Domains of estrogen receptor alpha (ERalpha) required for ERalpha/Sp1-mediated activation of GC-rich promoters by estrogens and antiestrogens in breast cancer cells

Estrogen receptor alpha (ERalpha)/Sp1 activation of GC-rich gene promoters in breast cancer cells is dependent, in part, on activation function 1 (AF1) of ERalpha, and this study investigates contributions of the DNA binding domain (C) and AF2 (DEF) regions of ERalpha on activation of ERalpha/Sp1. 17Beta-estradiol (E2) and the antiestrogens 4-hydroxytamoxifen and ICI 182,780 induced reporter gene activity in MCF-7 and MDA-MB-231 cells cotransfected with human or mouse ERalpha (hERalpha or MOR), but not ERbeta and GC-rich constructs containing three tandem Sp1 binding sites (pSp13) or other E2-responsive GC-rich promoters. Estrogen and antiestrogen activation of hERalpha/Sp1 was dependent on overlapping and different regions of the C, D, E, and F domains of ERalpha. Antiestrogen-induced activation of hERalpha/Sp1 was lost using hERalpha mutants deleted in zinc finger 1 [amino acids (aa) 185-205], zinc finger 2 (aa 218-245), and the hinge/helix 1 (aa 265-330) domains. In contrast with antiestrogens, E2-dependent activation of hERalpha/Sp1 required the C-terminal F domain (aa 579-595), which contains a beta-strand structural motif. Moreover, in peptide competition experiments overexpression of a C-terminal (aa 575-595) F domain peptide specifically blocked E2-dependent activation of hERalpha/Sp1, suggesting that F domain interactions with nuclear cofactors are required for ERalpha/Sp1 action.     

Activation of mouse RAG-2 promoter by Myc-associated zinc finger protein

Recombination activating gene-1 (RAG-1) and RAG-2 are expressed specifically in lymphocytes undergoing the antigen receptor gene rearrangement during the lymphocyte development. Our previous study showed that the -41 to -17 nucleotides (nt) 5' -upstream region of mouse RAG-2 were pre-requisite for the core promoter activity and that Pax-5/c-Myb/LEF-1 protein-protein complex was responsible for its activity in immature B cells. In this study, we show that the -65/-42 sequence, the non-conserved sequence between human and mouse RAG-2 promoter, is necessary for the full promoter activity for mouse RAG-2. Electrophoresis mobility shift assay revealed that Myc-associated zinc finger protein (MAZ) as well as SP1/3 binds a GA box in this region. Using chromatin immunoprecipitation, we show that MAZ binds the RAG-2 promoter region in pre-B cells. Furthermore, we show that MAZ synergistically activates the murine RAG-2 promoter with Pax-5/c-Myb/LEF-1 complex. These results first demonstrate that MAZ participates in activation of mouse RAG-2 promoter.