Developmental regulation of the chicken βB1-crystallin promoter

M. K. Duncan, S. I. Tomarev, Ales Cvekl, J. Piatigorsky

Research output: Contribution to journalArticle

Abstract

Purpose. To investigate the molecular basis of the lens-fiber cell-specific expression of the chicken βB1-crystallin gene. Methods. Various chicken βB1-crystallin promoter fragments were linked to the chloramphenicol acetyl transferase (CAT) reporter gene and their activities assayed in transgenic mice throughout lens development. Gel shifts were used to identify sequences in the chicken βB1-crystallin gene capable of binding lens nuclear proteins and purified recombinant transcription factors. Co-transfection of chicken βB1-crystallin promoter fragments with Pax-6 and Prox 1 expression vectors was performed in primary patched lens epithelial cells. Results. The chicken βB1-crystallin promoter (-434/+30) is able to drive the expression of a CAT reporter gene at levels approaching that of endogenous mouse βB1-crystallin specifically in the transgenic mouse lens. Direct CAT histochemistry demonstrated that this -434/+30 transgene is expressed in both the primary and secondary lens fibers of transgenic mice. Deletion of sequences from -434/-153 resulted in a 10-fold reduction in promoter activity in the lenses of adult transgenic mice. Direct CAT histochemistry demonstrated that this 10-fold reduction was due to both lower gene expression in primary fiber cells as well as a lack of detectable CAT expression in secondary fiber cells. The chicken βB1-crystallin promoter can bind the homeobox proteins Pax-6 and Prox 1 in gel shift studies. Cotransfection studies demonstrated that Pax-6, which is expressed at high levels in lens epithelial cells, represses the activity of the chicken βB1-crystallin promoter, while Prox 1 activates βB1-crystallin gene expression. Conclusions. Sequences from -434/+30 of the chicken βB1-crystallin promoter are sufficient to direct high level gene expression to lens fiber cells. Pax-6 can repress and Prox 1 can activate chicken βB1-crystallin promoter activity in transfected lens cells. The quantitative and spatial pattern of chicken βB1-crystallin may result from a balance between the amounts of Pax-6 and Prox 1 expressed by the cells of the lens.

Original languageEnglish (US)
JournalInvestigative Ophthalmology and Visual Science
Volume37
Issue number3
StatePublished - Feb 15 1996
Externally publishedYes

Fingerprint

Crystallins
Chickens
Lenses
Naproxen
Chloramphenicol
Transferases
Transgenic Mice
Reporter Genes
Gene Expression
Gels
Epithelial Cells
Homeodomain Proteins
Sequence Deletion
Nuclear Proteins
Transgenes
Genes
Transfection
Carrier Proteins

ASJC Scopus subject areas

  • Ophthalmology

Cite this

Developmental regulation of the chicken βB1-crystallin promoter. / Duncan, M. K.; Tomarev, S. I.; Cvekl, Ales; Piatigorsky, J.

In: Investigative Ophthalmology and Visual Science, Vol. 37, No. 3, 15.02.1996.

Research output: Contribution to journalArticle

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abstract = "Purpose. To investigate the molecular basis of the lens-fiber cell-specific expression of the chicken βB1-crystallin gene. Methods. Various chicken βB1-crystallin promoter fragments were linked to the chloramphenicol acetyl transferase (CAT) reporter gene and their activities assayed in transgenic mice throughout lens development. Gel shifts were used to identify sequences in the chicken βB1-crystallin gene capable of binding lens nuclear proteins and purified recombinant transcription factors. Co-transfection of chicken βB1-crystallin promoter fragments with Pax-6 and Prox 1 expression vectors was performed in primary patched lens epithelial cells. Results. The chicken βB1-crystallin promoter (-434/+30) is able to drive the expression of a CAT reporter gene at levels approaching that of endogenous mouse βB1-crystallin specifically in the transgenic mouse lens. Direct CAT histochemistry demonstrated that this -434/+30 transgene is expressed in both the primary and secondary lens fibers of transgenic mice. Deletion of sequences from -434/-153 resulted in a 10-fold reduction in promoter activity in the lenses of adult transgenic mice. Direct CAT histochemistry demonstrated that this 10-fold reduction was due to both lower gene expression in primary fiber cells as well as a lack of detectable CAT expression in secondary fiber cells. The chicken βB1-crystallin promoter can bind the homeobox proteins Pax-6 and Prox 1 in gel shift studies. Cotransfection studies demonstrated that Pax-6, which is expressed at high levels in lens epithelial cells, represses the activity of the chicken βB1-crystallin promoter, while Prox 1 activates βB1-crystallin gene expression. Conclusions. Sequences from -434/+30 of the chicken βB1-crystallin promoter are sufficient to direct high level gene expression to lens fiber cells. Pax-6 can repress and Prox 1 can activate chicken βB1-crystallin promoter activity in transfected lens cells. The quantitative and spatial pattern of chicken βB1-crystallin may result from a balance between the amounts of Pax-6 and Prox 1 expressed by the cells of the lens.",
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N2 - Purpose. To investigate the molecular basis of the lens-fiber cell-specific expression of the chicken βB1-crystallin gene. Methods. Various chicken βB1-crystallin promoter fragments were linked to the chloramphenicol acetyl transferase (CAT) reporter gene and their activities assayed in transgenic mice throughout lens development. Gel shifts were used to identify sequences in the chicken βB1-crystallin gene capable of binding lens nuclear proteins and purified recombinant transcription factors. Co-transfection of chicken βB1-crystallin promoter fragments with Pax-6 and Prox 1 expression vectors was performed in primary patched lens epithelial cells. Results. The chicken βB1-crystallin promoter (-434/+30) is able to drive the expression of a CAT reporter gene at levels approaching that of endogenous mouse βB1-crystallin specifically in the transgenic mouse lens. Direct CAT histochemistry demonstrated that this -434/+30 transgene is expressed in both the primary and secondary lens fibers of transgenic mice. Deletion of sequences from -434/-153 resulted in a 10-fold reduction in promoter activity in the lenses of adult transgenic mice. Direct CAT histochemistry demonstrated that this 10-fold reduction was due to both lower gene expression in primary fiber cells as well as a lack of detectable CAT expression in secondary fiber cells. The chicken βB1-crystallin promoter can bind the homeobox proteins Pax-6 and Prox 1 in gel shift studies. Cotransfection studies demonstrated that Pax-6, which is expressed at high levels in lens epithelial cells, represses the activity of the chicken βB1-crystallin promoter, while Prox 1 activates βB1-crystallin gene expression. Conclusions. Sequences from -434/+30 of the chicken βB1-crystallin promoter are sufficient to direct high level gene expression to lens fiber cells. Pax-6 can repress and Prox 1 can activate chicken βB1-crystallin promoter activity in transfected lens cells. The quantitative and spatial pattern of chicken βB1-crystallin may result from a balance between the amounts of Pax-6 and Prox 1 expressed by the cells of the lens.

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