Lens-specific activity of the mouse alpha A-crystallin promoter in the absence of a TATA box: functional and protein binding analysis of the mouse alpha A-crystallin PE1 region.
Lens-specific expression of the mouse alpha A-crystallin gene is regulated at the level of transcription. Here, we have studied the role of the PE1 region, which contains the TATA box (-31/-26) and the immediately adjacent PE1B sequence (-25/-12), in transcriptional regulation. Deletions within either the TATA box or PE1B sequence eliminated promoter activity in transfected lens cells. Surprisingly, these deletions did not eliminate lens-specific promoter activity of the transgene of transgenic mice. Transcription of the transgene with a TATA-deleted promoter initiated at multiple sites in the lenses of the transgenic mice. Footprint analysis revealed that the entire PE1 region was protected by nuclear extracts prepared from lens cells which express the alpha A-crystallin gene and from fibroblasts which do not express the gene. The -37/+3 region formed three specific EMSA complexes using lens cell nuclear extracts, while a similar but much less intense pattern was observed when a fibroblast nuclear extract was used. Competition experiments indicated that these complexes were not due to the binding of TBP to the TATA box, but rather to the binding of other nuclear proteins to the PE1B -25/-19 region. A series of co-transfection competition studies in vivo also suggested the functional importance of proteins binding in the -25/-19 region. The PE1B protein-DNA interactions appear to be conserved in the chicken, rodent and human alpha A-crystallin gene as well as within the alpha A- and alpha B-crystallin genes in the mouse. Our findings indicate that the PE1B region is important for mouse alpha A-crystallin promoter activity; the proximity of this site to the TATA box raises the possibility for cooperativity or competition between TBP and PE1B-bound proteins.
Sax
C M
CM
Laboratory of Molecular and Developmental Biology, NEI, NIH, Bethesda, MD 20892.
Cvekl
A
A
Kantorow
M
M
Gopal-Srivastava
R
R
Ilagan
J G
JG
Ambulos
N P
NP
Piatigorsky
J
J
D000818:Animals; D001483:Base Sequence; D001667:Binding, Competitive; D002457:Cell Extracts / Q000737:chemistry; D002460:Cell Line; D002467:Cell Nucleus / Q000737:chemistry; D017124:Conserved Sequence; D003459:Crystallins / Q000235:genetics*; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004847:Epithelial Cells; D007908:Lens, Crystalline / Q000166:cytology / Q000378:metabolism*; D051379:Mice; D008822:Mice, Transgenic; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D017384:Sequence Deletion / Q000502:physiology; D016385:TATA Box / Q000235:genetics; D014158:Transcription, Genetic / Q000235:genetics*
D002457:Cell Extracts; D003459:Crystallins; D004268:DNA-Binding Proteins; D011993:Recombinant Fusion Proteins; D004247:DNA