Elsevier

Genomics

Volume 86, Issue 3, September 2005, Pages 295-305
Genomics

Detection of potential GDF6 regulatory elements by multispecies sequence comparisons and identification of a skeletal joint enhancer

https://doi.org/10.1016/j.ygeno.2005.05.003Get rights and content

Abstract

The identification of noncoding functional elements within vertebrate genomes, such as those that regulate gene expression, is a major challenge. Comparisons of orthologous sequences from multiple species are effective at detecting highly conserved regions and can reveal potential regulatory sequences. The GDF6 gene controls developmental patterning of skeletal joints and is associated with numerous, distant cis-acting regulatory elements. Using sequence data from 14 vertebrate species, we performed novel multispecies comparative analyses to detect highly conserved sequences flanking GDF6. The complementary tools WebMCS and ExactPlus identified a series of multispecies conserved sequences (MCSs). Of particular interest are MCSs within noncoding regions previously shown to contain GDF6 regulatory elements. A previously reported conserved sequence at −64 kb was also detected by both WebMCS and ExactPlus. Analysis of LacZ-reporter transgenic mice revealed that a 440-bp segment from this region contains an enhancer for Gdf6 expression in developing proximal limb joints. Several other MCSs represent candidate GDF6 regulatory elements; many of these are not conserved in fish or frog, but are strongly conserved in mammals.

Section snippets

Multispecies sequences and alignments

The 209-kb mouse BAC clone RPCI23-117O7 contains the entire Gdf6 gene and extensive flanking regions [4]. Using the previously established sequence of this BAC as a reference, we have generated (∼2.7 Mb) or obtained (∼1.5 Mb) sequences of the orthologous genomic regions from 13 additional vertebrates (Fig. 1a). A previous PipMaker analysis of the mammalian GDF6 sequences has been described [16]. To examine the degree of noncoding conservation in other vertebrates, we obtained sequences from

Discussion

Previous human and mouse sequence comparisons suggested the presence of numerous conserved noncoding regions within and flanking the GDF6 gene [4], [16]. These regions represent tantalizing candidates for serving as cis-acting regulatory elements that mediate the complex expression of GDF6 [8], [9]. Here, we report an extension of those studies that has involved the generation and comparison of the sequence of the genomic region encompassing GDF6 in multiple additional vertebrates. Two

BAC clones and sequences

The Gdf6-containing mouse BAC RPCI23-117O7 sequence (GenBank No. AC058786) served as the reference for comparative analyses and corresponds to UCSC Genome Browser coordinates chr4:9,641,000–9,850,732 (mm4/NCBI build 32, October 2003). Orthologous human and rat genomic sequences were retrieved from the respective genome-wide data sets [16], [42], [43]. Minimally overlapping GDF6-containing BACs from rat, chimpanzee, baboon, cow, pig, cat, dog, platypus, and zebrafish were identified [17] and

Acknowledgments

We thank numerous people associated with the NISC Comparative Sequencing Program, in particular Robert Blakesley, Gerry Bouffard, Jennifer McDowell, Baishali Maskeri, Nancy Hanson, the many dedicated mapping and sequencing technicians, and other staff. We also thank Laura Selenke and Lissett Ramirez for expert technical assistance and Karen Deal, Maureen Gannon, Anna Means, and Laura Wilding for generously sharing equipment and advice. We also thank Ronald Chandler for helpful discussions.

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