title: Gene regulation by PAX6: structural-functional correlations of missense mutants and transcriptional control of Trpm3/miR-204.
abstract: OBJECTIVE
  Pax6 is a key regulatory gene for eye, brain, and pancreas development. It acts as a transcriptional activator and repressor. Loss-of-function of Pax6 results in down- and upregulation of a comparable number of genes, although many are secondary targets. Recently, we found a prototype of a Pax6-binding site that acts as a transcriptional repressor. We also identified the Trpm3 gene as a Pax6-direct target containing the miR-204 gene located in intron 6. Thus, there are multiple Pax6-dependent mechanisms of transcriptional repression in the cell. More than 50 Pax6 missense mutations have been identified in humans and mice. Two of these mutations, N50K (Leca4) and R128C (Leca2), were analyzed in depth resulting in different numbers of regulated genes and different ratios of down- and upregulated targets. Thus, additional studies of these mutants are warranted to better understand the molecular mechanisms of the mutants' action.
  
  
  METHODS
  Mutations in PAX6 and PAX6(5a), including G18W, R26G, N50K, G64V, R128C, and R242T, were generated with site-directed mutagenesis. A panel of ten luciferase reporters driven by six copies of Pax6-binding sites representing a spectrum of sites that act as repressors, moderate activators, and strong activators were used. Two additional reporters, including the Pax6-regulated enhancer from mouse Trpm3 and six copies of its individual Pax6-binding site, were also tested in P19 cells.
  
  
  RESULTS
  PAX6 (N50K) acted either as a loss-of-function or neutral mutation. In contrast, PAX6 (R128C) and (R242T) acted as loss-, neutral, and gain-of-function mutations. With three distinct reporters, the PAX6 (N50K) mutation broke the pattern of effects produced by substitutions in the surrounding helices of the N-terminal region of the paired domain. All six mutations tested acted as loss-of-function using the Trpm3 Pax6-binding site.
  
  
  CONCLUSIONS
  These studies highlight the complexity of Pax6-dependent transcriptional activation and repression mechanisms, and identify the N50K and R128C substitutions as valuable tools for testing interactions between Pax6, Pax6 (N50K), and Pax6 (R128C) with other regulatory proteins, including chromatin remodelers.
authors.affiliation: Department of Ophthalmology and Visual Sciences, Albert Einstein College of Medicine, Bronx, NY.
authors.forename: Qing
authors.initials: Q
authors.lastname: Xie
authors.affiliation: Department of Ophthalmology and Visual Sciences, Albert Einstein College of Medicine, Bronx, NY.
authors.forename: Devina
authors.initials: D
authors.lastname: Ung
authors.affiliation: Department of Biochemistry, Albert Einstein College of Medicine, Bronx, NY ; Department of Systems and Computational Biology, Albert Einstein College of Medicine, Bronx, NY.
authors.forename: Kamil
authors.initials: K
authors.lastname: Khafizov
authors.affiliation: Department of Biochemistry, Albert Einstein College of Medicine, Bronx, NY ; Department of Systems and Computational Biology, Albert Einstein College of Medicine, Bronx, NY.
authors.forename: Andras
authors.initials: A
authors.lastname: Fiser
authors.affiliation: Department of Ophthalmology and Visual Sciences, Albert Einstein College of Medicine, Bronx, NY ; Department of Genetics, Albert Einstein College of Medicine, Bronx, NY.
authors.forename: Ales
authors.initials: A
authors.lastname: Cvekl
mesh_terms: D000818:Animals; D001483:Base Sequence; D005136:Eye Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D018398:Homeodomain Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008156:Luciferases; D051379:Mice; D035683:MicroRNAs / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D020125:Mutation, Missense / Q000235:genetics*; D059372:Nucleotide Motifs / Q000235:genetics; D000071841:PAX6 Transcription Factor; D051761:Paired Box Transcription Factors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D012097:Repressor Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D040681:Structural Homology, Protein; D013329:Structure-Activity Relationship; D050053:TRPM Cation Channels / Q000235:genetics* / Q000378:metabolism; D014158:Transcription, Genetic; D015533:Transcriptional Activation / Q000235:genetics
chemical_list: D005136:Eye Proteins; D018398:Homeodomain Proteins; C574692:MIRN204 microRNA, mouse; D035683:MicroRNAs; D000071841:PAX6 Transcription Factor; C000605730:PAX6 protein, human; D051761:Paired Box Transcription Factors; C000605732:Pax6 protein, mouse; D012097:Repressor Proteins; D050053:TRPM Cation Channels; C533868:TRPM3 protein, mouse; D008156:Luciferases