A complex enhancer of the chicken βA3/A1-crystallin gene depends on an AP-1-CRE element for activity

Joan B. McDermott, Ales Cvekl, Joram Piatigorsky

Research output: Contribution to journalArticle

10 Citations (Scopus)

Abstract

Purpose. To define transcriptional regulatory elements of the chicken βA3/A1-crystallin gene. Methods. Reporter genes were made with fragments of the chicken βA3/A1-crystallin gene fused to the bacterial gene encoding chloramphenicol acetyltransferase (CAT). The reporter plasmids were transfected into primary cultures of chicken-patched lens epithelium of fibroblast cells, and the CAT activity of cellular extracts was measured. The binding of lens nuclear proteins to βA3/A1 sequences was tested in electrophoretic mobility shift assays. Results. Sequences from -287 to -254 bp relative to the transcriptional start site function as an enhancer in transfected lens and nonlens cells. The length of a T-rich sequence downstream of the enhancer influences its activity. Minimal enhancer activity depends on sequences between -270 and -254 bp, and full activity requires additional upstream sequences. The minimal enhancer includes a consensus sequence (TGAGTCA) for basic region-leucine zipper (bZIP) proteins of the AP-1-CREB superfamily. Lens nuclear proteins bind the enhancer sequences to form several specific complexes, some of which are related antigenically to members of the AP-1 and CREB families of proteins. Conclusions. An enhancer of the chicken βA3/A1-crystallin gene between - 287 and -254 bp functions in both lens and nonlens cells and binds multiple nuclear proteins. Temporal and spatial regulation of βA3/A1 expression in the lens may be regulated by the enhancer.

Original languageEnglish (US)
Pages (from-to)951-959
Number of pages9
JournalInvestigative Ophthalmology and Visual Science
Volume38
Issue number5
StatePublished - 1997
Externally publishedYes

Fingerprint

Crystallins
Transcription Factor AP-1
Chickens
Lenses
Nuclear Proteins
Chloramphenicol O-Acetyltransferase
Genes
Transcriptional Regulatory Elements
Basic-Leucine Zipper Transcription Factors
Bacterial Genes
Leucine Zippers
Cyclic AMP Response Element-Binding Protein
Consensus Sequence
Electrophoretic Mobility Shift Assay
Reporter Genes
Plasmids
Epithelium
Fibroblasts

Keywords

  • β-crystallins
  • AP-1
  • CREB
  • enhancer
  • gene regulation
  • lens

ASJC Scopus subject areas

  • Ophthalmology

Cite this

A complex enhancer of the chicken βA3/A1-crystallin gene depends on an AP-1-CRE element for activity. / McDermott, Joan B.; Cvekl, Ales; Piatigorsky, Joram.

In: Investigative Ophthalmology and Visual Science, Vol. 38, No. 5, 1997, p. 951-959.

Research output: Contribution to journalArticle

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T1 - A complex enhancer of the chicken βA3/A1-crystallin gene depends on an AP-1-CRE element for activity

AU - McDermott, Joan B.

AU - Cvekl, Ales

AU - Piatigorsky, Joram

PY - 1997

Y1 - 1997

N2 - Purpose. To define transcriptional regulatory elements of the chicken βA3/A1-crystallin gene. Methods. Reporter genes were made with fragments of the chicken βA3/A1-crystallin gene fused to the bacterial gene encoding chloramphenicol acetyltransferase (CAT). The reporter plasmids were transfected into primary cultures of chicken-patched lens epithelium of fibroblast cells, and the CAT activity of cellular extracts was measured. The binding of lens nuclear proteins to βA3/A1 sequences was tested in electrophoretic mobility shift assays. Results. Sequences from -287 to -254 bp relative to the transcriptional start site function as an enhancer in transfected lens and nonlens cells. The length of a T-rich sequence downstream of the enhancer influences its activity. Minimal enhancer activity depends on sequences between -270 and -254 bp, and full activity requires additional upstream sequences. The minimal enhancer includes a consensus sequence (TGAGTCA) for basic region-leucine zipper (bZIP) proteins of the AP-1-CREB superfamily. Lens nuclear proteins bind the enhancer sequences to form several specific complexes, some of which are related antigenically to members of the AP-1 and CREB families of proteins. Conclusions. An enhancer of the chicken βA3/A1-crystallin gene between - 287 and -254 bp functions in both lens and nonlens cells and binds multiple nuclear proteins. Temporal and spatial regulation of βA3/A1 expression in the lens may be regulated by the enhancer.

AB - Purpose. To define transcriptional regulatory elements of the chicken βA3/A1-crystallin gene. Methods. Reporter genes were made with fragments of the chicken βA3/A1-crystallin gene fused to the bacterial gene encoding chloramphenicol acetyltransferase (CAT). The reporter plasmids were transfected into primary cultures of chicken-patched lens epithelium of fibroblast cells, and the CAT activity of cellular extracts was measured. The binding of lens nuclear proteins to βA3/A1 sequences was tested in electrophoretic mobility shift assays. Results. Sequences from -287 to -254 bp relative to the transcriptional start site function as an enhancer in transfected lens and nonlens cells. The length of a T-rich sequence downstream of the enhancer influences its activity. Minimal enhancer activity depends on sequences between -270 and -254 bp, and full activity requires additional upstream sequences. The minimal enhancer includes a consensus sequence (TGAGTCA) for basic region-leucine zipper (bZIP) proteins of the AP-1-CREB superfamily. Lens nuclear proteins bind the enhancer sequences to form several specific complexes, some of which are related antigenically to members of the AP-1 and CREB families of proteins. Conclusions. An enhancer of the chicken βA3/A1-crystallin gene between - 287 and -254 bp functions in both lens and nonlens cells and binds multiple nuclear proteins. Temporal and spatial regulation of βA3/A1 expression in the lens may be regulated by the enhancer.

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