Identification of a DNA binding protein cooperating with estrogen receptor as RIZ (retinoblastoma interacting zinc finger protein).

Double-stranded DNA fragments were selected from a random pool by repeated cycles of estrogen receptor-specific immunoprecipitation in the presence of a nuclear extract and PCR amplification (cyclic amplification and selection of target, CAST, for multiple elements). Fragments were cloned and sequence analysis indicated the 5-nucleotide word TTGGC was the most recurrent sequence unrelated to the known estrogen responsive element. Screening a HeLa cell expression library with a probe designed with multiple repeats of this sequence resulted in the identification of a 1700-aa protein showing a complete homology with the product of the human retinoblastoma-interacting zinc-finger gene RIZ. In transfection experiments, RIZ protein was able to bestow estrogen inducibility to a promoter containing an incomplete estrogen responsive element and a TTGGC motif. RIZ protein present in MCF-7 cell nuclear extract retarded the TTGGC-containing probe in an EMSA. Estrogen receptor was co-immunoprecipitated from MCF-7 cell extract by antibodies to RIZ protein and vice versa, thus indicating an existing interaction between these two proteins.     

Delineation of sites mediating estrogen regulation of the rat creatine kinase B gene.

We have previously confirmed the estrogen-induced protein of rat uterus to be creatine kinase B (CKB), and demonstrated a 1.7-kilobase pair fragment encompassing the promoter and adjoining 5'-flank to be capable of conferring estrogen responsiveness in HeLa cells. In this study we find an element at -550, aGGTCAgaaCACCCt, with limited similarity to the estrogen response element consensus, to be involved in conferring estrogen responsiveness on the CKB promoter. This element can bind estrogen receptor (ER) and is flanked by two GC boxes, which we find capable of binding bacterially expressed Sp1. Additional responsiveness is found closely associated with the CKB promoter at high levels of cotransfected ER construct. No potential response element was identified in this region, but we find the ER DNA-binding domain to be required.