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This version published online on June 13, 2002
Molecular Endocrinology, doi:10.1210/me.2002-0064
Molecular Endocrinology Vol. 0, No. 2002 200200641-
doi:10.1210/me.2002-0064
Copyright © 2002 by the Endocrine Society.
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Submitted on February 8, 2002
Accepted on May 13, 2002

Identification of the RUSH Consensus-binding Site by CASTing: Demonstration that RUSH Mediates the Ability of PRL to Augment Progesterone-dependent Gene Expression

Aveline Hewetson1, Ericka C. Hendrix1, Malini Mansharamani1, Vaughan H. Lee1, and Beverly S. Chilton1*

1 Department of Cell Biology & Biochemistry (AH, EH, VHL, BSC), Texas Tech University Health Sciences Center, Lubbock, TX 79430; and Department of Cell Biology (MM), Johns Hopkins University School of Medicine, Baltimore, MD 21205 Key Words: RUSH transcription factors, Smarca3, Uteroglobin promoter, Secretoglobin, endometrium, CASTing, GATA-4, PRL signal transduction, progesterone, RUSH-binding site

* To whom correspondence should be addressed. E-mail: beverly.chilton{at}ttmc.ttuhsc.edu.

RUSH-1{alpha}(ß) 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 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-1{alpha}(ß) antibodies. Bound DNA was amplified by PCR. The consensus motif (MCWTDK) was identified after five rounds of CASTing, authenticated by CASTing with RUSH-1{alpha}-specific antibodies and recombinant protein, and refined with EMSA. Dissociation rate constants (Kd = 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.




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