Abstract
Purpose: :
The identification of the information content of eukaryotic promoters has thus far remained confined to universal landmarks and conserved sequence elements such as enhancers and transcription factor binding motifs, while sequences surrounding these motifs, which may be gene-specific, have largely been ignored. We wanted to ascertain the role of gene-specific sequences if any in the regulation of the expression from the αB-crystallin (cryab) heat shock promoter. αB-crystallin (cryab) is regulated by a heat shock promoter which is activated in a developmentally regulated fashion in the ocular lens, retina and the brain among other tissues and therefore presents an ideal paradigm for these investigations.
Methods: :
We used reporter constructs with various manipulations to understand the impact of genetic manipulations on expression both in the test tube as well as in cultured cells. More importantly we have now used transgenic mice to assess whether the sequences identified in cultured cell assays have a role in vivo, in transgenic mice. Quantitative real time polymerase chain reaction and immunoblotting was used to follow the reporter expression in the transgenic mice.
Results: :
The genome-wide search of the promoter sequences surrounding the heat shock promoter of the cryab ascertained that while heat shock elements (HSEs) are universal, the sequences surroundingthe heat shock elements (HSEs) are gene-specific. In vitro expression studies in cultured cells have shown that a 10 bp sequence adjoining the heat shock elements is required for reporter expression from the cryab promoter in cultured cells. We demonstrate here that without this 10 bp sequence the expression from this promoter is lost in all tissues examined (including the ocular lens, retina, brain, heart kidney and muscle) in the transgenic mice.
Conclusions: :
These data reveal an as yet unrecognized form of the control of promoter activation in eukaryotic cells, namely the critical importance of Gene-specific Promoter Sequence (GPS) elements in the activation of a eukaryotic promoter/gene(such as cryab).
Keywords: crystallins • gene/expression • transcription