Identification of TFII-I as the endoplasmic reticulum stress response element binding factor ERSF: its autoregulation by stress and interaction with ATF6.

When mammalian cells are subjected to Ca2+ depletion stress or glycosylation block, the transcription of a family of glucose-regulated protein (GRP) genes encoding endoplasmic reticulum (ER) chaperones is induced. The GRP promoters contain multiple copies of the ER stress response element (ERSE) (20, 26). The ERSE consists of a tripartite structure, CCAAT(N9)CCACG, with N being an invariant region of 9 bp. Through binding site competitions and immuno-cross-reactivity, two major transcription factors from HeLa cell nuclear extracts (NEs) that bind to the ERSE have been identified. They are NF-Y/CBF and YY1, and they interact with the CCAAT and CCACG elements, respectively (11, 21). Using −98 ERSE1 of the rat grp78 promoter as a model system, we discovered a novel complex (termed ERSF) in NE prepared from ER-stressed HeLa cells that exhibits enhanced binding to the ERSE (20). Optimal binding of the ERSF to the ERSE requires a conserved GGC motif within the internal 9-bp region. Specific mutation of the GGC motif results in substantial loss of stress inducibility mediated by the ERSE. However, ERSF binding to the ERSE alone is not sufficient to activate the ER stress response, since full stress inducibility of ERSE requires integrity of the CCAAT, GGC, and CCACG sequence motifs, as well as precise spacing among these sites. In addition, ATF6, a basic leucine zipper protein, activates the ERSE through the CCACG sequence (6, 26, 27). ATF6 itself undergoes ER stress-induced changes and has been shown to interact with ERSE binding factors YY1 and NF-Y (6, 9, 27). There is also evidence suggesting the existence of a mammalian homologue of the yeast Hac1 protein that binds selectively to a unfolded protein response element-related sequence within the −163 ERSE3 of the rat grp78 promoter (4). Collectively, these findings imply that multiple transcription factors are required for ERSE activation through direct or indirect interaction with DNA regulatory elements on the ERSE. The mechanism for these upstream regulatory complexes to communicate with the basal transcription machinery of the GRP promoters is presently unknown. The transcription factor TFII-I was isolated as a binding protein that binds to the initiator elements in core promoters of TATA-less promoters (18, 19). Subsequently, it has been shown that TFII-I can also bind to E-box elements and can interact with upstream regulatory factors, such as USF1 and c-myc (16, 17). In addition to the 957-amino-acid form of TFII-I (referred to as Δ), there are three other alternatively spliced forms of TFII-I (α, β, and γ) (3). The primary structure of the multiple forms of TFII-I, ranging in size from 120 to 150 kDa, contains six directly repeated 90-residue motifs that each possess a potential helix-loop–span-helix homology (5, 17). These unique structural features suggest that TFII-I may have the capacity for multiple protein-protein interactions and, potentially, multiple protein-DNA interactions. In support of this possibility, ectopic TFII-I can act synergistically with USF1 to activate transcription of the adenovirus major late promoter in vivo (17). Recent evidence suggests that TFII-I may also have a role in signal transduction. TFII-I is phosphorylated in vivo at serine, threonine, and tyrosine residues, and its activity is regulated by phosphorylation (14). Tyrosine phosphorylation of TFII-I is enhanced after epidermal growth factor stimulation (8). Also, the nuclear import of TFII-I could be regulated in response to antigenic stimuli (15). Thus, it has been proposed that TFII-I is a multifunctional protein that can coordinate the formation of an active promoter complex and provide linkage-specific signal-responsive activator complexes to the general transcription machinery (5, 17). TFII-I itself could also be a sensor of induction stimuli, resulting in specific modifications upon stimulation of gene expression. Through chromatographic purification and microsequencing, we have identified ERSF as TFII-I. In NIH 3T3 cells, TFII-I remains predominantly nuclear in both control cells and cells treated with thapsigargin (Tg), a potent inducer of the GRP stress response through depletion of the ER Ca2+ store. However, in Tg-treated cells, the level of TFII-I transcript was elevated, correlating with an overall increase in TFII-I protein level in the nuclei of Tg-stressed cells. Purified recombinant isoforms of TFII-I bind directly to the ERSE of the grp78 and ERp72 promoters. The stimulation of ERSE-mediated transcription by TFII-I requires the integrity of the consensus tyrosine phosphorylation site and the GGC sequence motif of the ERSE. We further discovered that TFII-I is an interactive protein partner of ATF6 and that optimal stimulation of ERSE by ATF6 requires TFII-I.