Abstract
BackgroundThe Dlc1 (deleted in liver cancer 1) tumour suppressor gene codes for a RhoGTPase activating protein that is found inactivated in many tumour types. Several transcriptional isoforms have been described but the functional significance and tissue distribution of each form is presently poorly understood. Also, differences in the number of isoforms and splice variants reported still exist between different mammalian species. In order to better understand the number and function of the different variants of the Dlc1 gene in the mouse, we have carried out a detailed analysis. Extensive 3' RACE experiments were carried out in order to identify all possible Dlc1 isoforms and splice variants in the mouse. In addition, we have generated a gene trapped mouse that targets one of these isoforms in order to study its biological function. The effect of this gene trap insertion on the splicing of other isoforms has also been studied.ResultsIn addition to the known 6.1 and 6.2 Kb transcripts of Dlc1, our study revealed the existence of a novel 7.6 Kb transcriptional isoform in the mouse, which corresponds to the human 7.4 Kb (KIAA1723) cDNA transcript. A gene trapped embryonic cell line, with an insertion between Exon 1 and 2 of the 6.1 Kb transcriptional isoform, was used to generate a transgenic mouse. This line showed a significant reduction in the expression of the trapped isoform. However, reduced expression of the other isoforms was not seen. Mice heterozygous for the gene trapped allele were phenotypically normal, but homozygous mutant embryos did not survive beyond 10.5 days post coitum. Dlc1gt/gt embryos showed defects in the brain, heart, and placental blood vessels. Cultured serum-free mouse embryo cells from Dlc1 deficient embryos had elevated RhoA activity and displayed alterations in the organization of actin filaments and focal adhesions. The Dlc1 deficient cells also exhibited increased wound closure in an in vitro scratch assay.ConclusionsThe mouse has three major transcriptional isoforms of the Dlc1 gene that are differentially expressed in various tissues. A mouse with exon 1 of the 6.1 Kb transcript gt resulted in hypomorphic expression of Dlc1 protein and an embryonic lethal phenotype in the homozygous condition, which indicates that this isoform plays a major role in mouse development. The Dlc1 deficient cells showed altered cytoskeleton structure, increased RhoA activity and cellular migration.
Highlights
The deleted in liver cancer 1 (Dlc1) tumour suppressor gene codes for a RhoGTPase activating protein that is found inactivated in many tumour types
The deleted in liver cancer 1 (Dlc1) gene encodes a RhoGTPase activating protein (RhoGAP) that has been found to be inactivated by deletion or promoter methylation in many tumours, resulting in alterations in cellular proliferation, cytoskeleton reorganization and gene expression in tumour cells [8,9,10,11,12,13,14,15]
Based on these complimentary DNAs and others, found in the databases, we predict that there is a 7.6 Kb transcript, which arises from a promoter greater than 220 Kb upstream from exon 1 of the 6.2 Kb Dlc1 transcript (Figure 1C)
Summary
The Dlc (deleted in liver cancer 1) tumour suppressor gene codes for a RhoGTPase activating protein that is found inactivated in many tumour types. In order to better understand the number and function of the different variants of the Dlc gene in the mouse, we have carried out a detailed analysis. The deleted in liver cancer 1 (Dlc1) gene encodes a RhoGTPase activating protein (RhoGAP) that has been found to be inactivated by deletion or promoter methylation in many tumours, resulting in alterations in cellular proliferation, cytoskeleton reorganization and gene expression in tumour cells [8,9,10,11,12,13,14,15]. Two principal transcripts of 6.5 and 5.5 Kb and a minor transcript of 7.6 Kb have been reported by Northern blotting [22], but the complete characterization and tissue specific expression of all the isoforms and splice variants of Dlc gene in the mouse has not yet been carried out
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