OXBOX, a Positive Transcriptional Element of the Heart-Skeletal Muscle ADP/ATP Translocator Gene*

Three positive transcriptional control regions have been identified in the promoter of the human heart- skeletal muscle adenine nucleotide translocator gene (ANTI). By transfecting promoter-chloramphenicol acetyltransferase fusion constructs into C2C12 myogenic cells, each positive region was found to 2-3-fold. The first region spans from -123 to -674 base pairs (bp), the second from -2.6 to -3.1 kilobases, and the third from -3.1 to -8.8 kilo-bases. Linker-scanning mutants generated using the polymerase reaction and modified oligonucleo- tides identified the OXBOX the element within the -123 to -674-bp

Three positive transcriptional control regions have been identified in the promoter of the human heartskeletal muscle adenine nucleotide translocator gene (ANTI).
By transfecting promoter-chloramphenicol acetyltransferase fusion constructs into C2C12 myogenic cells, each positive region was found to increase transcription 2-3-fold. The first region spans from -123 to -674 base pairs (bp), the second from -2.6 to -3.1 kilobases, and the third from -3.1 to -8.8 kilobases. Linker-scanning mutants generated using the polymerase chain reaction and modified oligonucleotides have identified the OXBOX (B'-GGCTCTAAA-GAGG) as the positive element within the -123 to -674-bp region. This element enhances transcription in muscle cells but not in HeLa cells, suggesting that it is muscle-specific.
Gel retardation experiments have revealed a factor from C2C12 cells which specifically binds to a 40-hp piece of the ANT1 promoter containing the OXBOX.
Since the OXBOX is also found in the promoter of the human ATP synthase @ subunit gene, it is the first tissue-specific element identified which could coordinately regulate mitochondrial oxidative phosphorylation genes.
The adenine nucleotide (ADP/ATP) translocator (ANT)' is a homodimeric protein of the mitochondrial inner membrane (1, 2) which transports ADP into and ATP out of the mitochondrion.
In eukaryotic cells, mitochondrial ATP production and cellular ATP consumption are linked through ANT, the kinetics of which have been proposed to play a major regulatory role in mitochondrial OXPHOS (3)(4)(5)(6)(7). Multiple ANT isoforms have been identified. These were initially recognized by antigenic and electrophoretic differences among heart, liver, and kidney ANTS (8,9) and subsequently confirmed by the isolation and characterization of three human and two bovine ANT cDNAs. We have characterized a complete human ANT cDNA (ANTl) (lo), which is expressed primarily in heart and skeletal muscle (11). A second human cDNA has been isolated from fibroblast cells (hp2Fl) or ANT2) (12)  cant levels in heart, skeletal muscle, liver, kidney, and brain (11). A third human cDNA has been isolated from liver (pHAT8 or ANT3) (13). The two published bovine cDNAs are homologues of ANT1 and ANT3 (14).
Recently, we have cloned and sequenced the human ANT1 gene (11). Comparison with the human ANT3 (T2) gene (15) revealed that the intron-exon structure of the two genes is the same, but that their gene regulatory sequences are markedly different. Classical TATA and CCAAT boxes are present in the ANT1 gene, but not in the ANT3 gene. Both of the genes have Spl binding motifs, GC boxes, in the first intron. However, the ANT3 gene has 12 additional GC boxes in the promoter region, whereas the ANT1 gene lacks these.
Comparison of the ANT1 and ATP synthase p subunit (ATPsynp) promoters revealed a 13-bp homologous sequence, the OXBOX (11,16,17). This sequence is located at a similar position in these genes, but is not found in ANT3 (15). We have postulated that the OXBOX might be a &-acting element regulating transcription of the ANT1 and ATPsynP genes (11,17).
To identify the transcriptional regulatory elements of the ANT1 promoter and to determine if the OXBOX is a transcriptional element, we prepared a series of promoter-reporter gene constructs using chloramphenicol acetyltransferase. The expression of these constructs was analyzed in mouse myogenic C2C12 cells (18) and HeLa cells, and three major positive muscle regulatory regions were mapped, one of which was identified as the OXBOX. Competitor oligonucleotides were added together with the labeled probe during the binding reaction.

Identification of Multiple
&-Acting Elements in ANT1 Promoter-The transcriptional regulatory elements of the ANT1 promoter were surveyed using an array of deletion promoter-CAT fusion plasmids (Fig. 1). Plasmid pBLANTCAT1 contains nucleotides at +42 to -123 bp relative to the transcriptional start site at np 0 (11)   Transient transfection chloramphenicol acetyltransferase assay of these plasmids revealed three discrete regions, in addition to the CCAAT and TATA elements, which increase transcription 2-3-fold in both myoblasts and myotubes (Fig.  1). The first region is located between -123 and -674 bp (pBLANTCAT1 uersus pBLANTCAT2). The second region is located between -2.6 and -3.1 kb (pBLANTCAT2 uersus pBLANTCAT5). The third region is located between -3.1 and -8.8 kb (pBLANTCAT5 uersuS pBLANTCAT6). A negative regulatory region may be located between -674 bp and -1.2 kb (pBLANTCAT2 uersus pBLANTCAT3) (Fig. 1).
The TATA and CCAAT elements were shown to be absolutely required for transcription initiation since their removal abolished all CAT expression (pBLANTCAT5a; Fig. 1). We also observed a 2-3-fold increase in CAT expression with all of the promoter constructs except pBLANTCAT1 in myotubes relative to myoblasts (Fig. 1).
Characterization of OBOX Function by PCR Linker Ablation-The OXBOX was shown to be the positive regulatory element between -123 and -674 bp by using PCR linker ablation mutants (Fig. 2). PCR was used to prepare a 721-bp fragment without (pBLOB-CAT) and with (pBLOB+CAT) the OXBOX, cloned into pBLCAT3. In pBLOB-CAT, the OXBOX was substituted with a BgZII site while the length of the DNA and all other sequences were preserved (Fig. 4). pBLOB-CAT and pBLOB+CAT were used to prepare a second set of plasmids, pBLfOB-CAT and pBLfOB+CAT, by the addition of the region -674 bp to -3.1 kb containing the second positive regulatory element (Fig. 4).
Transfections of the two sets of OXBOX-plus and OX-BOX-minus plasmids into mouse myogenic C2C12 cells revealed marked reduction in CAT activity when the OXBOX was ablated (Fig. 3). In multiple experiments, ablation of the OXBOX reduced transcription 2-3-fold (Fig. 4). This was true whether or not the second positive element was present.
Hence, the OXBOX is a positive transcriptional element in muscle cells, and its action is additive relative to the other promoter elements. The specific protein-OXBOX probe complex is indicated by an arrow. Lane 1, 40,000 cpm of OXBOX probe; lane 2,40,000 cpm of the probe, 1 rg of C2C12 nuclear proteins, and 4 pg of poly(dI-dC); lanes 3-8, same amounts of the probe, nuclear proteins, and poly(dI-dC) plus additional cold OXBOX probe and a 35-bp nonspecific oligonucleotide (shown on the top). minus plasmids into epithelially derived HeLa cells yielded very low CAT activity, and the level of expression was the same with or without the OXBOX. In three independent experiments using pSV&AT (27) as the positive control (lOO%), pBLCAT3 gave a mean value of 0.5 + 0.4% of control and pBLOB-CAT and pBLOB+CAT gave 3.8 f 2.4 and 3.7 + 1.9% and pBLfOB-CAT and pBLfOB+CAT gave 2.4 f 2.1 and 2.8 f 2.5%, respectively. These results demonstrate that the ANT1 promoter functions poorly in nonmuscle cells and proves that the OXBOX sequence is a muscle-specific positive regulatory element.

OXBOX
Binding Factors-To determine if the OXBOX interacts with specific nuclear factors, the 40-bp region of the ANT1 promoter containing the OXBOX was labeled and incubated with nuclear extract prepared from C2C12 cells. Several DNA-protein complexes were observed as bands in a gel mobility shift assay (Fig. 5). One of these complexes was shown to be specific for sequences containing the OXBOX (indicated by the arrow). The addition of small quantities of "cold" OXBOX probe removed this band (lanes 3-5), whereas a 35-bp nonspecific oligonucleotide which lacked the OXBOX sequence had less effect on the complex (lanes 6-8). Hence, mouse C2Cl2 nuclei appear to contain a nuclear factor which specifically binds to the OXBOX probe.
Elements of the ADPIATP Translocator Gene DISCUSSION Three major positive &-acting elements were identified in the ANT1 promoter which function in the CZC12 myoblast cells and result in a high level of reporter gene expression. Endogenous ANT1 mRNA was also detected in C2C12 myoblasts by Northern blot analysis.' CAT activity was also found to be 2-3-fold higher in C2C12 myotubes than in myoblasts. A similar increase was seen in endogenous ANT1 mRNA levels of C2C12 myoblasts versus myotubes by Northern blot.' The myoblast-to-myotube increases may result either from an increased transcription rate or simply a differential accumulation of mRNA.
The three positive elements were functionally additive and independent.
The first element was shown to be the OXBOX, located between -452 and -464 bp. Since the OXBOX is found in both the ANT1 and ATPsynp genes, it joins the recently identified enhancer-like consensus element (5'-TA-GAGACAAGGTTTCACCA) in ATPsyn& cytochrome cl, the pyruvate dehydrogenase El (Y subunit gene (28) and the NRF-1 element  in cytochrome c (29) as one of a family of transcriptional control elements specific for OXPHOS genes. Several lines of evidence suggest that the OXBOX is a tissue-specific regulatory element for expression of OXPHOS genes in heart and skeletal muscle. First, the ANT1 OXBOX element functions in C2C12 cells, but not in HeLa cells. Second, the OXBOX sequence has been found in human ANT1 and ATPsynfi genes, and both genes have maximal levels of expression in heart and skeletal muscle (11,14,17). Finally, the recently published sequence of the human ANT3 gene, a non-heart-skeletal muscle isoform, does not possess the OXBOX sequence (15). Thus, the OXBOX increases transcription of selected OXPHOS genes in heart and skeletal muscle to meet the higher ATP demand of these tissues (30) and is the first tissue-specific OXPHOS regulatory element to be identified.