Progesterone-dependent deoxyribonucleic acid looping between RUSH/SMARCA3 and Egr-1 mediates repression by c-Rel.
Steroids regulate alternative splicing of RUSH/SMARCA3. The full-length, progesterone-dependent alpha-isoform and the 3'-truncated, estrogen-dependent beta-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-1alpha 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-1beta replaces RUSH-1alpha binding, Egr-1 and c-Rel are unavailable as molecular ties, and DNA looping is disfavored.
Hewetson
Aveline
A
Department of Cell Biology and Biochemistry, Texas Tech University Health Sciences Center, Texas 79430, USA.
Chilton
Beverly S
BS
D000818:Animals; D001665:Binding Sites / Q000235:genetics; D047369:Chromatin Immunoprecipitation; D004247:DNA / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D051766:Early Growth Response Protein 1 / Q000235:genetics / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D008954:Models, Biological; D016133:Polymerase Chain Reaction; D011374:Progesterone / Q000494:pharmacology*; D011401:Promoter Regions, Genetic / Q000235:genetics; D011485:Protein Binding / Q000187:drug effects; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D020569:Proto-Oncogene Proteins c-rel / Q000235:genetics / Q000378:metabolism*; D011817:Rabbits; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic / Q000187:drug effects
D004268:DNA-Binding Proteins; D051766:Early Growth Response Protein 1; C100023:HLTF protein, human; D020033:Protein Isoforms; D020569:Proto-Oncogene Proteins c-rel; D014157:Transcription Factors; D011374:Progesterone; D004247:DNA