Purchase this article with an account.
FB Berry, RA Saleem, JS Friedman, MA Walter; Complex Transcriptional Activation Mediated By Foxc1 Regulatory Domains . Invest. Ophthalmol. Vis. Sci. 2002;43(13):2438.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
Purpose:To examine the mechanisms of intramolecular regulation of transcriptional activation by FOXC1. Methods:FOXC1 contains transcriptional activation domains in the N- and C-terminus of the FOXC1 protein as well as a highly phosphorylated inhibitory domain. These regions of the FOXC1 protein were fused to the GAL4 DNA-binding domain and their ability to activate transcription of a GAL4-Luciferase reporter was tested. Lastly regions of the FOXC1 forkhead domain were fused to green fluorescent protein (GFP) to identify nuclear localization signal in FOXC1. Results:Amino acid residues 436-553 in the C-terminal region of FOXC1 possesses strong transcriptional activation activity. Luciferase levels observed with this region were 10 fold higher than full-length FOXC1 and over 100 fold higher than baseline luciferase levels. The addition of residues 215-435 to the C-terminal activation domain reduced trans-activation by 3 fold. This transcriptional inhibitory activity was transferable as the association of residues 215-366 to the GAL4 activation domain (AD) impaired transcriptional activation of GAL4 AD by over five fold. However, this region does not contain intrinsic transcriptional repression activity as residues 215-366 did not affect expression of a constitutively active GAL4-SV40 luciferase reporter. Furthermore residues 215-366 lie within a highly phosporylated region of FOXC1 and their removal produced a transcriptionally hyperactive FOXC1 protein which displayed a reduced level of phosphorylation. Finally we determined that residues 168-176 in the FOXC1 forkhead domain was sufficient to target GFP to the nucleus suggesting that this region contains the FOXC1 nuclear localization signal. Conclusion:FOXC1 contains a potent C-terminal transcriptional activation as well as a highly phosphorylated inhibitory region. This study suggests that FOXC1 transcription activation is under complex control orchestrated through these regulatory elements. These regulatory regions are currently being used as bait to screen for interacting proteins in a human fetal eye library.
This PDF is available to Subscribers Only