Endoplasmic Reticulum Stress-Induced Formation of Transcription Factor Complex ERSF Including NF-Y (CBF) and Activating Transcription Factors 6α and 6β That Activates the Mammalian Unfolded Protein Response

Abstract

The levels of molecular chaperones and folding enzymes in the endoplasmic reticulum (ER) are controlled by a transcriptional induction process termed the unfolded protein response (UPR). The mammalian UPR is mediated by the cis-acting ER stress response element (ERSE), the consensus sequence of which is CCAAT-N 9 -CCACG. We recently proposed that ER stress response factor (ERSF) binding to ERSE is a heterologous protein complex consisting of the constitutive component NF-Y (CBF) binding to CCAAT and an inducible component binding to CCACG and identified the basic leucine zipper-type transcription factors ATF6␣ and ATF6␤ as inducible components of ERSF. ATF6␣ and ATF6␤ produced by ER stress-induced proteolysis bind to CCACG only when CCAAT is bound to NF-Y, a heterotrimer consisting of NF-YA, NF-YB, and NF-YC. Interestingly, the NF-Y and ATF6 binding sites must be separated by a spacer of 9 bp. We describe here the basis for this strict requirement by demonstrating that both ATF6␣ and ATF6␤ physically interact with NF-Y trimer via direct binding to the NF-YC subunit. ATF6␣ and ATF6␤ bind to the ERSE as a homo-or heterodimer. Furthermore, we showed that ERSF including NF-Y and ATF6␣ and/or ␤ and capable of binding to ERSE is indeed formed when the cellular UPR is activated. We concluded that ATF6 homo-or heterodimers recognize and bind directly to both the DNA and adjacent protein NF-Y and that this complex formation process is essential for transcriptional induction of ER chaperones.Two mammalian proteins with molecular masses of 78 and 94 kDa identified in the mid-1970s were named glucose-regulated proteins (GRP78 and GRP94, respectively) due to the marked increases in their levels on depletion of glucose from media for cell culture (24). Subsequent studies indicated that this phenomenon is part of the cellular response to the accumulation of unfolded proteins in the endoplasmic reticulum (ER) (8, 18). Not only glucose deprivation but also various physiological and environmental stress conditions (ER stress) cause unfolding or misfolding of proteins in the ER, where newly synthesized secretory and transmembrane proteins fold and assemble. As only correctly folded molecules are allowed to move along the secretory pathway, eukaryotic cells must deal with protein misfolding in the ER promptly and appropriately to avoid malfunction and/or mislocalization of proteins that are synthesized on membrane-bound ribosomes and translocated into the ER. A typical cellular strategy to cope with unfolded proteins in the ER is induction of molecular chaperones and folding enzymes localized in the lumen of the ER, such as BiP/GRP78 and GRP94, resulting in augmentation of the folding capacity of the ER. This homeostatic response is achieved by a transcriptional induction process coupled with signaling from the ER to the nucleus, now known as the unfolded protein response (UPR).The transcriptional apparatus responsible for the mammalian UPR was poorly understood until the cis-acting ER stress response element (ERSE), necessary and ...

17β-Estradiol inhibits ER stress-induced apoptosis through promotion of TFII-I-dependent Grp78 induction in osteoblasts

“…It can stabilize ATF6 binding to the ERSEs of Grp78 promoters, in cooperation with other binding factors such as YY1 and NF-Y. 39,40 We found that 17b-E 2 could promote TFII-I phosphorylation and nuclear localization and enhance the Grp78 promoter activity in a dose-dependent manner in the presence of TFII-I in response to ER stress. ChIP analysis further revealed that 17b-E 2 enhanced binding of TFII-I to the Grp78 promoter in ER stress.…”

“…NF-Y, YY1, ATF6 and TFII-I are four key factors that bind to the ERSEs of Grp78 promoter and subsequently promote the expression of Bip. 39,40 Therefore, we examined the expression levels of NF-Y, YY1 and ATF6 in MC3T3-E1 cells treated with or without 17b-E 2 . Unexpectedly, 17b-E 2 did not significantly change these proteins expression levels following Tg treatment (Figure 3a).…”

Roles of CHOP/GADD153 in endoplasmic reticulum stress

“…pATF6a(N) and pATF6b(N) contain a bZIP domain and bind to the ER stress response element (ERSE) consensus sequence 5 0 -CCAAT-N 9 -CCACG-3 0 as a homo-or heterodimer interacting with NF-Y trimer. 36,37 Activated Ire1a and Ire1b cleave the substrate precursor XBP-1 mRNA to mature XBP-1 mRNA by their endoribonuclease activity present in their cytosolic domains. [38][39][40] XBP-1 splicing removes a 26-nucleotide intron, which switches the open reading frames to yield its bZIP and transactivation domain.…”

Analysis of ATF6 Activation in Site-2 Protease-deficient Chinese Hamster Ovary Cells

“…Upon ER stress ATF6 is cleaved via a process termed regulated intramembrane proteolysis (Rip), resulting in the release of the N-terminal region of ATF6 from the membrane . This fragment, carrying the basic leucine zipper and transactivation domains, is translocated into the nucleus where it activates the transcription of ER-localized molecular chaperones and folding enzymes (collectively termed ER chaperones hereafter) by directly binding to the cis-acting ER stress-response element (ERSE) present in the ER chaperone promoters, in collaboration with the general transcription factor NF-Y Yoshida et al, 1998;Yoshida et al, 2000;Yoshida et al, 2001b). Induced ER chaperones help maintain the homeostasis of the ER.…”