An alternative Pit-1 RNA splicing product reveals modular binding and nonmodular transcriptional activities of the POU-specific domain.
We have identified a form of the pituitary-specific POU protein Pit-1 that results from deletion of the POU-specific (POUs) domain by alternative RNA splicing. This natural variant of Pit-1 (called delta 4Pit-1) has revealed several aspects of the function of the POUs domain. The delta 4Pit-1 protein was characterized using a delta 4Pit-1-specific antiserum. Further, selection assays of random oligonucleotide pools identified binding site preferences for both wild type and delta 4Pit-1. Methylation interference, copper phenanthrolene, and missing contact analyses were used to compare the binding characteristics of the two forms of Pit-1 on a selected site. DNA binding affinity assays on several DNA elements revealed that the POUs domain contains a modular DNA binding activity affecting the DNA binding affinity of the entire POU domain on some, but not on other, DNA sites. Functional analysis on such DNA elements has revealed that the POUs domain is an essential, but nonmodular, component of the Pit-1 trans-activation domain dependent on its natural context within the Pit-1 protein.
Voss
J W
JW
Howard Hughes Medical Institute, School and Department of Medicine, University of California, San Diego, La Jolla 92093-0648.
Wilson
L
L
Rhodes
S J
SJ
Rosenfeld
M G
MG
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002522:Chlorocebus aethiops; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D009841:Oligonucleotides / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012326:RNA Splicing; D012045:Regulatory Sequences, Nucleic Acid; D016415:Sequence Alignment; D017384:Sequence Deletion; D050817:Transcription Factor Pit-1; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D015533:Transcriptional Activation
D004268:DNA-Binding Proteins; D009841:Oligonucleotides; D050817:Transcription Factor Pit-1; D014157:Transcription Factors; D004247:DNA