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(381) THE RUSH PROMOTER CONTAINS A FUNCTIONAL Inr-DPE COMBINATION AND STEROID RESPONSE ELEMENTS. Chilton, Beverly1, Hendrix, Ericka1, Hewetson, Aveline1, 1 Department of Cell Biology & Biochemistry, Lubbock, TX ABSTRACT- RUSH, a member of the Smarca3 family of chromatin remodeling machines, is critical to PRL enhancement of progesterone-dependent uteroglobin gene transcription. To characterize the regulatory sequences responsible for RUSH expression, 2059-bp of genomic sequence (GenBank #AF481732) was cloned. A series of 5′-deletion constructs at 550-bp intervals beginning with a –1814/+90 fragment were cloned into a luciferase reporter plasmid (pGL3-Basic) and tested for transcriptional activity in two different SV-40 transformed rabbit uterine epithelial cell lines (HRE-H9, RBE-7) following transient transfection. Deletions from -1814 to -164 eliminated multiple GAGA-binding elements and alternating pyrimidine-purine elements (CA) that are anisotropically flexible, and known to promote DNA bending, and increased reporter gene activity 6-fold. Deletion analysis indicated the core elements essential for RUSH promoter activity are retained in the –164/+90 region. The size of the 5′-untranslated region (233-bp) and the transcription start site were determined by primer extension analysis. A single, 90-bp primer extension product was resolved by denaturing PAGE (7M urea). The transcription start site was mapped to a consensus initiator (Inr) element in a TATA-less region with a downstream promoter element (DPE) at position +29. Interrogation of the proximal-promoter with MatInspector and TRANSFAC resources led to the identification of two putative Sp1 binding sites centered at -58 and -130, plus a Y-box (-34/-26) that overlaps GR and ER half-sites. In addition to the classic Inr-DPE combination, a second putative Inr (-25/-20) was identified. Mutations in one or both Sp1 sites had little or no effect in HRE-H9 cells. In contrast, removal of both elements caused a 2-fold increase in promoter activity in RBE-7 cells. Mutation analysis confirmed the Inr at +1 is capable of directing accurate transcription initiation in conjunction with the DPE. However, the second Inr is also capable of directing basal transcription. Mutations in the GR binding site reduced promoter activity 12-13-fold in RBE-7 cells, and 2-5-fold in HRE-H9 cells. This steroid response element in the core promoter is likely to be an active contributor to the combinatorial regulation of RUSH transcription factors. Supported by NIH HD29457 to BSC. KEY WORDS: RUSH/Smarca3, Uteroglobin, Prolactin, Promoter |
