Upstream stimulatory factor 2 activates the mammalian F1F0 ATP synthase alpha-subunit gene through an initiator element

Abstract

The F1F0 ATP synthase is the central enzyme complex of the mitochondrial oxidative phosphorylation system synthesizing ATP from ADP and Pi. Our laboratory has been studying the transcriptional regulation of the nuclear gene that encodes the alpha-subunit of the mammalian mitochondrial ATP synthase complex (ATPA). We have previously identified an initiator element in the core promoter that plays an important role in expression of this gene. In this article, we demonstrate that ectopic expression of the transcription factor, upstream stimulatory factor 2 (USF2), transactivates the ATPA gene through this initiator element. Importantly, cotransfection of a dominant-negative USF2 mutant significantly reduces both the basal activity and the level of activation of the ATPA initiator by coexpressed USF2 demonstrating the role of endogenous USF2 proteins in this activation. We also identify several nucleotides in the ATPA initiator element that are important for both basal activity and USF2-dependent transactivation. We have also previously determined that the binding of the multifunctional regulatory protein, YY1, to this initiator element can positively regulate the ATPA gene. Here, we show that expression of YY1 together with USF2 results in a decreased level of activation of the ATPA initiator relative to expression of USF2 alone, suggesting competition between these two regulatory proteins.

FIG. 1

Sequence of a portion of the bovine ATPA gene. The nucleotide sequence of the +1 to +141 bp region of the bovine ATPA gene (25) is shown. The E-box element (3) and the YY1-binding site (4) are indicated. The initiator methionine is underlined. The +68 to +86 bp region of the ATPA promoter is boxed. The major sites of transcription initiation of the bovine ATPA gene (4,25) are indicated by arrowheads.

FIG. 2

USF2 transactivates the ATPA promoter through an initiator element. HeLa cells were cotransfected with 10 μg of either pATPA (+23/+136 bp)/CAT, pATPA (+237+96 bp)/CAT, or pATPA (+68/+86 bp)/CAT (37) together with 10 μg of pCMV (□) or pCMV-USF2b (▪). Cells were harvested approximately 48 h after transfection and the levels of CAT activity were determined.

FIG. 3

Transacting properties of USF2b, USF2a, and a mutant USF2a protein. (A) Transactivation of the ATPA initiator element by USF2b. Ten micrograms of the pATPA (+68/+86 bp)/CAT reporter plasmid was cotransfected with the indicated amounts (in μg) of an expression vector expressing isoform USF2b (22). (B) Transactivation of the ATPA initiator element by isoform USF2a. Ten micrograms of the pATPA (+68/+86 bp)/CAT plasmid was cotransfected into HeLa cells together with the indicated amounts (in μg) of an expression vector expressing USF2a (22). (C) A mutant form of USF2a lacking the transactivation domains does not activate the ATPA core promoter. Ten micrograms of the pATPA (+68/+86 bp)/CAT plasmid was cotransfected with the indicated amounts (in μg) of an expression vector expressing a mutant form of USF2a that lacks the amino-terminal transactivation domains, USF2aΔN (22).

FIG. 4

A dominant-negative mutant of USF2a reduces both the basal activity and the activation of the ATPA initiator by USF2a. HeLa cells were cotransfected with 10 μg of the pATPA (+68/+86 bp)/CAT reporter plasmid together with 5 μg of the expression plasmid, pSVUSF2a, or the equivalent amount of empty vector, and 10 or 15 μg of pSVTDUSF2aΔB or the equivalent amount of empty vector. TDUSF2aΔB is a dominant-negative mutant of USF2a that is defective in DNA binding but is still capable of dimerization with USF2 and USF1 (22). Cells were harvested after 48 h and assayed for CAT activity.

FIG. 5

The effect of mutations in the ATPA initiator element. (A) Diagram of the mutations in the ATPA initiator element. The sequence of the +68 to +86 bp fragment of the wild-type (WT) ATPA gene is shown on the top line. Mutations in this sequence (M1, M2, and M3) are indicated by lower case letters. (B) Diagram of the basal activity and the stimulatory effects of USF2a. HeLa cells were cotransfected with 1 μg of either pSV ((□) or pSVUSF2a (▪) together with 1 μg of either the wild-type pATPA (+68/+86 bp)/CAT vector (WT) or the mutant pATPA/CAT vectors (M1, M2, and M3). Cells were harvested after 48 h and the levels of CAT activity were determined.

FIG. 6

Expression of YY1 together with USF2 reduces the activation of the ATPA initiator element. HeLa cells were cotransfected with 10 μg of the pATPA (+68A+86 bp)/CAT reporter plasmid together with either pCMV, pCMV-USF2b (15 μg), pCMV-YYl (5 μg), or pCMV-YY1 (5 μg) plus pCMV-USF2b (15 μg). The total amount of DNA in each transfection was adjusted to 32 μg using pCMV DNA.