RESUMO
The retinoblastoma protein-interacting zinc-finger (RIZ) gene, also known as PRDM2, encodes two protein products, RIZ1 and RIZ2, differing for the presence of a 202 aa domain, called PR domain, at the N-terminus of the RIZ1 molecule. While the histone H3 K9 methyltransferase activity of RIZ1 is associated with the negative control of cell proliferation, no information is currently available on either expression regulation of the RIZ2 form or on its biological activity. RIZ proteins act as ER co-activators and promote optimal estrogen response in female reproductive tissues. In estrogen-responsive cells, 17-ß estradiol modulates RIZ gene expression producing a shift in the balanced expression of the two forms. Here, we demonstrate that an estrogen-responsive element (ERE) within the RIZ promoter 2 is regulated in a ligand-specific manner by ERα, through both the AF1 and AF2 domains. The pattern of ERα binding, histone H4 acetylation, and histone H3 cyclical methylation of lysine 9 was comparable to other estrogen-regulated promoters. Association of topoisomerase IIß with the RIZ promoter 2 confirmed the transcriptional activation induced by estrogen. We hypothesize that RIZ2, acting as a negative regulator of RIZ1 function, mediates the proliferative effect of estrogen through regulation of survival and differentiation gene expression.
Assuntos
Neoplasias da Mama/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/fisiologia , Elementos Facilitadores Genéticos/fisiologia , Estradiol/metabolismo , Histona-Lisina N-Metiltransferase/genética , Histona-Lisina N-Metiltransferase/fisiologia , Proteínas Nucleares/genética , Proteínas Nucleares/fisiologia , Fatores de Transcrição/genética , Fatores de Transcrição/fisiologia , Animais , Sequência de Bases , Neoplasias da Mama/patologia , Células COS , Diferenciação Celular/genética , Linhagem Celular Tumoral , Sobrevivência Celular/genética , Chlorocebus aethiops , Estradiol/farmacologia , Feminino , Regulação Neoplásica da Expressão Gênica/fisiologia , Humanos , Dados de Sequência Molecular , Regiões Promotoras Genéticas/fisiologiaRESUMO
The retinoblastoma protein-interacting zinc-finger (RIZ) gene, a member of the nuclear protein methyltransferase superfamily, is characterized by the presence of the N-terminal PR domain. The RIZ gene encodes for two proteins, RIZ1 and RIZ2. While RIZ1 contains the PR (PRDI-BF1 and RIZ homologous) domain, RIZ2 lacks it. RIZ gene expression is altered in a variety of human cancers and RIZ1 is now considered to be a candidate tumor suppressor. Estradiol treatment of MCF-7 cells produced a selective decrease of RIZ1 transcript and an increase of total RIZ mRNA. Experiments of chromatin immunoprecipitation indicated that RIZ2 protein expression was controlled by estrogen receptor and RIZ1 had a direct repressor function on c-myc gene expression. To investigate the role of RIZ gene products as regulators of the proliferation/differentiation transition, we analyzed the effects of forced suppression of RIZ1 induced in MCF-7 cells by siRNA of the PR domain-containing form. Silencing of RIZ1 expression stimulated cell proliferation, similar to the effect of estradiol on these cells, associated with a transient increase of c-myc expression.
Assuntos
Neoplasias da Mama/genética , Proliferação de Células , Proteínas de Ligação a DNA/genética , Estradiol/farmacologia , Expressão Gênica , Proteínas Nucleares/genética , Fatores de Transcrição/genética , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Imunoprecipitação da Cromatina , Proteínas de Ligação a DNA/metabolismo , Feminino , Inativação Gênica , Genes myc/fisiologia , Histona-Lisina N-Metiltransferase , Humanos , Proteínas Nucleares/metabolismo , RNA Mensageiro , RNA Interferente Pequeno/farmacologia , Receptores de Estrogênio , Proteína do Retinoblastoma/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Transcrição/metabolismo , Transcrição Gênica , Células Tumorais CultivadasRESUMO
In order to understand the oncogenic properties of retinoblastoma-interacting zinc-finger (RIZ) gene products, we produced an MCF-7-derived cell line expressing a fusion protein containing the zinc-finger (aa 359-497) domain of RIZ protein (MCF-7/znf). The Zn-finger domain contains three of the eight putative Zn-finger motifs and is located in proximity of the E1A-like domain containing the Rb protein-binding motif. The MCF-7/znf cells showed a higher growth rate than the parental or the control cell lines, both in hormone-deprived conditions or upon estrogen stimulation. Furthermore, they were less sensitive to the growth inhibitory effect of anti-estrogens and showed a higher level of expression of cyclin D1 and A. The expressed Zn-finger domain recombinant product was localized in the nucleus and in the nucleoli and its expression modified the pattern of actin staining in the cytoplasm. In conclusion the presented results indicated that the Zn-finger domain could be endowed with the putative oncogenic activity of RIZ2 gene product.
