Pax-6 interactions with TATA-box-binding protein and retinoblastoma protein.
OBJECTIVE
To identify proteins that physically interact with Pax-6, a paired domain- and homeodomain (HD)-containing transcription factor that is a key regulator of eye development.


METHODS
Protein-protein interactions involving Pax-6, TATA-box-binding protein (TPB), and retinoblastoma protein were studied using affinity chromatography with Pax-6 as ligand, glutathione-S-transferase (GST) pull-down assays, and immunoprecipitations.


RESULTS
The authors have shown that Pax-6 is a sequence-specific activator of many crystallin genes, all containing a TATA box, in the lens. Others have shown that lens fiber cell differentiation, characterized by temporally and spatially regulated crystallin gene expression, depends on retinoblastoma protein. In the present study it was shown that Pax-6 interacted with the TBP, the DNA-binding subunit of general transcription complex TFIID. GST pull-down assays indicated that this interaction was mediated by the Pax-6 HD, with a substantial role for its N-terminal arm and first two alpha-helices. The experiments also indicated a binding role for the C-terminal-activation domain of the protein. In addition, the present study showed that the HD of Pax-6 interacted with retinoblastoma protein. Immunoprecipitation experiments confirmed retinoblastoma protein/Pax-6 complexes in lens nuclear extracts.


CONCLUSIONS
Blending the present results with those in the literature suggests that Pax-6 and retinoblastoma protein participate in overlapping regulatory pathways controlling epithelial cell division, fiber cell elongation, and crystallin gene expression during lens development.
Cvekl
A
A
Laboratory of Molecular and Developmental Biology, National Eye Institute, National Institutes of Health, Bethesda, Maryland 20892-2730, USA.
Kashanchi
F
F
Brady
J N
JN
Piatigorsky
J
J
D000595:Amino Acid Sequence; D000818:Animals; D002455:Cell Division; D002846:Chromatography, Affinity; D003459:Crystallins / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004847:Epithelial Cells / Q000502:physiology; D005136:Eye Proteins / Q000378:metabolism*; D005982:Glutathione Transferase; D018398:Homeodomain Proteins; D007908:Lens, Crystalline / Q000502:physiology; D008969:Molecular Sequence Data; D000071841:PAX6 Transcription Factor; D051761:Paired Box Transcription Factors; D010957:Plasmids; D011233:Precipitin Tests; D011817:Rabbits; D012097:Repressor Proteins; D016160:Retinoblastoma Protein / Q000378:metabolism*; D016385:TATA Box; D035181:TATA-Box Binding Protein; D014157:Transcription Factors / Q000378:metabolism*
D003459:Crystallins; D004268:DNA-Binding Proteins; D005136:Eye Proteins; D018398:Homeodomain Proteins; D000071841:PAX6 Transcription Factor; D051761:Paired Box Transcription Factors; D012097:Repressor Proteins; D016160:Retinoblastoma Protein; D035181:TATA-Box Binding Protein; D014157:Transcription Factors; D005982:Glutathione Transferase