Abstract
Mutations in the FOXC1 gene result in Axenfeld-Rieger malformations of the anterior segment of the eye and lead to an increased susceptibility of glaucoma. To understand how the FOXC1 protein may function in contributing to these malformations, we identified functional regions in FOXC1 required for nuclear localization and transcriptional regulation. Two regions in the FOXC1 forkhead domain, one rich in basic amino acid residues, and a second, highly conserved among all FOX proteins, were necessary for nuclear localization of the FOXC1 protein. However, only the basic region was sufficient for nuclear localization. Two transcriptional activation domains were identified in the extreme N- and C-terminal regions of FOXC1. A transcription inhibitory domain was located at the central region of the protein. This region was able to reduce the trans-activation potential of the C-terminal activation domain, as well as the GAL4 activation domain. Lastly, we demonstrate that FOXC1 is a phosphoprotein, and a number of residues predicted to be phosphorylated were localized to the FOXC1 inhibitory domain. Removal of residues 215-366 resulted in a transcriptionally hyperactive FOXC1 protein, which displayed a reduced level of phosphorylation. These results indicate that FOXC1 is under complex regulatory control with multiple functional domains modulating FOXC1 transcriptional regulation.
Highlights
FOX (Forkhead Box [1]) proteins are a family of transcription factors that contain an evolutionarily conserved 110-amino acid DNA-binding domain known as the forkhead domain (FHD).1 This DNA-binding domain was first identified as a region of homology between the Drosophila fork head protein [2] and the rat hepatocyte nuclear factor 3 protein [3]
Analysis of the amino acid sequence of the FOXC1 FHD revealed a span of basic amino acids at positions 168 –176 that may serve as a potential nuclear localization signals (NLSs)
In this report we describe the molecular dissection of FOXC1 to identify important functional regions required for nuclear localization and regulation of transcription
Summary
FOX (Forkhead Box [1]) proteins are a family of transcription factors that contain an evolutionarily conserved 110-amino acid DNA-binding domain known as the forkhead domain (FHD).1 This DNA-binding domain was first identified as a region of homology between the Drosophila fork head protein [2] and the rat hepatocyte nuclear factor 3 protein [3]. Two regions in the FOXC1 forkhead domain, one rich in basic amino acid residues, and a second, highly conserved among all FOX proteins, were necessary for nuclear localization of the FOXC1 protein. Two transcriptional activation domains were identified in the extreme N- and C-terminal regions of FOXC1.
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