Dual roles for Pax-6: A transcriptional repressor of lens fiber cell- specific β-crystallin genes

Melinda K. Duncan, John I. Haynes, Ales Cvekl, Joram Piatigorsky

Research output: Contribution to journalArticle

109 Citations (Scopus)

Abstract

It has been demonstrated previously that Pax-6, a paired domain (PD)/homeodomain (HD) transcription factor critical for eye development, contributes to the activation of the αB-, αA-, δ1-, and ζ-crystallin genes in the lens. Here we have examined the possibility that the inverse relationship between the expression of Pax-6 and β-crystallin genes within the developing chicken lens reflects a negative regulatory role of Pax-6. Cotransfection of a plasmid containing the βB1-crystallin promoter fused to the chloramphenicol acetyltransferase reporter gene and a plasmid containing the full-length mouse Pax-6 coding sequences into primary embryonic chicken lens epithelial cells or fibroblasts repressed the activity of this promoter by as much as 90%. Pax-6 constructs lacking the C-terminal activation domain repressed βB1-crystallin promoter activity as effectively as the full- length protein, but the PD alone or Pax-6 (5a), a splice variant with an altered PD affecting its DNA binding specificity, did not. DNase footprinting analysis revealed that truncated Pax-6 (PD+HD) binds to three regions (-183 to -152, -120 to -48, and -30 to +1) of the βB1-crystallin promoter. Earlier experiments showed that the βB1-crystallin promoter sequence from -120 to - 48 contains a cis element (PL2 at -90 to -76) that stimulates the activity of a heterologous promoter in lens cells but not in fibroblasts. In the present study, we show by electrophoretic mobility shift assay and cotransfection that Pax-6 binds to PL2 and represses its ability to activate promoter activity; moreover, mutation of PL2 eliminated binding by Pax-6. Taken together, our data indicate that Pax-6 (via its PD and HD) represses the βB1-crystallin promoter by direct interaction with the PL2 element. We thus suggest that the relatively high concentration of Pax-6 contributes to the absence of βB1-crystallin gene expression in lens epithelial cells and that diminishing amounts of Pax-6 in lens fiber cells during development allow activation of this gene.

Original languageEnglish (US)
Pages (from-to)5579-5586
Number of pages8
JournalMolecular and Cellular Biology
Volume18
Issue number9
StatePublished - 1998
Externally publishedYes

Fingerprint

Crystallins
Lenses
Genes
Chickens
Plasmids
Fibroblasts
Epithelial Cells
Chloramphenicol O-Acetyltransferase
Deoxyribonucleases
Electrophoretic Mobility Shift Assay
Reporter Genes
Transcriptional Activation
Transcription Factors
Gene Expression
Mutation
DNA

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics
  • Cell Biology

Cite this

Dual roles for Pax-6 : A transcriptional repressor of lens fiber cell- specific β-crystallin genes. / Duncan, Melinda K.; Haynes, John I.; Cvekl, Ales; Piatigorsky, Joram.

In: Molecular and Cellular Biology, Vol. 18, No. 9, 1998, p. 5579-5586.

Research output: Contribution to journalArticle

Duncan, Melinda K. ; Haynes, John I. ; Cvekl, Ales ; Piatigorsky, Joram. / Dual roles for Pax-6 : A transcriptional repressor of lens fiber cell- specific β-crystallin genes. In: Molecular and Cellular Biology. 1998 ; Vol. 18, No. 9. pp. 5579-5586.
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