Nuclear factor YY1 activates the mammalian F0F1 ATP synthase alpha-subunit gene

Abstract

Analysis of the promoters of the bovine and human nuclear-encoded mitochondrial F0F1 ATP synthase alpha-subunit genes (ATPA) has identified several positive cis-acting regulatory regions that are important for basal promoter activity in human HeLa cells. We have previously determined that the binding of a protein factor, termed ATPF1, to an E-box sequence (CANNTG) located within one of these cis-acting regions is critical for transcriptional activation of the ATPA gene. In this article, we describe a second positive cis-acting regulatory element of the ATPA gene that is important for expression of the ATPA gene. We show that this cis-acting element also contains a binding site for a protein present in HeLa cells. On the basis of electrophoretic mobility shift patterns, oligonucleotide competition assays, and immunological cross-reactivity, we conclude that this protein factor is Yin-Yang 1 (YY1). Experiments carried out to examine the functional role of YY1 within the context of the ATPA promoter demonstrated that YY1 acts as a positive regulator of the ATPA gene. For example, when the YY1 binding site of the ATPA promoter was placed upstream of a reporter gene it was found to activate transcription in transient transfection assays. In addition, disruption of the YY1 binding site in the ATPA gene resulted in a loss of transcriptional activity. Furthermore, in cotransfection experiments overexpression of YY1 in trans was found to activate transcription of ATPA promoter-CAT constructs. Thus, at least two positive trans-acting regulatory factors, ATPF1 and YY1, are important for expression of the bovine and human F0F1 ATP synthase alpha-subunit genes.

FIG. 1

Sequence of a region of the bovine ATPA promoter. The nucleotide sequence of the +25 to +96 bp region of the bovine ATPA gene (31) is shown. The putative binding sites for the regulatory factors, ATPF1 (43) and YY1 (this article) are underlined. The sites of transcription initiation that map within this region of the bovine ATPA gene are indicated by arrowheads [(31); D. J. Pierce, unpublished observations). The sites of initiation that are located at identical positions in the bovine and human (1) ATPA genes are indicated by large arrowheads.

FIG. 2

Binding of a HeLa nuclear factor(s) to the ATPA promoter. Electrophoretic mobility shift DNA-protein binding assays were carried out using ³²P-labeled probes containing fragments of the bovine ATPA promoter (31,43) from +49 to +96 bp (lanes 1–5) or from +68 to +86 bp (lanes 6–10), together with HeLa cell nuclear extracts. Where indicated, a 100-fold molar excess of unlabeled DNA was added as a competitor. The nonspecific competitor DNA used in this experiment was sheared E. coli DNA. Lanes 1 and 6 are control reactions to which no HeLa nuclear extract was added.

FIG. 3

Oligonucleotides containing YY1 binding sites effectively compete for ATPA-HeLa. complexes. Electrophoretic mobility shift assays were carried out using a ³²P-labeled fragment (+49 to +96 bp) of the ATPA gene as a probe, together with HeLa nuclear extracts. For competition assays, a 100-fold molar excess of various oligonucleotides was added as indicated: ATPA (+49 to +96 bp) (lane 3), P5 +1 (lane 4), rpL30 (lane 5), NF-E1 (lane 6), and mutant NF-E1 (lane 7). The sequences of the competitor oligonucleotides are listed in the Materials and Methods section. Lane 1 indicates labeled probe to which no HeLa cell extract was added.

FIG. 4

YY1 is present in HeLa-ATPA complexes. A fragment containing the +49 to +96 bp region of the ATPA gene was end-labeled and used as a probe in electrophoretic mobility shift assays. The binding reactions contained either no extract (lane 1), purified YY1 (Upstate Biotechnology) (lane 2), or HeLa nuclear extracts (lanes 3–5). In some reactions, preimmune serum (PI; 2 μl) (lane 3) or anti-YY1 antiserum (2 μl) (lanes 4 and 5) was added. In lane 5, a 100-fold molar excess of the NF-E1 oligonucleotide was added as a competitor.

FIG. 5

The YY1 binding site in the ATPA gene acts as an activator of transcription. The +25 to +136 bp or the +68 to +86 bp region of the ATPA gene was cloned into the vector, pCAT-Basic (Promega). Similarly, the same fragments of the ATPA gene containing mutations (M1, M2) in the YY1 element were cloned into the pCAT-Basic vector. In addition, the P5 (+1) region of the adeno-associated virus P5 (37) was cloned into pCAT-Basic. Wild-type (WT) YY1 elements are indicated by closed boxes and mutated YY1 elements by hatched boxes. HeLa cells were transfected with each of the indicated CAT reporter plasmids, together with pCMV-β-gal plasmid DNA. Cells were harvested after 48 h and assayed for CAT and β-galactosidase activities as described in the Materials and Methods section. The CAT activities of the transfected cells were normalized relative to the β-galactosidase activities.

FIG. 6

Sites of transcription initiation of DNA constructs transfected into HeLa cells. HeLa cells were transfected with either the ATPA (+68 to +86 bp) fragment cloned into the pCAT-Basic vector or the pCAT-Basic vector DNA alone. Total RNA was isolated from cells approximately 48 h after transfection. A 5′ end-labeled CAT primer DNA was annealed to approximately 50 μg of RNA from the transfected cells and extended with Moloney murine leukemia virus reverse transcriptase. Lane 1 represents primer extension products from cells transfected with the ATPA (+68/+86 bp)–CAT DNA and lane 2 with the pCAT-Basic DNA. The sequencing ladder (labeled G, A, T, C) was generated by using the same CAT primer and the ATPA (+68/+86 bp)-CAT plasmid DNA as the template. The transcription start sites are indicated by arrowheads on the antisense sequence.

FIG. 7

Mutant forms of the ATPA promoter do not bind YY1. Electrophoretic mobility shift DNA-protein binding assays were carried out using labeled oligonucleotides containing either the wild-type ATPA (+68 to +86 bp) sequence (lanes 1–5), the mutant 1 ATPA (+68 to +86 bp) sequence (lanes 6 and 7), or the mutant 2 ATPA (+68 to +86 bp) sequence (lanes 8 and 9). Where indicated, a 100-fold molar excess of either wild-type, mutant 1, or mutant 2 ATPA oligonucleotide was added as a competitor. Lanes 1, 6, and 8 represent control reactions to which no HeLa nuclear extract was added. Wild-type ATPA (+68 to +86 bp) sequence: CTCGGCCATTTTGTCCCAG; mutant 1 ATPA sequence: CTCAATTATTTTGTCCCAG; mutant 2 ATPA sequence: CTCGGCCAGGAAGTCCAG.

FIG. 8

YY1 transactivates ATPA–CAT reporters. HeLa cells were cotransfected with either 10 μg of pATPA wild-type (+25/ +136 bp)/CAT, pATPA wild-type (+68/+86 bp)/CAT, pATPA mutant 1 (+68/+86 bp)/CAT, or pSVCAT plasmid DNAs, together with 10 μg of either pCMV (open bars) or pCMV-YY1 (closed bars), and 2 μg of pCMV-β-gal DNA. Cells were harvested after 48 h and assayed for β-galactosidase and CAT activities as described in the Materials and Methods section. The CAT activities of the cells that were transfected with pCMV-YY1 DNA were compared to those with pCMV DNA (fold activation).