Expression of the adenine nucleotide translocator 2 (ANT2) gene is growth regulated. We report a feature of the ANT2 promoter that involves a novel regulatory function for the Sp1 transfactor. We show that expression from the ANT2 proximal promoter is modulated through three Sp1 elements, two of which activate and one of which partially inhibits transcription. The inhibitor site, box C, is juxtaposed to transcription start (nucleotides ؊7 to ؊2). Sp1 bound to box C decreases transcription initiation. This was demonstrated by introducing mutations in box C which (a) increased chloramphenicol acetyltransferase expression in the transient transfection assay and (b) inhibited binding of both purified Sp1 and Sp1 in crude nuclear extracts. The activating elements (A and B boxes) are located at adjacent sites in the distal region of the proximal promoter. Mutation of either box inhibits transfection by 90%, indicating that they act in a synergistic manner. Supershift experiments with crude nuclear extracts showed that only Sp1 was bound to the three GC boxes. The finding that Sp1 acts as an activator/inhibitor within the same promoter region was verified in NIH3T3, HeLa, JEG3, and COS-1, indicating that this dual effect of Sp1 is widely preserved. These data suggest a unique role for Sp1 and raise the possibility that growth activation of the ANT2 gene is regulated by the interaction of Sp1 on the A, B, and C boxes. ANT2 is one of three genes that encodes mammalian adenine nucleotide translocator (ANT, ATP/ADP translocase) proteins (1-4). The three genes are differentially expressed in a tissuespecific manner (5-7), and during cellular differentiation (6 -9). The ANT2 isoform is unique since it is expressed in a growthdependent fashion. ANT2 cDNA clones were first isolated as differentially expressed transcripts from growth-activated (10) and serum-stimulated (8) cells. Subsequently, growth-related expression of ANT2 was demonstrated in several cell lines (6,8) and in activated human peripheral lymphocytes (11). Induction of ANT2 transcripts is rapid (8, 12); this, together with the conditions under which the original clones were isolated (10), suggests that it might belong to the group of early-immediate genes. This conclusion is supported by the finding that ANT2 transcripts are induced by mitogens such as platelet-derived growth factor and epidermal growth factor and that induction is not inhibited by cycloheximide (8).To understand the growth-dependent control of ANT2 expression, we characterized the promoter region of the human gene. We report a unique role for the transactivating Sp1 protein. Sp1 is a ubiquitously expressed factor that is required for expression of a large number of constitutive and regulated genes. Sp1 has been described only as a transcription activator. However, Sp1 activation can be suppressed by a variety of mechanisms. For example, Sp3, a member of the Sp1 family (13, 14), competes for Sp1 binding sites (15) but is not able to activate transcription (15). Activation by Sp1 can also be supp...

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Expression of adenine nucleotide translocator isoform 2 (ANT2) is growth regulated. In the present study, we report the presence of a silencer region in the human ANT2 promoter and the purification of a two-component factor that recognizes a specific hexanucleotide element, GTCCTG, of the silencer. Transfection of deletion constructs shows that ANT2 silencer activity extends over a region of at least 310 nts. However, mutating the GTCCTG element completely relieves silencing activity in the context of the human ANT2 promoter. The data suggest that the GTCCTG element might be required for maintaining silencer activity of the extended silencer region. The ANT2 silencer region cloned in front of the herpes simplex virus thymidine kinase promoter confers nearly complete inhibition to the heterologous promoter. However, unlike the ANT2 promoter, mutating the GTCCTG element restores only partial activity to the herpes simplex virus thymidine kinase promoter. A protein complex consisting of two major polypeptides of 37 and 49 kDa was isolated from HeLa nuclear extracts by affinity chromatography using the GTCCTG element as the affinity resin. Cross-linking studies and Southwestern analysis indicate that p37 binds DNA. p49 appears to be loosely associated with the p37/DNA complex but is necessary for strong binding of p37. Our data implicating the GTCCTG element directly in silencing of the ANT2 promoter, together with data from the literature reporting the presence of this element within the silencer region of several additional promoters, suggest a general role of the GTCCTG element in transcriptional silencing.The adenine nucleotide translocator (ANT) 1 proteins exchange cytosolic ADP for mitochondrial ATP, thereby playing an essential role in maintaining cell metabolism and growth. Recent findings also implicate ANT in the initiation of events leading to apoptosis (1). Mammalian ANT is encoded in three genes, ANT1, ANT2, and ANT3 (2-5), that are expressed in a tissue-specific manner (6). ANT1 mRNA is expressed predominantly in heart and skeletal muscle (7, 8) whereas ANT2 mRNA is expressed in a broad range of tissues (6, 7, 9), but predominantly those tissues that undergo rapid proliferation (6, 10). However, the ANT2 isoform is unique in that it is also expressed in a growth-dependent manner (11) in a wide variety of cell lines. ANT2 expression is low in quiescent cells and is substantially increased by factors that induce entrance into G 1 and subsequent cell growth (11). ANT2 expression in serumactivated NIH3T3 cells is inhibited by actinomycin D 2 but not cycloheximide (12), indicating that expression is regulated at the level of transcription but does not require new protein synthesis.The physiological significance of expression of just the ANT2 isoform in a growth-dependent manner has not been delineated, although it is possible that slight kinetic differences in ADP/ATP exchange catalyzed by the isoforms (13) might provide the growth-activated cell with an energetic advantage. To understand the complex regula...

Adenine nucleotide translocase-2 (ANT2) catalyzes the exchange of ATP for ADP across the mitochondrial membrane, thus playing an important role in maintaining the cytosolic phosphorylation potential required for cell growth. Expression of ANT2 is activated by growth stimulation of quiescent cells and is down-regulated when cells become growth-arrested. In this study, we address the mechanism of growth arrest repression. Using a combination of transfection, in vivo dimethyl sulfate mapping, and in vitro DNase I mapping experiments, we identified two protein-binding elements (Go-1 and Go-2) that are responsible for growth arrest of ANT2 expression in human diploid fibroblasts. Proteins that bound the Go elements were purified and identified by matrix-assisted laser desorption ionization time-offlight mass spectrometry as members of the NF1 family of transcription factors. Chromatin immunoprecipitation analysis showed that NF1 was bound to both Go-1 and Go-2 in quiescent human diploid cells in vivo, but not in the same cells stimulated to growth by serum. NF1 binding correlated with the disappearance of ANT2 transcripts in quiescent cells. Furthermore, overexpression of NF1-A, -C, and -X in NIH3T3 cells repressed expression of an ANT2-driven reporter gene construct. Two additional putative repressor elements in the ANT2 promoter, an Sp1 element juxtaposed to the transcription start site and a silencer centered at nucleotide ؊332, did not appear to contribute to growth arrest repression. Thus, enhanced binding of NF1 is a key step in the growth arrest repression of ANT2 transcription. To our knowledge, this is the first report showing a role for NF1 in growth arrest.