Molecular cloning and characterization of the 5′ region of the mouse trkA proto-oncogene

Abstract

The trkA proto-oncogene encodes a high-a nity NGF receptor that is essential for the survival, di erentiation and maintenance of many neural and non-neural cell types. Altered expression of the trkA gene or trkA receptor malfunction have been implicated in neurodegeneration, tumor progression and oncogenesis. We have cloned and characterized the 5' region of the mouse trkA gene and have identi®ed its promoter. trkA promoter sequences are GC-rich, lack genuine TATA or CAAT boxes, and are contained within a CpG island which extends over the entire ®rst coding exon. The mouse trkA transcription start site is located 70/71 bp upstream to the AUG translation initiation codon. Sequence analysis showed that the gene encoding the insulin receptor-related receptor, IRR, is located just 1.6 kbp upstream to the trkA gene and is transcribed in the opposite direction. We have used trkA-CAT transcriptional fusions to study trkA promoter function in transient transfection experiments. RNase protection assays and CAT protein ELISA analyses showed that a 150 bp long DNA segment, immediately upstream to the start site, is su cient to direct accurate transcription in trkA-expressing cells. Dissection of this fragment allowed us to identify a 13 bp cis-regulatory element essential for both promoter activity and cell-type speci®c expression. Deletion of this 13 bp segment as well as modi®cation of its sequence by site-directed mutagenesis led to a dramatic decline in promoter activity. Gel mobility shift assays carried out with double-stranded oligonucleotides containing the 13 bp element revealed several speci®c DNA-protein complexes when nuclear extracts from trkA-expressing cells were used. Supershift experiments showed that the Sp1 transcription factor was a component of one of these complexes. Our results identify a minimal trkA gene promoter, located very close to the transcription start site, and de®ne a 13 bp enhancer within this promoter sequence.

Molecular basis for keratoconus: Lack of TrkA expression and its transcriptional repression by Sp3

“…Whitelock et al (4) analyzed a panel of transcription factors and found an enhanced level of Sp1 expression in corneal epithelium of patients affected by keratoconus compared with normal individuals. Interestingly, Sp1 is known to bind ciselements in the promoter region of TrkA NGFR , which contains several putative binding sites for Sp1͞Sp3 factors (6,27). Moreover, Gaudreault et al (28) reported that alterations in Sp1 and Sp3 expression may trigger cells to express a set of keratins typical of the terminal differentiated state of corneal epithelial cells.…”

“…Some have proposed that an increase in Sp1 transcriptional activity is responsible for the reduced expression of ␣1-PI (5). Interestingly, Sacristan et al (6) reported that Sp1 regulates expression of the NGF-receptor TrkA (TrkA NGFR ) through interactions with a cis-regulatory element in its promoter.…”

Transcriptional regulation of Trk family neurotrophin receptors

“…Many putative transcription-factorbinding sites have been found within the 5'-flanking region of the human TrkA gene. The 5' region of the mouse TrkA gene was characterized by reporter assays in PC12 and N2a neuroblastoma cells which normally express TrkA [51]. The mouse TrkA promoter sequence is GC -rich and is contained within a CpG island that extends over the entire first coding exon.…”

Genes for neurotrophic factors and their receptors: structure and regulation

“…The only experimental data available concern the proximal region of the TrkA promoter [22]. It contains a 13-nucleotide regulatory element essential for cell type-specific activity of the TrkA promoter in transient assays and binds a protein complex containing the SP1 transcription factor [23]. All Trk gene promoters are TATA-less and relatively GC rich.…”

“…They have significant sequence similarity [24]. Surprisingly, analysis of the TrkA locus demonstrated that the gene encoding the insulin receptor-related receptor (IRR) is located just 1.6 kb upstream of the TrkA gene and is transcribed in the opposite direction, suggesting shared regulatory elements that partially explain their coordinated regulation [23]. For the TrkB gene, two alternative promoters have been described, but their biological significance or alternative expression capacity have not been elucidated [24].…”