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Comparative Study
, 99 (2), 780-5

Genomic Structure and Functional Control of the Dlx3-7 Bigene Cluster

Affiliations
Comparative Study

Genomic Structure and Functional Control of the Dlx3-7 Bigene Cluster

Kenta Sumiyama et al. Proc Natl Acad Sci U S A.

Abstract

The Dlx genes are involved in early vertebrate morphogenesis, notably of the head. The six Dlx genes of mammals are arranged in three convergently transcribed bigene clusters. In this study, we examine the regulation of the Dlx3-7 cluster of the mouse. We obtained and sequenced human and mouse P1 clones covering the entire Dlx3-7 cluster. Comparative analysis of the human and mouse sequences revealed several highly conserved noncoding regions within 30 kb of the Dlx3-7-coding regions. These conserved elements were located both 5' of the coding exons of each gene and in the intergenic region 3' of the exons, suggesting that some enhancers might be shared between genes. We also found that the protein sequence of Dlx7 is evolving more rapidly than that of Dlx3. We conducted a functional study of the 79-kb mouse genomic clone to locate cis-element activity able to reproduce the endogenous expression pattern by using transgenic mice. We inserted a lacZ reporter gene into the first exon of the Dlx3 gene by using homologous recombination in yeast. Strong lacZ expression in embryonic (E) stage E9.5 and E10.5 mouse embryos was found in the limb buds and first and second visceral arches, consistent with the endogenous Dlx3 expression pattern. This result shows that the 79-kb region contains the major cis-elements required to direct the endogenous expression of Dlx3 at stage E10.5. To test for enhancer location, we divided the construct in the mid-intergenic region and injected the Dlx3 gene portion. This shortened fragment lacking Dlx7-flanking sequences is able to drive expression in the limb buds but not in the visceral arches. This observation is consistent with a cis-regulatory enhancer-sharing model within the Dlx bigene cluster.

Figures

Figure 1
Figure 1
Dot plot analysis between mouse Dlx3–7 cluster (P1–972: horizontal line) and human DLX3–7 cluster (P1–1490: vertical line). Black boxes indicate exons. Exon and intron positions of mouse Dlx3 and Dlx7 are color coded as green and yellow, respectively. Dlx7- and Dlx3-flanking conservation motifs (F7–1, F3–1,2) are indicated in red. Intergenic conservation motifs (from I37–1 to 5) are shown in orange.
Figure 2
Figure 2
Percentage identity plot of mouse Dlx3–7 cluster (horizontal line) to human DLX3–7 cluster. Vertical axis shows percentage of sequence identity from 50% (bottom) to 100% (top). Large black and gray boxes on the plot represent coding and untranslated region, respectively. Small rectangles and triangles are repetitive elements (see percentage identity plot legend). Exon and intron positions are color coded as green and yellow, respectively. Dlx7 and Dlx3 flanking conservation motifs (F7–1, F3–1, 2) are indicated in red. Intergenic conservation motifs (from I37–1 to 5) are shown in orange.
Figure 3
Figure 3
Diagram showing locations and information of five highest sequence similarities in the human and mouse Dlx3–7 intergenic region (from I37–1 to 5). Numbers below are length of conservation and the percentage of sequence identity within the alignment between human and mouse. A 40-bp region with 80% sequence identity is found between I37–1 and I37–5, as noted.
Figure 4
Figure 4
Procedure diagram to make the Dlx37-lacZ-79kb construct. (a) Linearized pPAC-ResQ yeast-bacteria shuttle vector captures P1–972 insert by homologous recombination in yeast. Further homologous recombination makes lacZ-URA3 insertion after eighth amino acid from first methyonine. (b) EcoRI restriction enzyme digestion patterns of original P1–972 (lane 1), pPAC-ResQ with P1–972 insert (lane 2), and with lacZ-URA3 insertion (lane 3). A 5.7-kb fragment is a diagnostic fragment for lacZ-URA3 insertion (lane 3, indicated by arrow).
Figure 5
Figure 5
(a) Diagram of transgenes and summary of reporter expression patterns in transgenic animals. Blue boxes and open circles indicate exons and conserved motifs, respectively. Dlx37-lacZ-79kb has the full-length of P1–972, whereas Dlx37-lacZ-19kb is truncated at the middle of the intergenic region, retaining 7 kb upstream and 4 kb downstream of Dlx3. (b) In situ hybridization of Dlx3 (A and D) and β-galactosidase staining showing transgene expression (B, C, E, and F). (AC) Stage E9.5. (DF) Stage E10.5. (B and E) β-galactosidase staining of Dlx37-lacZ-79kb transgenic embryos. Note staining in visceral arches (indicated by red arrow) and limbs (black filled arrow) detected in in situ hybridization. (C and F) β -galactosidase staining in Dlx37-lacZ-19kb transgenic embryos. Note loss of staining in visceral arches (red arrow). Frontonasal ectoderm expression detected in in situ hybridization (white open arrow) is not detected in transgenic animals.

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