Identification of growth factor independent-1 (GFI1) as a repressor of 25-hydroxyvitamin D 1-alpha hydroxylase (CYP27B1) gene expression in human prostate cancer cells

Abstract

The hormone 1,25-dihydroxyvitamin D (1,25D) may play a protective role in prostate cancer. 25-hydroxyvitamin D 1-a hydroxylase (CYP27B1) is the enzyme responsible for the regulation of cellular 1,25D levels. CYP27B1 is substantially repressed in prostate cancer cells. We have investigated the molecular basis for this inhibition. First, we identify a repressive region between -997 and -1200 in the human CYP27B1 promoter following transient transfection analysis in the prostate cancer cell lines DU145, PC3 and LNCaP. Next, we demonstrate a role for the transcription factor growth factor independent-1 (GFI1) in the repression of CYP27B1. Electrophoretic mobility assays with nuclear extracts from prostate cancer cell lines established binding of GFI1 to the sequence 5k-TGGTACAATCATAACTCACTGCAG-3k present at -997 to -1200 in the repressive region. Site directed mutagenesis of the core GFI1 binding sequence (5k-AATC-3k) substantially increased while forced expression of GFI1 decreased the expression of the CYP27B1 reporter construct. Importantly, GFI1 repression is dependent on an intact GFI1 binding site in the -997 to -1200 region. GFI1 is an oncoprotein known to form a large protein complex with co-repressors that recruit histone deacetylases. We propose that the formation of such a repressive complex on the inhibitory domain of the CYP27B1 gene in prostate cancer cells could lead to silencing of either the nearby enhancer or proximal promoter domains and lead to cancer progression by reducing local production of 1,25D. These studies provide the basis for a more detailed understanding of CYP27B1 repression in prostate cancer cells and could provide a novel insight in future diagnosis and treatment.

ZNF652, A Novel Zinc Finger Protein, Interacts with the Putative Breast Tumor Suppressor CBFA2T3 to Repress Transcription

“…CBFA2T1 and CBFA2T3 have previously been shown to interact with GFI-1, a zinc-finger transcription repressor (14). We recently showed that GFI-1 represses 25-hydroxyvitamin D 1a-hydroxylase (CYP27B1) expression in human prostate cancer cells, and that this repression is mediated through GFI-1 -specific, DNA-binding sequences within the CYP27B1 promoter (28). Reporter assays were done to investigate if the ETO proteins differ in their ability to further repress the GFI-1 -mediated transcriptional repression from the CYP27B1 promoter.…”

“…To generate pGL2-GAL4-TK-luciferase construct, 5 ¶ and 3 ¶ ends of the XbaIBamHI fragment carrying 5Â GAL4 DNA-binding sequences from pG5CAT (Promega, Madison, WI) were filled-in using Klenow DNA polymerase and cloned at blunted XhoI site of pGL2-TK-luciferase. Constructs used for GFI-1 -mediated repression assays were pCYP27B1WT(À1200)-Luc that contained a 1,244 bp region of the CYP27B1 promoter (À1200 together with 44 bp of CYP27B1 5 ¶ untranslated region), pCYP27B1Enh(À997)-Luc that was deleted for the region containing the GFI-1 consensus binding site, and pCYP27B1mGFI (-1200)-Luc that contained mutated GFI-1 binding site (28). The construct expressing FLAG-GFI-1 was kindly provided by H.L.…”

Gfi-1 represses CDKN2B encoding p15 INK4B through interaction with Miz-1

“…Gfi-1 has further been shown to cooperate with c-Myc and Pim1 in lymphomagenesis. In addition, Gfi-1 may play a role in lung and prostate cancers (3,4).…”

A Genetic Screen in Drosophila for Genes Interacting With senseless During Neuronal Development Identifies the Importin moleskin

“…Gfi-1 and its paralog Gfi-1b are also involved in hematopoiesis (Zeng et al 2004;Duan and Horwitz 2005) and immune system function (Schmidt et al 1998b;Karsunky et al 2002;Zhu et al 2002;Hock et al 2003;Yucel et al 2003;Zeng et al 2004;Rathinam et al 2005) and have been implicated in disease processes including neutropenia (Karsunky et al 2002;Person et al 2003), carcinogenesis (Gilks et al 1993;Schmidt et al 1996Schmidt et al , 1998aHock et al 2004;Kazanjian et al 2004;Shin et al 2004;Dwivedi et al 2005), and neurodegenerative disease (Moroy 2005;Tsuda et al 2005). The expression of both sens and Gfi-1 is positively regulated by the bHLH proneural gene atonal/Math-5.…”