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Progesterone-Dependent Deoxyribonucleic Acid Looping between RUSH/SMARCA3 and Egr-1 Mediates Repression by c-Rel

Department of Cell Biology and Biochemistry, Texas Tech University Health Sciences Center (TTUHSC), Lubbock, Texas 79430

Address all correspondence and requests for reprints to: Beverly S. Chilton, Ph.D., Texas Tech University Health Sciences Center, 3601 4th Street, MS6540, Lubbock, Texas 79430. E-mail: beverly.chilton{at}ttuhsc.edu.

Steroids regulate alternative splicing of RUSH/SMARCA3. The full-length, progesterone-dependent -isoform and the 3'-truncated, estrogen-dependent β-isoform have identical DNA-binding domains, nuclear localization signals, and RING fingers. Transcription of RUSH/SMARCA3 is mediated by a bipartite progesterone receptor half-site/overlapping Y-box combination (–38/–26), where progesterone activation is attenuated by nuclear factor Y binding. Regulation also involves two GC-rich sequences in the proximal promoter (–162/+90) and a RUSH/SMARCA3 site (–616/–611) in the 5'-untranslated region. Isoform-specific binding to the RUSH/SMARCA3 site is dictated by the hormonal milieu, as is the availability of factors that bind to the distal GC-rich site (–131/–126), a composite binding site for Egr-1/specific protein-1/3/Myc-associated zinc finger protein/myeloid zinc finger-1/c-Rel, and the proximal GC-rich site (–62/–53), which binds only Sp1/3. TransSignal TF-TF interaction arrays, supershift assays, and chromatin immunoprecipitation analyses confirmed strong physical interactions between RUSH/Egr-1 and RUSH/c-Rel that were visualized with fluorescent microscopy. Higher-order, long-range interactions between RUSH and Egr-1/c-Rel derivative of the anisotropic flexibility of the intervening DNA sequence were shown by Chromosome Conformation Capture assay. Glutathione S-transferase pull-downs confirmed that the RING finger is the protein-binding domain, suggesting that the RUSH isoforms have equivalent potential for protein interactions. Transient transfection assays showed that the cooperative interaction between RUSH and Egr-1 mediates repression by c-Rel. Thus, progesterone-induced transcription is fine-tuned by isoform-specific autoregulation, in which newly synthesized RUSH-1 binds DNA and interacts physically with liganded Egr-1 in the proximal promoter via a DNA-looping mechanism to mediate repression by c-Rel. In the absence of progesterone induction, RUSH-1β replaces RUSH-1 binding, Egr-1 and c-Rel are unavailable as molecular ties, and DNA looping is disfavored.

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Ligands:   Progesterone  |  R5020