Pax-6 and alphaB-crystallin/small heat shock protein gene regulation in the murine lens. Interaction with the lens-specific regions, LSR1 and LSR2.
We have demonstrated previously that a transgene comprising the -164/+44 fragment of the murine alphaB-crystallin gene fused to the bacterial chloramphenicol acetyltransferase (cat) gene is lens-specific in transgenic mice. The -147 to -118 sequence was identified as a lens-specific regulatory region and is called here LSR1 for lens-specific region 1. In the present experiments, a -115/+44-cat transgene was also lens-specific in transgenic mice, although the average activity was 30 times lower than that derived from the -164/+44-cat transgene. The -115/+44 alphaB-crystallin fragment contains a highly conserved region (-78 to -46) termed here LSR2. A -68/+44-cat transgene, in which LSR2 is truncated, was inactive in transgenic mice. DNase I footprinting indicated that LSR1 and LSR2 bind partially purified nuclear proteins from either alphaTN4-1 lens cells or the mouse lens as well as the purified paired domain of Pax-6. Site-specific mutation of LSR1 eliminated both Pax-6 binding and promoter activity of the -164/+44-cat transgene in transgenic mice. Finally antibody/electrophoretic mobility shift assays and cotransfection experiments indicated that Pax-6 can activate the alphaB-crystallin promoter via LSR1 and LSR2. Our data strengthen the idea that Pax-6 has had a major role in recruiting genes for high expression in the lens.
Gopal-Srivastava
R
R
Laboratory of Molecular and Developmental Biology, National Eye Institute, National Institutes of Health, Bethesda, Maryland 20892-2730, USA.
Cvekl
A
A
Piatigorsky
J
J
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D003459:Crystallins / Q000096:biosynthesis* / Q000235:genetics; D018983:DNA Footprinting; D004268:DNA-Binding Proteins / Q000378:metabolism*; D005136:Eye Proteins; D018507:Gene Expression Regulation, Developmental; D006360:Heat-Shock Proteins / Q000378:metabolism*; D018398:Homeodomain Proteins; D007908:Lens, Crystalline / Q000378:metabolism*; D051379:Mice; D008822:Mice, Transgenic; D008969:Molecular Sequence Data; D000071841:PAX6 Transcription Factor; D051761:Paired Box Transcription Factors; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D012097:Repressor Proteins; D014157:Transcription Factors / Q000378:metabolism*; D019076:Transgenes
D003459:Crystallins; D004268:DNA-Binding Proteins; D005136:Eye Proteins; D006360:Heat-Shock Proteins; D018398:Homeodomain Proteins; D000071841:PAX6 Transcription Factor; D051761:Paired Box Transcription Factors; C000605732:Pax6 protein, mouse; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; D014157:Transcription Factors