Reed NP, Mortlock DP. Identification of a distant cis-regulatory element controlling pharyngeal arch-specific expression of zebrafish gdf6a/radar. (2010) Dev Dyn 239: 1047-60
Show Abstract · Added June 9, 2010Skeletal formation is an essential and intricately regulated part of vertebrate development. Humans and mice deficient in growth and differentiation factor 6 (Gdf6) have numerous skeletal abnormalities, including joint fusions and cartilage reductions. The expression of Gdf6 is dynamic and in part regulated by distant evolutionarily conserved cis-regulatory elements. radar/gdf6a is a zebrafish ortholog of Gdf6 and has an essential role in embryonic patterning. Here, we show that radar is transcribed in the cells surrounding and between the developing cartilages of the ventral pharyngeal arches, similar to mouse Gdf6. A 312 bp evolutionarily conserved region (ECR5), 122 kilobases downstream, drives expression in a pharyngeal arch-specific manner similar to endogenous radar/gdf6a. Deletion analysis identified a 78 bp region within ECR5 that is essential for transgene activity. This work illustrates that radar is regulated in the pharyngeal arches by a distant conserved element and suggests radar has similar functions in skeletal development in fish and mammals. | Publication | 20201106 (PMID)
10.1002/dvdy.22251 (DOI) |
Pregizer S, Mortlock DP. Control of BMP gene expression by long-range regulatory elements. (2009) Cytokine Growth Factor Rev 20: 509-15
Show Abstract · Added June 9, 2010Much evidence suggests that "developmental regulator" genes, like those encoding transcription factors and signaling molecules, are typically controlled by many modular, tissue-specific cis-regulatory elements that function during embryogenesis. These elements are often far from gene coding regions and promoters. Bone morphogenetic proteins (BMPs) drive many processes in development relating to organogenesis and differentiation. Four BMP family members, Bmp2, Bmp4, Bmp5, and Gdf6, are now known to be under the control of distant cis-regulatory elements. BMPs are thus firmly placed in the category of genes prone to this phenomenon. The analysis of distant BMP regulatory elements has provided insight into the many pleiotropic effects of BMP genes, and underscores the biological importance of non-coding genomic DNA elements. | Publication | 19900834 (PMID)
PMC2787762 (PMCID)
10.1016/j.cytogfr.2009.10.011 (DOI) |
Chandler KJ, Chandler RL, Mortlock DP. Identification of an ancient Bmp4 mesoderm enhancer located 46 kb from the promoter. (2009) Dev Biol 327: 590-602
Show Abstract · Added June 9, 2010Bone morphogenetic protein 4 (Bmp4) is a multi-functional, developmentally regulated gene that is essential for mouse development, as most Bmp4-null mouse embryos die at the onset of gastrulation and fail to develop mesoderm. Little is known about the transcriptional regulation of Bmp4. To identify potential long-range cis-regulatory elements that direct its complex spatiotemporal expression patterns, we surveyed the mouse Bmp4 locus using two overlapping bacterial artificial chromosome (BAC) reporter transgenes. Our findings indicate that tissue-specific cis-regulatory elements reside greater than 28 kb 5' or 3' to the mouse Bmp4 transcription unit. In addition, comparative analyses identified three noncoding evolutionarily conserved regions (ECRs), spaced around the gene and conserved from mammals to fish, that are maintained in a syntenic group across vertebrates. Deletion of one of these conserved sequences (ECR2) from a BAC transgene revealed a tissue-specific requirement for ECR2 in driving Bmp4 expression in extraembryonic and embryonic mesoderm. Furthermore, a 467 bp mouse sequence containing ECR2 reproducibly directed lacZ minigene expression in mesoderm. Taken together, this shows that an ancient, mesoderm-specific cis-regulatory element resides nearly 50 kb 5' to mouse Bmp4. | Publication | 19159624 (PMID)
PMC2846791 (PMCID)
10.1016/j.ydbio.2008.12.033 (DOI) |
Chandler KJ, Chandler RL, Broeckelmann EM, Hou Y, Southard-Smith EM, Mortlock DP. Relevance of BAC transgene copy number in mice: transgene copy number variation across multiple transgenic lines and correlations with transgene integrity and expression. (2007) Mamm Genome 18: 693-708
Show Abstract · Added April 7, 2010Bacterial artificial chromosomes (BACs) are excellent tools for manipulating large DNA fragments and, as a result, are increasingly utilized to engineer transgenic mice by pronuclear injection. The demand for BAC transgenic mice underscores the need for careful inspection of BAC integrity and fidelity following transgenesis, which may be crucial for interpreting transgene function. Thus, it is imperative that reliable methods for assessing these parameters are available. However, there are limited data regarding whether BAC transgenes routinely integrate in the mouse genome as intact molecules, how BAC transgenes behave as they are passed through the germline across successive generations, and how variation in BAC transgene copy number relates to transgene expression. To address these questions, we used TaqMan real-time PCR to estimate BAC transgene copy number in BAC transgenic embryos and lines. Here we demonstrate the reproducibility of copy number quantification with this method and describe the variation in copy number across independent transgenic lines. In addition, polymorphic marker analysis suggests that the majority of BAC transgenic lines contain intact molecules. Notably, all lines containing multiple BAC copies also contain all BAC-specific markers. Three of 23 founders analyzed contained BAC transgenes integrated into more than one genomic location. Finally, we show increased BAC transgene copy number correlates with increased BAC transgene expression. In sum, our efforts have provided a reliable method for assaying BAC transgene integrity and fidelity, and data that should be useful for researchers using BACs as transgenic vectors. | Publication | 17882484 (PMID)
10.1007/s00335-007-9056-y (DOI) |
Eeds AM, Mortlock D, Wade-Martins R, Summar ML. Assessing the functional characteristics of synonymous and nonsynonymous mutation candidates by use of large DNA constructs. (2007) Am J Hum Genet 80: 740-50
Show Abstract · Added June 9, 2010As we identify more and more genetic changes, either through mutation studies or population screens, we need powerful tools to study their potential molecular effects. With these tools, we can begin to understand the contributions of genetic variations to the wide range of human phenotypes. We used our catalogue of molecular changes in patients with carbamyl phosphate synthetase I (CPSI) deficiency to develop such a system for use in eukaryotic cells. We developed the tools and methods for rapidly modifying bacterial artificial chromosomes (BACs) for eukaryotic episomal replication, marker expression, and selection and then applied this protocol to a BAC containing the entire CPSI gene. Although this CPSI BAC construct was suitable for studying nonsynonymous mutations, potential splicing defects, and promoter variations, our focus was on studying potential splicing and RNA-processing defects to validate this system. In this article, we describe the construction of this system and subsequently examine the mechanism of four putative splicing mutations in patients deficient in CPSI. Using this model, we also demonstrate the reversible role of nonsense-mediated decay in all four mutations, using small interfering RNA knockdown of hUPF2. Furthermore, we were able to locate cryptic splicing sites for the two intronic mutations. This BAC-based system permits expression studies in the absence of patient RNA or tissues with relevant gene expression and provides experimental flexibility not available in genomic DNA or plasmid constructs. Our splicing and RNA degradation data demonstrate the advantages of using whole-gene constructs to study the effects of sequence variation on gene expression and function. | Publication | 17357079 (PMID)
PMC1852709 (PMCID)
10.1086/513287 (DOI) |
Chandler RL, Chandler KJ, McFarland KA, Mortlock DP. Bmp2 transcription in osteoblast progenitors is regulated by a distant 3' enhancer located 156.3 kilobases from the promoter. (2007) Mol Cell Biol 27: 2934-51
Show Abstract · Added April 7, 2010Bone morphogenetic protein 2 (encoded by Bmp2) has been implicated as an important signaling ligand for osteoblast differentiation and bone formation and as a genetic risk factor for osteoporosis. To initially survey a large genomic region flanking the mouse Bmp2 gene for cis-regulatory function, two bacterial artificial chromosome (BAC) clones that extend far upstream and downstream of the gene were engineered to contain a lacZ reporter cassette and tested in transgenic mice. Each BAC clone directs a distinct subset of normal Bmp2 expression patterns, suggesting a modular arrangement of distant Bmp2 regulatory elements. Strikingly, regulatory sequences required for Bmp2 expression in differentiating osteoblasts, as well as tooth buds, hair placodes, kidney, and other tissues, are located more than 53 kilobases 3' to the promoter. By testing BACs with engineered deletions across this distant 3' region, we parsed these regulatory elements into separate locations and more closely refined the location of the osteoblast progenitor element. Finally, a conserved osteoblast progenitor enhancer was identified within a 656-bp sequence located 156.3 kilobases 3' from the promoter. The identification of this enhancer should permit further investigation of upstream regulatory mechanisms that control Bmp2 transcription during osteoblast differentiation and are relevant to further studies of Bmp2 as a candidate risk factor gene for osteoporosis. | Publication | 17283059 (PMID)
PMC1899916 (PMCID)
10.1128/MCB.01609-06 (DOI) |
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