Our research is focused on the transcription factor Pax6, with the overall aim of detecting new target genes in order to understand Pax6’s role in normal development and cancer.
Pax6 is an evolutionary conserved transcription factor, expressed in the central nervous system, eye, nose and pancreas during vertebrate embryo development. Pax6 heterozygous mutants lead to the eye phenotype Aniridia in humans, and are coupled to glucose intolerance and the lack of pineal gland. Several cancer cell lines show deregulated expression of Pax6. None of the published Pax6 targets genes are able to fully explain the phenotypes observed when Pax6 is mutated, or what Pax6 possible could contribute to in tumors.
To identify new target genes we use:
- Chromatin immunoprecipitation (ChIP) and gene expression microarray of; 1) genetically engineered Pax6 inducible cell lines and 2) cancer cell lines endogenously expressing Pax6 treated with Pax6 siRNA or stably transfected with shRNA. Several Crispr/Cas cell lines with Pax6 knock-out are generated.
- 2D gel-electrophoresis, Mass-spechtrometry and SILAC on protein extracts from stably transfected cell-lines that show morphological changes upon Pax6 and Pax6(5a) expression.
- Verification of the most interesting target genes are done by electromobility shift assay (EMSA), as well as RT-PCR, qPCR and transient transfection/reporter-gene assay.
We have shown that the two isoforms Pax6 and Pax6(5a) regulate common and individual target genes when expressed in fibroblast cell lines that normally do not express Pax6/Pax6(5a) (Kiselev et al 2018). A high percentage of the target genes are associated with the membrane and extracellular matrix. A group of glycosylases are also regulated, indicating that Pax6 can modify glycosylation of membrane proteins. Further, manipulation of Pax6 expression show that Pax6 also affect cell proliferation and migration. We have identified DKK3 (a component of the Wnt signaling pathway) as a target gene in several mouse and human cell lines, and have identified the Pax6 binding site in the promoter (Forsdahl et al 2014). Our current work is with pancreatic adenocarcinoma cells and glioblastoma cells, where shRNA and Crispr/Cas have been used to knock out PAX6. The glioblastoma cell line U251 show increased proliferation, changes in the cell cycle distribution and increased migration when PAX6 is knocked out (Hegge et al 2018). The cells were also more resilient to oxidative stress, which is an important observation since chemotherapeutic treatment often induces oxidative stress.