Biomedical subjects
J Gilthorpe
Publications and source records attributed to J Gilthorpe.
Detecting conserved regulatory elements with the model genome of the Japanese puffer fish, Fugu rubripes.
Comparative vertebrate genome sequencing offers a powerful method for detecting conserved regulatory sequences. We propose that the compact genome of the teleost Fugu rubripes is well suited for this purpose. The evolutionary distance of teleosts from other vertebrates offers the maximum stringency for such evolutionary comparisons. To illustrate the comparative genome approach for F. rubripes, we use sequence comparisons between mouse and Fugu Hoxb-4 noncoding regions to identify conserved sequence blocks. We have used two approaches to test the function of these conserved blocks. In the first, homologous sequences were deleted from a mouse enhancer, resulting in a tissue-specific loss of activity when assayed in transgenic mice. In the second approach, Fugu DNA sequences showing homology to mouse sequences were tested for enhancer activity in transgenic mice. This strategy identified a neural element that mediates a subset of Hoxb-4 expression that is conserved between mammals and teleosts. The comparison of noncoding vertebrate sequences with those of Fugu, coupled to a transgenic bioassay, represents a general approach suitable for many genome projects.
Multiple positive and negative regulatory elements in the promoter of the mouse homeobox gene Hoxb-4.
Mouse Hoxb-4 (Hox-2.6) is a homeobox gene that belongs to a family which also includes Hoxa-4, Hoxc-4, and Hoxd-4 and that is related to the Deformed gene in Drosophila melanogaster. We have determined the sequence of 1.2 kb of 5' flanking DNA of mouse Hoxb-4 and by nuclease S1 and primer extension experiments identified two transcription start sites, P1 and P2, 285 and 207 nucleotides upstream of the ATG initiator codon, respectively. We have shown that this region harbors two independent promoters which drive CAT expression in several different cell lines with various efficiencies, suggesting that they are subject to cell-type-specific regulation. Through detailed mutational analysis, we have identified several cis-regulatory elements, located upstream and downstream of the transcription start sites. They include two cell-type-specific negative regulatory elements, which are more active in F9 embryonal carcinoma cells than in neuroblastoma cells (regions a and d at -226 to -186 and +169 to +205, respectively). An additional negative regulatory element has been delimited (region b between +22 and +113). Positive regulation is achieved by binding of HoxTF, a previously unknown factor, to the sequence GCCATTGG (+148 to +155) that is essential for efficient Hoxb-4 expression. We have also defined the minimal promoter sequences and found that they include two 12-bp initiator elements centered around each transcription start site. The complex architecture of the Hoxb-4 promoter provides the framework for fine-tuned transcriptional regulation during embryonic development.