Identification of the RUSH consensus-binding site by cyclic amplification and selection of targets: demonstration that RUSH mediates the ability of prolactin to augment progesterone-dependent gene expression.
RUSH-1alpha(beta) transcription factors were cloned by recognition site screening with an 85-bp region (-170/-85) of the rabbit uteroglobin gene. Deletion analysis showed this region was essential to prolactin (PRL) action, but conclusions were limited by the complexity of the large deletion. Cyclic amplification and selection of targets (CASTing) was used to identify the RUSH-binding site (-126/-121). Endometrial nuclear proteins were incubated with a pool of degenerate oligonucleotides and immunoprecipitated with RUSH-1alpha(beta) antibodies. Bound DNA was amplified by PCR. The consensus motif (MCWTDK) was identified after five rounds of CASTing, authenticated by CASTing with RUSH-1alpha-specific antibodies and recombinant protein, and refined with EMSA. Dissociation rate constants (K(d) = 0.1-1.0 nM; r = 0.99) revealed high-affinity binding. Chromatin immunoprecipitation confirmed in vivo binding of RUSH to the transcriptionally active uteroglobin promoter. CASTing also revealed RUSH-GATA transcription factor interactions. Endometrial GATA-4 expression is progesterone dependent (Northern analysis) and preferentially localized in the epithelium (in situ hybridization). Although physically affiliated with RUSH, uterine forms of GATA-4 were not required for RUSH-DNA binding. Site-directed mutagenesis and transient transfection assays showed the RUSH motif mediates the ability of PRL to augment progesterone-dependent uteroglobin transcription. RUSH is central to the mechanism whereby PRL augments progesterone-dependent gene transcription.
Department of Cell Biology and Biochemistry, Texas Tech University Health Sciences Center, Lubbock, Texas 79430, USA.
Aveline
A
Hewetson
Ericka C
EC
Hendrix
Malini
M
Mansharamani
Vaughan H
VH
Lee
Beverly S
BS
Chilton
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D016384:Consensus Sequence; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D024202:Electrophoretic Mobility Shift Assay; D004717:Endometrium / Q000378:metabolism; D005260:Female; D050991:GATA4 Transcription Factor; D005786:Gene Expression Regulation / Q000187:drug effects*; D017403:In Situ Hybridization; D008969:Molecular Sequence Data; D011374:Progesterone / Q000494:pharmacology*; D011388:Prolactin / Q000494:pharmacology*; D011485:Protein Binding; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011817:Rabbits; D020218:Response Elements / Q000235:genetics; D017384:Sequence Deletion / Q000235:genetics; D013816:Thermodynamics; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism; D014598:Uteroglobin / Q000235:genetics*
D004268:DNA-Binding Proteins; D050991:GATA4 Transcription Factor; D012333:RNA, Messenger; C104270:SMARCA3 protein, Oryctolagus cuniculus; D014157:Transcription Factors; D011374:Progesterone; D011388:Prolactin; D014598:Uteroglobin