Genomic structure and functional control of the Dlx3-7 bigene cluster Kenta Sumiyama*, Steven Q. Irvine*, David W. Stock†, Kenneth M. Weiss‡, Kazuhiko Kawasaki‡, Nobuyoshi Shimizu§, Cooduvalli S. Shashikant¶, Webb Millerʈ, and Frank H. Ruddle*,** *Department of Molecular, Cellular, and Developmental Biology, Yale University, New Haven, CT 06511; †Department of Biology, University of Colorado, Boulder, CO 80309; Departments of ‡Anthropology, ¶Dairy and Animal Science, and ʈComputer Science, Pennsylvania State University, University Park, PA 16802; and §Department of Molecular Biology, Keio University School of Medicine, Tokyo 160, Japan Contributed by Frank H. Ruddle, November, 1, 2001 The Dlx genes are involved in early vertebrate morphogenesis, Dlx3 is expressed in the first and second visceral arches and notably of the head. The six Dlx genes of mammals are arranged fronto-nasal ectoderm at E9.5 (9). Later in development, Dlx3 is in three convergently transcribed bigene clusters. In this study, we also expressed in the external respiratory epithelium of the nares, examine the regulation of the Dlx3-7 cluster of the mouse. We in whisker follicles, taste bud primordia, dental and mammary obtained and sequenced human and mouse P1 clones covering the gland epithelia, apical ectodermal ridge (AER) of limb buds, entire Dlx3-7 cluster. Comparative analysis of the human and genital primordia, and in several additional sites of epithelial- mouse sequences revealed several highly conserved noncoding mesenchymal interaction (8, 9). By the end of embryonic devel- regions within 30 kb of the Dlx3-7-coding regions. These conserved opment, Dlx3 is down-regulated, except in the skin, where it is elements were located both 5؅ of the coding exons of each gene transcribed in stratified epidermis and in the matrix cells of the and in the intergenic region 3؅ of the exons, suggesting that some hair follicles (10). The Dlx3 pattern of expression is different in enhancers might be shared between genes. We also found that the some respects in comparison with the bigene clusters Dlx2–1 and protein sequence of Dlx7 is evolving more rapidly than that of Dlx3. Dlx5–6 in that Dlx3 is not expressed in the central nervous system We conducted a functional study of the 79-kb mouse genomic and is largely expressed in recently acquired structures common clone to locate cis-element activity able to reproduce the endog- to the mammals. Dlx7 has an overlapping expression pattern with enous expression pattern by using transgenic mice. We inserted a the Dlx3 gene in the visceral arches and limb buds before E10.5 lacZ reporter gene into the first exon of the Dlx3 gene by using (ref. 11 and unpublished data). homologous recombination in yeast. Strong lacZ expression in The Dlx genes provide an interesting model for the regulation embryonic (E) stage E9.5 and E10.5 mouse embryos was found in of clustered genes because their arrangement in pairs is the the limb buds and first and second visceral arches, consistent with simplest case of gene clustering. The fact that all of the mam- the endogenous Dlx3 expression pattern. This result shows that malian clusters have the same genomic arrangement suggests the 79-kb region contains the major cis-elements required to direct that the clusters have not undergone extensive rearrangement the endogenous expression of Dlx3 at stage E10.5. To test for since their formation and may help elucidate the fate of cis- enhancer location, we divided the construct in the mid-intergenic regulatory elements after gene duplication. The Dlx3–7 cluster region and injected the Dlx3 gene portion. This shortened frag- is of special interest to us because its expression pattern lacks the ment lacking Dlx7-flanking sequences is able to drive expression in complication of CNS expression. In this report, we provide the limb buds but not in the visceral arches. This observation is detailed information on the structural organization of the Dlx3–7 consistent with a cis-regulatory enhancer-sharing model within the bigene cluster, identify conserved sequence elements in the Dlx bigene cluster. coding and noncoding domains, and by means of functional transgenic analysis test the enhancer activity for Dlx3–7 expres- he mammalian Dlx family consists of six genes with ho- sion control. Tmeoboxes related to that of Drosophila Distal-less (Dll). The mammalian genes take the form of bigene clusters. The paired Materials and Methods genes, termed Dlx2–1, Dlx5–6, and Dlx3–7, are organized in an P1 Artificial Chromosome (PAC) Clones and Sequencing. The clones inverted, convergently transcribed manner (1–3). It has been P1–972 and P1–1490 were isolated from Incyte Genomics (Palo proposed that a single Dlx gene duplicated to form an ancestral Alto, CA) mouse and human PAC libraries, respectively, by PCR inverted bigene cluster during the evolution of the early chor- screening of pooled libraries by using primers for the 3Ј end of dates and subsequent duplications gave rise to multiple bigene the Dlx3͞DLX3 homeodomain (3, 12). clusters (4). This hypothesis is based on phylogenetic clustering Nucleotide sequences were primarily determined by using the of Dlx2, 3, 5 to the exclusion of Dlx1, 6, 7. It is likely that the three shotgun method, and unsequenced gaps were filled by primer Dlx clusters underwent duplication together with the Hox gene walking (see ref. 13 for methods). The sequence was confirmed clusters, because Dlx2–1, 5–6, and 3–7 are closely linked to the by comparing a restriction map deduced from genomic sequence Hox D, A, and B clusters, respectively (1, 3, 5). There is a certain with an experimentally constructed restriction map. degree of spatial and temporal expression overlap between the linked Dlx genes around embryonic stage (E) E10.5 (6). Models Cluster Alignments and Sequence Comparisons. Alignments were are proposed to explain this overlapping pattern, including computed with PIPMAKER (14) (available at http:͞͞bio.cse. cis-regulatory sharing between paired genes (7). In this report, we will focus on the Dlx3–7 bigene cluster with the ultimate goal Abbreviations: E, embryonic stage; AER, apical ectodermal ridge; PAC, P1 artificial chro- of testing the shared enhancer hypothesis of bigene expression mosome. control. Data deposition: The sequences reported in this paper have been deposited in the GenBank The mouse Dlx3 gene is first expressed weakly in the rostral database (accession nos. AF452637 and AF452638). ectoderm, anterior to the neural plate during the head fold stage **To whom reprint requests should be addressed. E-mail: [email protected]. (6). Dlx3 is expressed by E8.5 in the ectoplacental cone and The publication costs of this article were defrayed in part by page charge payment. This chorionic plate, developing into a high level of expression by day article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. E10.5 in the labyrinthine layer of the embryonic placenta (8). §1734 solely to indicate this fact. 780–785 ͉ PNAS ͉ January 22, 2002 ͉ vol. 99 ͉ no. 2 www.pnas.org͞cgi͞doi͞10.1073͞pnas.012584999 Downloaded by guest on October 2, 2021 psu.edu͞pipmaker͞). The BLAST 2 sequences program accessed agreement with our earlier studies (3). Thus, we can divide the through the National Center for Biotechnology Information bigene clusters into three domains: an intergenic 3Ј region website (15) was also used for identifying local sequence shared by both genes, a flanking 5Ј upstream region unique to conservation. the Dlx7 gene, and a flanking 5Ј region unique to the Dlx3 gene (Figs. 1 and 2). The intergenic regions measure 17 kb in both Construction of the Mouse Dlx3-lacZ Reporter Gene. The 80-kb insert species. At least 7 kb of sequence upstream of the Dlx3 trans- of the P1–972 was captured into the pPAC-ResQ vector by using lation start site and at least 37 kb upstream of the Dlx7 homologous recombination in yeast (16) to make construct translation start site are captured in both the human and mouse Dlx37-A8. In brief, 50 ng of linearized pPAC-ResQ vector and clones. A close comparison of the human and mouse clones 400 ng of circular PAC P1–972 were cotransformed into yeast showed no other genes in the P1 clones, although several strain Y724 (17). More than 60% of the 200 clones obtained were expressed sequence tag sequences show similarity in the first 20 identified as recombinant by hybridization tests by using the Dlx3 kb upstream of Dlx7. 5Ј upstream region as probe. Yeast genomic DNA from recom- We compared the mouse Dlx3-7 bigene cluster to its human binant clones was isolated by using the PureGene kit (Gentra paralogs, namely, DLX2–1 and DLX5–6 (data not shown). The Systems), transformed into Escherichia coli DH10B cells by general genomic organization is similar among the clusters. All electroporation (16), and checked for rearrangement by restric- show convergent translation, three exons, and 5Ј-flanking and tion analysis. 3Ј-intergenic domains. The DLX2–1 and DLX5–6 intergenic The lacZ reporter gene was inserted in-frame into the first regions both measure 10 kb. Sequence similarities are found only exon of the mouse Dlx3 gene by using another round of homol- in the homeodomains, whereas other coding and noncoding ogous recombination in yeast. A targeting vector (pLZFSV- domains show low sequence conservation. The absence of Dlx3AB) was generated in which a lacZ-Ura3 cassette (18) was sequence similarity in the noncoding domains is interesting, flanked by PCR generated recombinogenic ends (Fig. 4a) from considering the paralogous nature of the bigene clusters and Ј Ј 5 of the Dlx3-coding region [primers KL001 (5 -GGT AAC their overlapping patterns of expression during development. AAC AAA GAG GGT TGA GA-3Ј) and KL 002 (5Ј-GAA Ј GGA GCC GCT CAT GCT-3 )], and portions of exon 1 and the Protein Sequence Comparisons Between Dlx7 and Dlx3.