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CALTECH CS 191A - Genomic Cis-Regulatory Logic

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Genomic Cis-Regulatory Logic:Experimental and ComputationalAnalysis of a Sea Urchin GeneChiou-Hwa Yuh, Hamid Bolouri, Eric H. Davidson*The genomic regulatory network that controls gene expression ultimately determinesform and function in each species. The operational nature of the regulatory programmingspecified in cis-regulatory DNA sequence was determined from a detailed functionalanalysis of a sea urchin control element that directs the expression of a gene in theendoderm during development. Spatial expression and repression, and the changingrate of transcription of this gene, are mediated by a complex and extended cis-regulatorysystem. The system may be typical of developmental cis-regulatory apparatus. All of itsactivities are integrated in the proximal element, which contains seven target sites forDNA binding proteins. A quantitative computational model of this regulatory element wasconstructed that explicitly reveals the logical interrelations hard-wired into the DNA.The genomic organization of cis-regulatorysystems lies at the nexus of developmentand evolution. Regulated transcription ofthousands of genes controls the mecha-nisms by which morphological form anddifferentiated cell function are spatially or-ganized during development, and the se-quences of the transcription factor targetsites in the regulatory DNA of each of thesegenes determines the inputs to which it willrespond (1). Reorganization of develop-mental cis-regulatory systems and of thenetworks in which they are linked musthave played a major role in metazoan evo-lution, because differences in the genetical-ly controlled developmental process under-lie the particular morphologies and func-tional characteristics of diverse animals. Forboth developmental and evolutionary bio-science, understanding genomic cis-regula-tory systems is a central necessity.We now present an experimental analy-sis of the multiple functions of a well de-fined cis-regulatory element that controlsthe expression of a gene during the devel-opment of the sea urchin embryo. The out-come is a computational model of the ele-ment, in which the logical functions medi-ated through its DNA target site sequencesare explicitly represented. The regulatoryDNA sequences of the genome may specifythousands of such information-processingdevices.The Endo16 cis-regulatory system.Endo16 is a gene that encodes a polyfunc-tional secreted protein (2) of the midgut inthe late embryo and larva. Transcription ofthe gene is activated soon after the primor-dial endoderm lineages are specified (in latecleavage), long before the gut forms (3, 4).Endo16 transcription is specifically re-pressed in the embryonic cell lineages thatare adjacent to the primordial endoderm orvegetal plate, that is, in cells that will giverise to ectoderm above the vegetal plate andto skeletogenic mesenchyme below (5). Inthe late blastula, all cells of the vegetalplate express Endo16, and after invagina-tion this gene is expressed throughout thearchenteron (5, 6). During gastrulation, thegene is activated in an additional ring ofprospective endoderm cells surrounding theblastopore; soon after this gene is activated,these cells invaginate as well to form thehindgut (6). Endo16 expression is thus anexcellent marker of endoderm cell fatespecification, in both the initial and laterphases of that process. Toward the end ofembryogenesis, Endo16 expression becomesconfined to the differentiating cells of themidgut (4). Transcription is extinguished inthe foregut and the delaminating mesodermin the late gastrula, and thereafter in thehindgut; however, there is an increase inthe rate of transcription in the midgut,where it can still be detected in advancedfeeding larval stages.Earlier results have indicated the func-tional and structural organization of theEndo16 cis-regulatory system (Fig. 1) (5, 7,8). When introduced into sea urchin eggs,the DNA sequence extending about 2300base pairs upstream from the transcriptionstart site is necessary and sufficient to re-create the expression of a linked reportergene in the same developmental and spatialpattern as displayed by the endogenousEndo16 gene (7). Within this cis-regulatorydomain (Fig. 1A), target sites have beenmapped for 15 different proteins that bindwith high specificity, that is, $104timestheir affinity for synthetic double-strandedcopolymer of deoxyinosine and deoxycyti-dine [poly(dI-dC)zpoly(dI-dC)] (7). Thoughsome have been identified and cloned, mostof these proteins are known only by theirmolecular mass, their DNA binding proper-ties, and their site specificity.We have unraveled the functional orga-nization of the 2300–base pair cis-regulato-ry system by determining the expression ofconstructs that include different subregionsof the sequence, normal or mutated, orsynthetic oligonucleotides representing ver-sions of the specific target sites [see also (5,8)]. Like other cis-regulatory systems thatmediate complex developmental patterns ofexpression, the Endo16 system is modular inorganization (1, 9). That is, it consists ofsubelements of the DNA sequence, each ofwhich can execute a certain regulatoryfunction when included in a construct bear-ing either its own or a heterologous frag-ment of DNA on which the basal transcrip-tion apparatus will assemble. Each such sub-element or regulatory module contains mul-tiple target sites for DNA binding factors;there are typically four to eight differentfactors per module (1), and Endo16 con-forms to this expectation. The modular el-ements indicated by these experiments (5,8) are indicated by the capital letters (G toA) in Fig. 1A. However, upstream of mod-ule B the boundaries of the subelements areas yet poorly defined.When tested individually, the most distalelement, module G, has the capacity tocause expression in the endoderm, as domodules B and A (5). However, their func-tions differ: Module G is relatively weak andappears to act throughout as an ancillaryelement; module B functions mainly in laterdevelopment (5, 8), and after gastrulation italone suffices to produce accurate midgutexpression (5). Module A is probably respon-sible for initiating expression in the vegetalplate in the early embryo. In a construct thatincludes no other cis-regulatory subelements,the transcription-enhancing activity of mod-ule A rises early in development, but it thendeclines and disappears when expression isbecoming confined to the midgut and mod-ule B becomes dominant (5, 8).Under normal conditions, the


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