Brega, Paola:

Identification of downstream genes of the TRPS1 transcription factor

Duisburg-Essen (2005), III, 99 Bl.
Dissertation / Fach: Biologie
Fakultät für Biologie
Dissertation
Abstract:
Mutations in the TRPS1 gene cause the tricho-rhino-phalangeal-syndrome (TRPS) type I and III, which are characterized by craniofacial and skeletal defects. The gene encodes a transcription factor that has a GATA-binding zinc finger and represses GATA-regulated gene expression. Mutation analysis of TRPS type I patients helped to identify the nuclear localization signal of TRPS1. To study the pathogenesis of TRPS, a TRPS1 transgenic mouse model expressing a potentially dominant negative TRPS1 allele in chondrocytes was generated. However, the mice expressed the transgene at a low rate only, and even adult mice did not show the typical brachydactyly or growth retardation. For identifying genes that are directly and indirectly regulated by TRPS1 two different strategies were followed. (I) A combination of RNA interference-mediated Trps1 silencing and cDNA microarray analysis allowed the identification of putative downstream genes of Trps1 in mouse NIH3T3 cells. Several genes overexpressed in cells where Trps1 was silenced are involved in the control of cell cycle progression or apoptosis. (II) Expression profiles of normal mouse embryonic limbs and snouts were compared with embryonic limbs and snouts of mice carrying a targeted deletion of the Trps1 GATA zinc finger. Several downstream genes implicated in cell cycle control, signal transduction, cell organization and transcription regulation were identified, and their relative expression values were verified by real-time PCR. Luciferase reporter-gene assays allowed the identification of the first potential TRPS1 target genes, LAPTM4b and HSPG2. Both promoters contain GATA motifs and can be repressed by TRPS1. LAPTM4b (lysosomal-associated transmembrane protein 4b) is probably involved in cell cycle control. HSPG2 codes for perlecan, the major heparan sulfate proteoglycan in the cartilage matrix. These findings suggest a role for Trps1 in the regulation of cell cycle control and in the maintenance of cell-matrix interactions.

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