Comparative sequence analysis of the Gdf6 locus reveals a duplicon-mediated chromosomal rearrangement in rodents and rapidly diverging coding and regulatory sequences☆
Introduction
It has been estimated that 3.5–5% of the human genome contains highly similar segmental duplicated regions or “duplicons” [1]. Through recombination, duplicated genomic segments can lead to chromosomal rearrangements such as deletions, inversions, and translocations. Segmental duplications are associated with several human “genomic disorders” that are characterized by rearrangements [2]. Segmental duplications are also associated with certain chromosomal rearrangements that characterize mammalian species [3], [4], [5]. These data suggest that segmental duplications play a significant role in mediating chromosomal rearrangements, including those driving evolutionary changes.
In addition, duplications that include entire genes can allow gene diversification and/or selection for novel functional roles by relaxing selective constraints on one or more gene copies [6]. Along with coding exons, cis-acting transcriptional regulatory elements such as promoters, enhancers, insulators, or “boundary” elements are presumably often present in segmental duplications. This raises the possibility that mobilization of such sequences could increase functional diversity by conferring novel regulatory abilities on genes near the relocation site. Numerous regulatory sequences have been documented that can exert effects across tens or even hundreds of kilobases, such as those controlling Shh [7], HoxD genes [8], Bmp5 [9], [10], interleukins [11], and globins [12], [13]. These examples of long-range regulatory sequences support the possibility that segmental duplications and/or rearrangements could disrupt existing gene/cis-element interactions, or create new ones.
Gdf6 is a member of the BMP (Bone Morphogenetic Protein) family of secreted signaling molecules [14]. Like its close paralog Gdf5, Gdf6 is expressed in mouse embryonic limb buds in stripes that mark the locations of future skeletal joints just prior to the initial stages of joint formation. Mice with mutations in either Gdf5 or Gdf6 have fusions of specific limb joints [14], [15]. Gdf6 mutant mice and/or Gdf5/Gdf6 double mutant animals also suffer fusions or malformations in cranial sutures, middle ear joints, and spinal joints [15]. These and other data strongly indicate that Gdf genes are key patterning molecules that control skeletal joint patterning [16], [17]. The regulatory mechanisms that control Gdf transcription in developing joint regions are currently not understood, but may provide insights into signaling events that direct specification or differentiation of articular cartilage from precursor cells. Recently, we used a BAC-transgene approach to identify several cis-regulatory elements of the mouse Gdf6 gene [18]. Those studies revealed distant intergenic sequences that drive Gdf6 transcription in embryonic elbow and knee joint primordia, the larynx, retina, digit tips, genitalia, and other anatomical locations. More detailed analysis of a region approximately 60 kb 5′ of the Gdf6 promoter revealed an enhancer that can drive reporter gene transcription specifically in the elbow and knee joints of transgenic mouse embryos [18]. Mouse-human sequence comparisons revealed that this and other flanking regions contain numerous islands of striking noncoding conservation, suggesting individual Gdf6 enhancers. Surprisingly, not all aspects of endogenous Gdf6 regulation were recapitulated by BAC transgenes that span almost 250 kb around the gene. For example, enhancers that drive Gdf6 expression in embryonic carpal, tarsal, or spinal joints were not detected, even though Gdf6 is also transcribed in these joints [14], [15], [18]. This suggests that additional Gdf6 enhancers exist but are probably far (>130 kb) from the Gdf6 promoter. The structure of neighboring genes and degree of cross-species conservation in distant flanking regions could therefore guide future efforts to locate distant Gdf6 regulatory sequences.
To extend our analysis of Gdf6 intergenic sequences, we have used a comparative sequence approach. Here we report the sequencing of Gdf6 in multiple vertebrates [19] and data indicating that evolutionary rearrangements occurred near Gdf6 in the rodent lineage. In particular, we describe a segmental duplication that likely mediated a chromosomal rearrangement and mobilized a copy of a tissue-specific enhancer.
Section snippets
Different structures of genes upstream of Gdf6 in mouse and human
Previous analysis revealed that a 2.9-kb fragment of the Gdf6 locus contains enhancer ability sufficient to drive expression of an hsp-LacZ minigene in proximal limb joints (elbow, knee, shoulder, and hip) in patterns that closely resemble normal Gdf6 expression [18]. We refer to the regulatory module in this fragment as the Gdf6 Proximal Joint Element (PJE). The large distance separating the PJE and the Gdf6 promoter [18] led us to examine the arrangement of neighboring genes in more detail.
Discussion
Here we describe a rodent-specific duplication that catalyzed a chromosomal rearrangement at the ancestral Uqcrb/Gdf6 locus. By detailed comparison of orthologous sequences from several mammals, we have defined a rodent-specific segmental duplication that spanned the Uqcrb gene and a distant cis-regulatory element for Gdf6. One duplicon maintained the intact Uqcrb gene, but its linked copy of the Gdf6 PJE conserved sequence is accumulating divergent mutations (in mouse) or has been lost
Isolation of Gdf6 BACs by overgo hybridization
Orthologous Gdf6 BACs were isolated by cross-hybridization to their respective BAC libraries using a set of conserved universal overgo probes [41] designed from regions of human/mouse homology in the Gdf6 gene region, using previously described methods [19]. Orthologous BACs were sequenced to advanced phase 2 quality using a shotgun approach by the NISC Comparative Sequencing Program.
Sequences used for analysis
The mouse Gdf6 BAC sequence used for analyses in Fig. 2, Fig. 3 is the reverse complement of finished BAC
Acknowledgments
We thank numerous people associated with the NISC Comparative Sequencing Program, in particular the dedicated technicians and other staff involved in BAC isolation, mapping, and sequencing. Ronald Chandler was supported by the Vanderbilt University Developmental Biology Training Grant. We gratefully acknowledge the Baylor Genome Center for generating the rat BAC CH230-258L8 draft sequence. We thank Michelle Johnson and David Kingsley (Stanford University) for human GDF6 coding sequences.
References (44)
Recent segmental duplications in the human genome
Science
(2002)- et al.
Segmental duplications: an ‘expanding’ role in genomic instability and disease
Nature Rev. Genet.
(2001) - et al.
Enrichment of segmental duplications in regions of breaks of synteny between the human and mouse genomes suggest their involvement in evolutionary rearrangements
Hum. Mol. Genet.
(2003) - et al.
The evolutionary chromosome translocation 4;19 in Gorilla gorilla is associated with microduplication of the chromosome fragment syntenic to sequences surrounding the human proximal CMT1A-REP
Genome Res.
(2001) Pericentromeric duplications in the laboratory mouse
Genome Res.
(2003)Evolution by Gene Duplication
(1970)A long-range Shh enhancer regulates expression in the developing limb and fin and is associated with preaxial polydactyly
Hum. Mol. Genet.
(2003)- et al.
A global control region defines a chromosomal regulatory landscape containing the HoxD cluster
Cell
(2003) - et al.
An extensive 3′ regulatory region controls expression of Bmp5 in specific anatomical structures of the mouse embryo
Genetics
(1998) - et al.
Efficient studies of long-distance Bmp5 gene regulation using bacterial artificial chromosomes
Proc. Natl. Acad. Sci. USA
(2000)
Identification of a coordinate regulator of interleukins 4, 13, and 5 by cross-species sequence comparisons
Science
A major positive regulatory region located far upstream of the human alpha-globin gene locus
Genes Dev.
A large upstream region is not necessary for gene expression or hypersensitive site formation at the mouse beta-globin locus
Proc. Natl. Acad. Sci. USA
Limb alterations in brachypodism mice due to mutations in a new member of the TGF beta-superfamily [see comments]
Nature
Multiple joint and skeletal patterning defects caused by single and double mutations in the mouse Gdf6 and Gdf5 genes
Dev. Biol.
Mechanisms of GDF-5 action during skeletal development
Development (Suppl.)
GDF5 coordinates bone and joint formation during digit development
Dev. Biol.
A general approach for identifying distant regulatory elements applied to the Gdf6 gene
Genome Res.
Comparative analyses of multi-species sequences from targeted genomic regions
Nature
Crystal structure of the cytochrome bc1 complex from bovine heart mitochondria
Science
Localization of the human gene encoding the 13.3-kDa subunit of mitochondrial complex III (UQCRB) to 8q22 by in situ hybridization
Cytogenet. Cell Genet.
Genome sequence of the Brown Norway rat yields insights into mammalian evolution
Nature
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