Positive autoregulation of the transcription factor Pax6 in response to increased levels of either of its major isoforms, Pax6 or Pax6(5a), in cultured cells.
BACKGROUND
Pax6 is a transcription factor essential for normal development of the eyes and nervous system. It has two major isoforms, Pax6 and Pax6(5a), and the ratios between their expression levels vary within narrow limits. We tested the effects of overexpressing either one or other isoform on endogenous Pax6 expression levels in Neuro2A and NIH3T3 cells.


RESULTS
We found that both isoforms caused an up-regulation of endogenous Pax6 expression in cells with (Neuro2A) or without (NIH3T3) constitutive Pax6 expression. Western blots showed that cells stably transfected with constructs expressing either Pax6 or Pax6(5a) contained raised levels of both Pax6 and Pax6(5a). Quantitative RT-PCR confirmed an increase in levels of Pax6(5a) mRNA in cells containing Pax6-expressing constructs and an increase in levels of Pax6 mRNA in cells containing Pax6(5a)-expressing constructs. The fact that the introduction of constructs expressing only one isoform increased the cellular levels of not only that isoform but also the other indicates that activation of the endogenous Pax6 locus occurred. The ratio between the levels of the two isoforms was maintained close to physiological values. The overexpression of either isoform in neuroblastoma (Neuro2A) cell lines also promoted morphological change and an increase in beta-III-tubulin expression, indicating an increase in neurogenesis.


CONCLUSIONS
Our results demonstrate that Pax6 can up-regulate production of Pax6 protein from an entire intact endogenous Pax6 locus in its genomic environment. This adds to previous studies showing that Pax6 can up-regulate reporter expression driven by isolated Pax6 regulatory elements. Furthermore, our results suggest that an important function of positive feedback might be to stabilise the relative levels of Pax6 and Pax6(5a).
Pinson
Jeni
J
Genes and Development Group, Centre for Integrative Physiology, University of Edinburgh, Hugh Robson Building, George Square, Edinburgh EH8 9XD, UK.
Simpson
T Ian
TI
Mason
John O
JO
Price
David J
DJ
D000818:Animals; D045744:Cell Line, Tumor; D005136:Eye Proteins / Q000235:genetics / Q000378:metabolism*; D018398:Homeodomain Proteins / Q000235:genetics / Q000378:metabolism*; D006706:Homeostasis; D051379:Mice; D041681:NIH 3T3 Cells; D000071841:PAX6 Transcription Factor; D051761:Paired Box Transcription Factors / Q000235:genetics / Q000378:metabolism*; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism*; D014162:Transfection; D015854:Up-Regulation
D005136:Eye Proteins; D018398:Homeodomain Proteins; D000071841:PAX6 Transcription Factor; D051761:Paired Box Transcription Factors; C000605732:Pax6 protein, mouse; D020033:Protein Isoforms; D012097:Repressor Proteins