Identification of TFII-I as the Endoplasmic Reticulum Stress Response Element Binding Factor ERSF: Its Autoregulation by Stress and Interaction with ATF6

Abstract

When mammalian cells are subjected to stress targeted to the endoplasmic reticulum (ER), such as depletion of the ER Ca²⁺ store, the transcription of a family of glucose-regulated protein (GRP) genes encoding ER chaperones is induced. The GRP promoters contain multiple copies of the ER stress response element (ERSE), consisting of a unique tripartite structure, CCAAT(N₉)CCACG. Within a subset of mammalian ERSEs, N₉ represents a GC-rich sequence of 9 bp that is conserved across species. A novel complex (termed ERSF) exhibits enhanced binding to the ERSE of the grp78 and ERp72 promoters using HeLa nuclear extracts prepared from ER-stressed cells. Optimal binding of ERSF to ERSE and maximal ERSE-mediated stress inducibility require the conserved GGC motif within the 9-bp region. Through chromatographic purification and subsequent microsequencing, we have identified ERSF as TFII-I. Whereas TFII-I remains predominantly nuclear in both nontreated NIH 3T3 cells and cells treated with thapsigargin (Tg), a potent inducer of the GRP stress response through depletion of the ER Ca²⁺ store, the level of TFII-I transcript was elevated in Tg-stressed cells, correlating with an increase in TFII-I protein level in the nuclei of Tg-stressed cells. Purified recombinant TFII-I isoforms bind directly to the ERSEs of grp78 and ERp72 promoters. The stimulation of ERSE-mediated transcription by TFII-I requires the consensus tyrosine phosphorylation site of TFII-I 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.

ACKNOWLEDGMENTS

We are greatly indebted to Ebrahim Zandi for helpful discussion and assistance with protein fractionation. We thank Stephen Desiderio for TFII-I expression vectors and antibodies, Ron Prywes for the ATF6 expression vector, and Mengyin Liu for assistance with Western blotting. The microsequencing was performed by Nicholas Sherman at the University of Virginia Biomedical Research Facility.

The University of Virginia Biomedical Research Facility is funded by a grant from the University of Virginia Pratt Committee. The microscopy was performed at the Electron Microscopy Core Facility at the Doheny Eye Institute, University of Southern California, supported by NEI/NIH core grant EY03040 and the USC/Norris Cancer Center. This work was supported by Public Health Service grants CA27607 from the NCI, National Institutes of Health, to A.S.L. and AI45150 from NIH to A.L.R.