Identification and characterization of Dlc1 isoforms in the mouse and study of the biological function of a single gene trapped isoform

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.

DLC1 is the principal biologically-relevant down-regulated DLC family member in several cancers

“…Despite this wide range, it was higher than DLC2 in each of the tissues except for colorectum, where expression of DLC1 and DLC2 was similar, and it was substantially higher than DLC3 in all four tissues. Taken together, these observations suggest DLC1 may be the most critical of the three genes for normal physiology, which is consistent with its requirement for fetal development [28–29], in contrast to DLC2 [30] and DLC3 [31]. However, DLC2 and DLC3 may have cell type-specific functions, such as the role of DLC2 in pancreatic physiology [32].…”

Using extended pedigrees to identify novel autism spectrum disorder (ASD) candidate genes

“…Examples included DLC1, which is a member of the rhoGAP family, a group of genes involved in signaling pathways that regulate cell processes involved in cytoskeletal change. Moreover, different isoforms of DLC1 have been observed to cause multiple phenotypes in mice (Sabbir et al 2010), and have previously been described in association with ASD ). Another gene is TRIM37, notable for its involvement in developmental patterning, and its association with 'mulibrey nanism', an autosomal recessive disorder of muscle, liver, brain and eye development (Hämäläinen et al 2004).…”

Inactivation of the Dlc1 Gene Cooperates with Downregulation of p15INK4b and p16Ink4a, Leading to Neoplastic Transformation and Poor Prognosis in Human Cancer

“…Conditional Dlc1 disruption induces increased RhoGTP and increased susceptibility to ras transformation Constitutive disruption of mouse Dlc1 leads to embryonic lethality (5,19). To develop an endogenous Dlc1 allele that can be conditionally disrupted, we used gene targeting to introduce LoxP sites on both sides of exon 4 of the Dlc1 gene (Fig.…”