Progesterone-dependent DNA looping between RUSH/SMARCA3 and Egr-1 mediates repression by c-Rel Abbreviated title: Progesterone-dependent DNA looping

Steroids regulate alternative splicing of RUSH/SMARCA3. The full-length, progesterone-dependent α isoform and the 3′-truncated, estrogen-dependent β isoform have identical DBDs, NLSs and RING fingers. Transcription of RUSH/SMARCA3 is mediated by a bipartite progesterone receptor halfsite/overlapping Y-box combination (-38/-26), where progesterone activation is attenuated by NF-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′-UTR. 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/Sp1/3/MAZ/MZF1/c-Rel, and the proximal GC-rich site (62/-53), which binds only Sp1/3. TransSignal TF-TF Interaction arrays, supershift assays and ChIP 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 Egr1/c-Rel derivative of the anisotropic flexibility of the intervening DNA sequence were shown by 3C assay. GST-pull-downs confirmed the RING finger is the protein-binding domain suggesting the RUSH isoforms have equivalent potential for protein interactions. Transient transfection assays showed 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-1α binds DNA, 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-1α binding, Egr-1 and c-Rel are unavailable as molecular ties, and DNA looping is disfavored. 1 Molecular Endocrinology. First published ahead of print January 3, 2008 as doi:10.1210/me.2007-0432 Copyright (C) 2008 by The Endocrine Society