Identification of NF1 as a silencer protein of the human adenine nucleotide translocase‐2 gene

Abstract

The human adenine nucleotide translocase-2 (ANT2) promoter contains a silencer region that confers partial repression on the heterologous herpes simplex virus thymidine kinase (HSVtk) promoter [Barath, P., Albert-Fournier, B., Luciakova, K., Nelson, B.D. (1999) J. Biol. Chem. 274, 3378-3384]. Two sequences in the silencer (Site-2 and Site-3) are protected in the DNase I assay in vitro, and one of these is a repeated GTCCTG element previously shown to act as the active repressor element. We have now purified the DNA binding protein, and identified it using MALDI-TOF MS as a 33-kDa member of the nuclear factor 1 (NF1) family of transcription factors. NF1 purified from rat liver and HeLa cell nuclei bind to both silencer Site-2 and Site-3, resulting in a DNase I footprint identical to that obtained with purified recombinant NF1. Furthermore, transient transfection experiments with reporter constructs containing mutated silencer Site-2 and/or Site-3 show that both sites contribute to repression of the HSVtk promoter. Finally, chromatin immunoprecipitation analysis reveals that NF1 is bound to both elements on the endogenous HeLa cell ANT2 promoter. Our data support the belief that NF1 acts as a repressor when bound to silencing Site-2 and Site-3 of the ANT2 gene.Keywords: adenine nucleotide translocase; NF1; promoter regulation; silencer protein; transcription.Two major isoforms of the adenine nucleotide translocase (ANT) are expressed in mammalian cells. Both catalyse the exchange of mitochondrial ATP for cytosolic ADP, thereby playing key roles in maintaining the cytosolic phosphorylation potential, adenylate charge, and the energy status of the cell. The two forms, ANT1 and ANT2 [1][2][3][4], are differentially expressed in mammalian tissue. ANT1 is expressed predominately in heart and skeletal muscle [5,6] whereas ANT2 is more widely expressed [5,7,8]. ANT2 is strongly growth regulated [9,10], and expression of the gene is downregulated in growth arrested cells [10] and re-activated in cells entering the G 1 growth phase. Activation of ANT2 expression is mediated at the level of transcription [10].To understand ANT2 expression, we have undertaken a study of its promoter. Constitutive ANT2 expression is maintained by two synergistically interacting Sp1 elements (the AB boxes) in the proximal promoter [11]. However, activation via the AB box Sp1 is modulated by three separate repressor regions. One of these is an Sp1 binding element (C box) juxtaposed to the transcriptional start site, which, when occupied, decreases ANT2 expression severalfold [11]. Repression appears to involve a direct interaction between Sp1 bound to the AB and C boxes [12]. A second repressor region, that is responsible for ANT2 down regulation in growth-arrested cells, has recently been identified in the distal promoter [10]. This region contains two DNA elements, Go-1 and Go-2, that bind nuclear factor 1 (NF1) in growth-arrested cells, but not in growthactivated cells [13]. NF1 binding is associated with growth arrest repression of ANT2 tr...

Growth-dependent repression of human adenine nucleotide translocator-2 (ANT2) transcription: evidence for the participation of Smad and Sp family proteins in the NF1-dependent repressor complex

“…Cells from starved and serum-induced human diploid fibroblasts were harvested, the nuclei were prepared [22] and nuclear proteins were extracted as described in [23]. The protein was measured using the Bio-Rad Protein Assay (Bio-Rad).…”

Transcriptional regulation of MICA and MICB: A novel polymorphism in MICB promoter alters transcriptional regulation by Sp1

“…The binding reactions were electrophoresed on 6% native acrylamide gels in Tris-Borate-EDTA Buffer. For competition experiments, a 100-fold molar excess of commercial competitor oligonucleotides CCAAT-binding factor (CBF/NF-Y) (sc-2591), Sp1 (sc-2502), C/EBP (sc-2525), NF-1 (sc-2553), CCAAT-displacement protein (CDP) (sc-2593) (Santa Cruz Biotechnology, Santa Cruz, CA, USA), an NF-1-B competitor sequence previously described [79], and the MHC-II-Y box sequence (which binds NF-Y protein (NF-Y/CBF) [80], were incubated with the extracts at 4 C for 15 min before adding the radioactive probe. To prove the specificity of binding, it was necessary for the reactions to be carried out in the presence of a 100-fold molar excess of unlabelled wild-type or mutant, MICA-emsa and MICB-(-66AG) probes.…”

Yin Yang 1 Is a Critical Repressor of Matrix Metalloproteinase-9 Expression in Brain Neurons

“…Using the DNase I footprinting, a method for an identification of novel DNA regulatory proteins (17,18), we have analyzed herein the rat Mmp-9 promoter fragments overlapping the Ϫ557/ϩ18-bp region of the gene in unstimulated neurons to look for repressive transcription factors.…”