Endoplasmic reticulum stress induction of the Grp78/BiP promoter: activating mechanisms mediated by YY1 and its interactive chromatin modifiers

Abstract

The unfolded protein response is an evolutionarily conserved mechanism whereby cells respond to stress conditions that target the endoplasmic reticulum (ER). The transcriptional activation of the promoter of GRP78/BiP, a prosurvival ER chaperone, has been used extensively as an indicator of the onset of the UPR. YY1, a constitutively expressed multifunctional transcription factor, activates the Grp78 promoter only under ER stress conditions. Previously, in vivo footprinting analysis revealed that the YY1 binding site of the ER stress response element of the Grp78 promoter exhibits ER stress-induced changes in occupancy. Toward understanding the underlying mechanisms of these unique phenomena, we performed chromatin immunoprecipitation analyses, revealing that YY1 only occupies the Grp78 promoter upon ER stress and is mediated in part by the nuclear form of ATF6. We show that YY1 is an essential coactivator of ATF6 and uncover their specific interactive domains. Using small interfering RNA against YY1 and insertional mutation of the gene encoding ATF6alpha, we provide direct evidence that YY1 and ATF6 are required for optimal stress induction of Grp78. We also discovered enhancement of the ER-stressed induction of the Grp78 promoter through the interaction of YY1 with the arginine methyltransferase PRMT1 and evidence of its action through methylation of the arginine 3 residue on histone H4. Furthermore, we detected ER stress-induced binding of the histone acetyltransferase p300 to the Grp78 promoter and histone H4 acetylation. A model for the ER stress-mediated transcription factor binding and chromatin modifications at the Grp78 promoter leading to its activation is proposed.

FIG. 1.

YY1 selectively activates the Grp78 promoter in Tg-stressed cells and is required for its full stress induction. (A) Summary of ER stress-induced changes in the DMS methylation pattern of the Grp78 promoter as revealed by in vivo footprinting (27). The sequence of the highly conserved human and mouse Grp78 promoter containing ERSE#3 where most of the ER stress-induced site occupancy changes occur is aligned. The solid arrow indicates constitutive protection from DMS methylation of the G residue. The open arrows and star indicate ER stress-inducible DMS methylation protection and hypersensitivity, respectively. The binding sites for the transcription factors NF-Y, TFII-I, and ATF6/YY1 are underlined. (B) CV-1 cells were transfected with the -169/Luc reporter plasmid and with either CMV-driven YY1 full-length expression plasmid or empty vector (V). Twenty-four hours after transfection, the cells were treated with 300 nM Tg for 16 h, harvested, and assayed for luciferase activity. (C) 293T cells were transfected with the EGFP vector and either U6 siControl or U6 siYY1. After 96 h, the cells were treated with Tg for 4 h and subjected to cell sorting by fluorescence. RNA was isolated from each sample, and equal amounts were used as template in RT-PCRs with oligo(dT) primers for cDNA synthesis and gene-specific primers as indicated. PCRs were carried out with 30, 26, 22, and 18 amplification cycles for determination of linear ranges. Data shown were subjected to 22 cycles of PCR. (D) Quantitation of Grp78 PCR products in panel C is shown, corrected for GAPDH levels.

FIG. 2.

ER stress-induced YY1 binding to the Grp78 promoter in vivo. (A) A total of 20 μg of HeLa nuclear extract from control and cells treated with Tg for 16 h was loaded onto a SDS-polyacrylamide gel, and the subsequent blot was reacted with anti-YY1 polyclonal and anti-GAPDH monoclonal antibodies. (B) NIH 3T3 cells were grown to 50% confluence in chamber slides and treated as indicated with 300 nM Tg for 8 h. The cells were fixed and stained with anti-YY1 monoclonal antibody and counterstained with propidium iodide. The cells were visualized with a Zeiss LSM510 confocal microscope (magnification, ×300). (C) NIH 3T3 cells were grown to 80 to 90% confluence; treated with 300 nM Tg for 0, 2, or 4 h; and then cross-linked with 1% formaldehyde. Chromatin extracts were prepared, and immunoprecipitation reactions were carried out with antibodies against YY1 or NF-Y. After reversal of cross-links, DNA was subjected to PCRs to amplify a 223-bp region of the Grp78 promoter. (D) Threefold dilutions of the DNA from the YY1 ChIP assay as shown in panel C were subjected to PCR for confirmation of linear range. (E) DNA collected from the YY1 and NF-Y ChIP assays were subjected to PCR with primers for a 248-bp region of the Grp78 exon VIII coding region.

FIG. 3.

ATF6α is required for full induction of Grp78 and its nuclear form co-localizes with YY1. (A) Primary MEFs (wild type) or with a βgeo gene trap mutation in the ATF6α gene were cultured and treated with Tg for 16 h. Western blotting of protein extracts from control cells and cells with Tg was performed with antibody against ATF6 (C1.12) (24). The bands corresponding to the wild-type ATF6 (p90) and its nuclear form ATF6(N), as well as the ATF6/βgeo fusion protein (p250), are shown. (B) RNA was isolated from the wild-type and mutant mouse ES cells treated with Tg for 16 h. Northern blotting was performed to probe for Grp78 transcript level with GAPDH as a control. The results were quantitated on a phosphorimager and are shown in graph format. (C) Cos-7 cells were grown to 50% confluence and transfected with either full-length HA-ATF6 or HA-ATF6(373) plasmids. After a 24-h incubation, the cells were fixed and stained with anti-HA monoclonal antibody (red) and anti-YY1 (green) polyclonal antibody. The cells were visualized on a Zeiss LSM510 confocal microscope (magnification, ×380). The merged image shows colocalization between HA-ATF6(373) and YY1 in the transfected cells (yellow). (D) 293T cells were transfected with either HA-ATF6(273) or HA-ATF6(373), and equal amounts of whole-cell extracts were subjected to sequential Western blotting to determine HA-ATF6 expression levels, with the GAPDH level serving as a loading control. (E) The transfected cells from the results shown in panel D were concurrently cross-linked with formaldehyde, and chromatin preparations were subjected to the ChIP assay with antibody against YY1 performed in duplicate and normal IgG as control. PCR primers for the 21-bp region of the human Grp78 promoter encompassing the three ERSEs were used, and the products are shown. Cells transfected with HA-ATF6(273) (top) and cells transfected with HA-ATF6(373) (bottom) are shown.

FIG. 4.

Mapping of YY1 and ATF6α interactive domains. (A) Schematic drawings of the nuclear form of the HA-tagged ATF6 (aa 1 to 373) and the truncated mutant (aa 1 to 273). Locations of the transactivation and the b-Zip domains are indicated. The domain required for interaction with YY1 is bracketed on top. (B) 293T cells were transfected with either the empty vector or the HA-ATF6 plasmids (top), and whole-cell extracts were immunoprecipitated with antibody against HA. Immunoprecipitated protein preparations (top two panels) and whole-cell extract (bottom) were run on 8% SDS-polyacrylamide gel electrophoresis gels and Western blotted with either anti-YY1 or anti-HA antibodies as indicated. (C) Schematic representation of the full-length FLAG-YY1(1-414) with its transcriptional activation, spacer, and zinc finger domains indicated. The deletion mutants used for immunoprecipitation assays are shown below. The zinc finger domain is indicated. The region (aa 261 to 333) required for interaction with ATF6 is bracketed on top. (D) FLAG-YY1 constructs as indicated (top) were transfected into 293T cells. Whole-cell extracts were prepared and subjected to immunoprecipitation with anti-FLAG antibody-conjugated agarose. Immunoprecipitated proteins resolved on a 4 to 15% gradient-denaturing gel were analyzed by Western blotting to detect HA-tagged ATF6(373) (top), FLAG-tagged YY1, and Western blots of whole-cell extracts with anti-HA antibodies (bottom).

FIG. 5.

YY1 is required for optimal activity of nuclear ATF6. (A) 293T cells were transfected with ATF6(373), -169/Luc, CMV β-Gal and increasing amounts of either FLAG-YY1(1-170) or FLAG-YY1(260-414). Cell extracts were prepared and assayed for luciferase activity. Values corrected for transfection efficiency by β-Gal activity are plotted. The standard deviations are shown. (B) 293T cells were transfected with EGFP vector and either U6 siControl or U6 siYY1. Transfection efficiency was determined to be at least 70% by GFP. Extracts were prepared 96 h after transfection and analyzed by Western blotting with antibodies against YY1 and GAPDH. (C) 293T cells transfected with U6 siControl or U6 siYY1, -169/Luc, and CMV β-Gal vectors were grown for 96 h. Cells were then transfected with ATF6(373); 24 h later, cell extracts were prepared and assayed for luciferase activity. Fold induction for each condition after normalization for β-Gal activity is plotted with standard deviations. The amount of transfected ATF6(373) in micrograms is shown at the bottom.

FIG. 6.

PRMT1 is recruited to the Grp78 promoter in Tg-stressed cells and enhances activation mediated by YY1 and ATF6(373). (A) Cos-7 cells were stably transfected with F-YY1 and CMV-Bsd. Cells were either cultured under normal conditions or treated with Tg for 3 h and harvested. Whole-cell extracts were immunoprecipitated with either normal mouse IgG or anti-Flag M2 agarose. Immunoprecipitates were then run on an 8% polyacrylamide gel and probed with antibodies against either Flag or PRMT1. A31 cell extract was used as a positive control for the presence of PRMT1. *, nonspecific band. (B) HeLa cells were treated with Tg for 3 h, chromatin extracts were prepared as shown in Fig. 4, and immunoprecipitation was carried out with antibodies against YY1, PRMT1, and the methylated arginine 3 residue of histone H4, as well as normal IgG. Products of the PCR using primers for the 213-bp region of the Grp78 promoter are shown. In each transfection experiment, CV-1 cells were transfected with -169/Luc reporter vector and CMV-Renilla luciferase control vector; pBluescript was used as an empty vector. Cells were harvested 24 h after transfection and assayed for dual-luciferase activity. (C) Increasing amounts of PRMT1 expression plasmid (in nanograms) were cotransfected as indicated. (D) Increasing amounts of YY1 expression plasmid (in nanograms) were cotransfected to establish the synergistic induction of the Grp78 promoter by YY1 in the presence of ATF6(373). (E) Increasing amounts of PRMT1 (in nanograms) were cotransfected with YY1 and ATF6(373) expression plasmids. The luciferase activity of the -169/Luc reporter gene alone is set at 1. *, P value of <0.05.

FIG. 7.

p300 is recruited to the Grp78 promoter in Tg-stressed cells, and its enhancing activity is dependent on YY1. (A) The ChIP assays were performed with HeLa cells as described in the legend to Fig. 6B. The immunoprecipitation assays were carried out with normal IgG or antibodies against p300, acetylated histone H4, and histone H3, and the primers for PCR are identical to the ones used in the experiment shown in Fig. 6B. (B) CV-1 cells were transfected and assayed for luciferase activity as described in the legend to Fig. 6E, except 0.1 μg of p300 expression plasmid was cotransfected as indicated.

FIG. 8.

Model of ER stress-inducible changes in transcription factor occupancy and chromatin remodeling of the Grp78 promoter. In nonstressed cells, NF-Y is in contact with the Grp78 promoter. Upon ER stress, while NF-Y binding remains intact and TFII-I binding is enhanced, ATF6 is cleaved to produce a nuclear form, ATF6(N), which associates with YY1 and enhances its binding to the Grp78 promoter. The YY1-interacting proteins PRMT1 and p300 are also recruited to the Grp78 promoter. Additional chromatin changes of histone H4 include acetylation and arginine 3 methylation.