The role of the transcription/repair factor TFIIH in the intestinal dysplasia of the zebrafish mutant sycorax

© 2012 Dr. Elizabeth Louise ChristieThe gene expression profiles of various cancer types show significant similarities to those of their developing tissue of origin, with a number of genes that are expressed in the developing intestine but not the adult intestine being re-expressed in colorectal cancer (CRC) cells. This study utilised a zebrafish intestinal mutant, sycorax, to further the current understanding of the genetics underlying vertebrate intestinal development and development and progression of CRC. The sycorax zebrafish mutant displays a dysplastic intestinal epithelium, with multilayering and disorganisation, reminiscent of a pre-neoplastic lesion. The mutation underlying the sycorax phenotype is a premature stop codon in the general transcription 2 H, polypeptide 4 (gtf2h4) gene, which is predicted to generate a truncated Gtf2h4 protein. Gtf2h4 is one of the components of the multisubunit complex, general transcription factor IIH (TFIIH), which is integral to a number of cellular processes. TFIIH is one of the general transcripton factors involved in transcription initation of all protein coding genes via RNA polymerase II. It also fine tunes the expression of a small proportion of genes by integrating signals from gene specific activators and repressors. TFIIH is involved in the regulation of transcription of rRNA by RNA polymerase I, in addition to being a crucial component of the DNA repair pathway known as nucleotide excision repair. Finally, TFIIH plays a role in the regulation of cell cycle progression. Northern blot analysis and metabolic labeling of newly synthesized rRNA in wild type and sycorax embryos demonstrated an increase in rRNA transcription in sycorax embryos. sycorax embryos were also shown to have reduced DNA repair capabilities following UV irradiation. Increased levels of proliferation in the intestine, as demonstrated by EdU incorporation, are hypothesised to contribute to the multilayering of the sycorax intestinal epithelium. Gene expression analysis of wild type and sycorax intestines identified a number of differentially expressed genes, many of which are involved in the regulation of cell proliferation. Upregulated genes in the sycorax intestine include c-myc and c-myb, which have previously been shown to be directly transcriptionally regulated by TFIIH, and are proposed to contribute to the increased levels of proliferation observed in the sycorax intestine. Furthermore, knockdown of c-myb in sycorax embryos rescues the intestinal dysplasia. These results demonstrate that sycorax embryos display abnormalities in all the processes in which TFIIH plays a role; therefore, the sycorax mutant provides a novel opportunity to further study the function of TFIIH in an entire vertebrate organism. Strikingly, as the sycorax intestinal phenotype is reminiscent of a pre-neoplastic lesion, and the oncogenes c-myc and c-myb are upregulated in the sycorax intestine, this study has identified a possible role for altered TFIIH function in CRC