Structure-function analysis of the rat prolactin promoter: phasing requirements of proximal cell-specific elements.
Expression of PRL, a member of the GH family of genes, is restricted to the lactotroph cells of the anterior pituitary. The proximal promoter of the rat PRL (rPRL) gene contains four factor-binding sites. Three nonadjacent elements, footprints (FP) I, III, and IV, are separated by an integral number of helical turns and bind a pituitary-specific factor, LSF-1. FP II binds another factor present in pituitary and nonpituitary cells. The mechanisms by which DNA-bound proteins influence RNA polymerase-II activity over large distances are not fully understood, but protein-protein interactions, with looping of intervening DNA, may bring distant sites into close proximity. Here, we demonstrate, using protein titration studies, that LSF-1 binds to the most proximal FP I element with the highest affinity, whereas it binds the more distal elements, FP III and FP IV, with progressively lower affinities. Time-course and salt-sensitivity studies reveal that binding of LSF-1 to all three pituitary-specific rPRL promoter sites occurs rapidly (less than or equal to 1 min) and requires fairly high salt concentrations (greater than or equal to 300 mM KCl) to destabilize protein-DNA interactions. Moreover, once bound, the pituitary nuclear factor(s) induces a conformational change in rPRL DNA structure with greatly delayed kinetics (greater than 15 min) and at a different salt concentration than are required for simply factor binding. Taken together, these data suggest a model in which LSF-1 initially binds fairly rapidly to multiple nonadjacent elements and then interacts with itself or other DNA-bound proteins much more slowly, possibly looping or bending the rPRL promoter.(ABSTRACT TRUNCATED AT 250 WORDS)
Department of Medicine, University of Colorado Health Sciences Center, Denver 80262.
C
C
Harvey
S M
SM
Jackson
S K
SK
Siddiqui
A
A
Gutierrez-Hartmann
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002460:Cell Line; D004268:DNA-Binding Proteins / Q000378:metabolism; D003850:Deoxyribonuclease I; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D015345:Oligonucleotide Probes; D010911:Pituitary Neoplasms; D011388:Prolactin / Q000235:genetics*; D011401:Promoter Regions, Genetic; D012319:RNA Polymerase II / Q000378:metabolism; D051381:Rats; D014158:Transcription, Genetic
D004268:DNA-Binding Proteins; D015345:Oligonucleotide Probes; D011388:Prolactin; D012319:RNA Polymerase II; D003850:Deoxyribonuclease I