Progesterone regulation of RUSH/SMARCA3/HLTF includes DNA looping

RUSH/SMARCA3 (SWI/SNF-related matrix-associated actin-dependent regulator of chromatin subfamily A, member 3) is capable of sequence-selective DNA binding and ATP-dependent DNA unwinding. In rabbit uterine epithelial cells, RUSH-1α (113 kDa) is the progesterone-dependent splice variant and RUSH-1β (95 kDa) is the oestrogen-dependent splice variant. Rabbit RUSH/SMARCA3 mRNA is primarily regulated at the proximal promoter (−162/+90) via a PRE (progesterone-response element) half-site/overlapping Y-box domain (−38/−26) and two Sp (specificity protein) 3 sites centred at −128 and −58. We investigated hormone regulation by exploring binding of transcription factors to a putative RUSH/SMARCA3 site (−616/−611) and the distal Sp3 (−131/−126) site. In response to progesterone, RUSH-1α binds the RUSH site and the Sp3 site becomes a functional binding site for Egr-1 (early growth-response gene product 1)/Sp (specificity protein)1/3/MAZ (Myc-associated zinc-finger protein)/MZF1 (myeloid zinc finger 1)/c-Rel. TransSignal TF–TF Interaction Arrays, supershift assays and ChIP (chromatin immunoprecipitation) analyses confirmed strong physical interactions between RUSH and Egr-1/c-Rel. Higher-order long-range interactions between RUSH and the Egr-1/c-Rel derivative of the anisotropic flexibility of the intervening DNA sequence were shown with 3C (chromosome conformation capture) assays. Transient transfection assays with mutant constructs showed the co-operative interaction between RUSH and Egr-1 mediates repression by c-Rel. Thus DNA-bound RUSH/SMARCA3 communicates with its own proximal promoter by looping the intervening DNA. Moreover, progesterone-dependent DNA looping is an adjunct to progesterone induction of the RUSH/SMARCA3 gene because the availability of RUSH isoforms and relevant binding partners is progesterone-regulated.