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Identification and characterization of Dlc1 isoforms in the mouse and study of the biological function of a single
Mohammad G Sabbir1, Nichola Wigle, Shauna Loewen
1Manitoba Institute of Cell Biology, CancerCare Manitoba, 675 McDermot Avenue, Winnipeg, MB R3E 0V9, Canada.
Background:
The 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.
Results:
In 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.
Conclusions:
The 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.
Insights
The deleted in liver cancer 1 (Dlc1) gene has three mouse isoforms, with one crucial for embryonic development. Dlc1 deficiency leads to developmental defects and altered cell migration, highlighting its role in tumor suppression.
Area of Science:
- Molecular Biology
- Genetics
- Developmental Biology
Background:
- The Dlc1 (deleted in liver cancer 1) gene encodes a RhoGTPase activating protein crucial for tumor suppression.
- Understanding the functional significance and tissue distribution of Dlc1's multiple transcriptional isoforms is limited.
- Species-specific variations in Dlc1 isoforms and splice variants necessitate detailed investigation in model organisms like the mouse.
Purpose of the Study:
- To comprehensively identify and characterize all Dlc1 isoforms and splice variants in mice.
- To investigate the biological function of a specific Dlc1 isoform using a gene-trapped mouse model.
- To analyze the impact of gene trapping on the expression of other Dlc1 isoforms.
Main Methods:
- Extensive 3' RACE (Rapid Amplification of cDNA Ends) experiments to identify Dlc1 isoforms.
- Generation of a gene-trapped mouse model targeting a specific Dlc1 isoform.
- Phenotypic analysis of heterozygous and homozygous mutant mice.
- Assessment of RhoA activity, actin cytoskeleton, and focal adhesions in Dlc1-deficient cells.
- In vitro scratch assay to evaluate cellular migration.
Main Results:
- Discovery of a novel 7.6 Kb Dlc1 transcriptional isoform in mice, homologous to human KIAA1723.
- Generation of a gene-trapped mouse line with reduced expression of the targeted 6.1 Kb Dlc1 isoform.
- Homozygous Dlc1gt/gt embryos exhibited embryonic lethality by 10.5 days post coitum with defects in brain, heart, and placental vasculature.
- Dlc1-deficient cells displayed elevated RhoA activity, altered actin and focal adhesion organization, and increased migration.
Conclusions:
- The mouse genome contains at least three major Dlc1 transcriptional isoforms with differential tissue expression.
- Targeting exon 1 of the 6.1 Kb Dlc1 transcript resulted in hypomorphic expression and embryonic lethality in homozygous mutants.
- This specific Dlc1 isoform is essential for mouse development, with deficiency impacting cytoskeleton, RhoA signaling, and cell migration.
