Pax-6 and alpha B-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 alpha B-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 alpha B-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 alpha TN4-1 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 alpha B-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.