Gene regulation by PAX6

Structural-functional correlations of missense mutants and transcriptional control of Trpm3/miR-204

Qing Xie, Devina Ung, Kamil Khafizov, Andras Fiser, Ales Cvekl

Research output: Contribution to journalArticle

9 Citations (Scopus)

Abstract

Purpose: 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.

Original languageEnglish (US)
Pages (from-to)270-282
Number of pages13
JournalMolecular Vision
Volume20
StatePublished - Mar 6 2014

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Mutation
Binding Sites
Genes
Missense Mutation
Regulator Genes
Site-Directed Mutagenesis
Luciferases
Introns
Transcriptional Activation
Chromatin
Pancreas
Up-Regulation
Brain
Proteins

ASJC Scopus subject areas

  • Ophthalmology

Cite this

Gene regulation by PAX6 : Structural-functional correlations of missense mutants and transcriptional control of Trpm3/miR-204. / Xie, Qing; Ung, Devina; Khafizov, Kamil; Fiser, Andras; Cvekl, Ales.

In: Molecular Vision, Vol. 20, 06.03.2014, p. 270-282.

Research output: Contribution to journalArticle

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abstract = "Purpose: 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.",
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T2 - Structural-functional correlations of missense mutants and transcriptional control of Trpm3/miR-204

AU - Xie, Qing

AU - Ung, Devina

AU - Khafizov, Kamil

AU - Fiser, Andras

AU - Cvekl, Ales

PY - 2014/3/6

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