors.lastname: Couronne
authors.forename: Michael B
authors.initials: MB
authors.lastname: Eisen
authors.forename: Axel
authors.initials: A
authors.lastname: Visel
authors.forename: Edward M
authors.initials: EM
authors.lastname: Rubin
mesh_terms: D000818:Animals; D001483:Base Sequence; D002885:Chromosomes, Human, Pair 16; D017124:Conserved Sequence; D004622:Embryo, Mammalian / Q000378:metabolism; D004625:Embryo, Nonmammalian; D004742:Enhancer Elements, Genetic; D015870:Gene Expression; D015894:Genome, Human; D023281:Genomics / Q000379:methods; D006801:Humans; D051379:Mice; D008822:Mice, Transgenic; D009420:Nervous System / Q000196:embryology / Q000378:metabolism; D016548:Prosencephalon / Q000196:embryology / Q000378:metabolism; D023741:Takifugu / Q000235:genetics; D014157:Transcription Factors / Q000235:genetics
chemical_list: C104146:SALL1 protein, human; D014157:Transcription Factors