Identification of a DNA binding protein cooperating with estrogen receptoras 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 selectio n of target, CAST, for multiple elements). Fragments were cloned and sequen ce analysis indicated the 5-nucleotide word TTGGC was the most recurrent se quence unrelated to the known estrogen responsive element. Screening a HeLa cell expression library with a probe designed with multiple repeats of thi s sequence resulted in the identification of a 1700-aa protein showing a co mplete homology with the product of the human retinoblastoma-interacting zi nc-finger gene RIZ. In transfection experiments, RIZ protein was able to be stow estrogen inducibility to a promoter containing an incomplete estrogen responsive element and a TTGGC motif. RIZ protein present in MCF-7 cell nuc lear extract retarded the TTGGC-containing probe in an EMSA. Estrogen recep tor was co-immunoprecipitated from MCF-7 cell extract by antibodies to RIZ protein and vice versa, thus indicating an existing interaction between the se two proteins. (C) 1999 Academic Press.