Autoregulation of co-chaperone BAG3 gene transcription

Abstract

The Bcl-2-associated athanogene, BAG, protein family through their BAG domain associates with the heat shock protein 70 (HSP-70) and modulates its chaperone activity. One member of this family, BAG3, appears to play an important role in protein homeostasis, as its expression promotes cell survival. Expression of BAG3 is enhanced by a variety of stress-inducing agents. Here we describe a role for BAG3 to modulate transcription of its own promoter through a positive feedback loop involving its 5'-UTR sequence. Activation of the BAG3 promoter is mediated by the BAG domain and is independent of BAG3 association with the UTR sequence. Autoactivation of the BAG3 gene is observed in several cultures of human glial cells including gliomas, but not in several other non-glial cell lines such as He La and others. Results from cell fractionation and immunocytochemistry showed BAG3 in the cytoplasm as well as the nuclei of glial cells. These observations suggest that BAG3 gene expression is controlled by its own product and that this may be critical for the biological activity of BAG3 in some cell types.

Figure 1. Activation of the BAG3 promoter by BAG3 protein overexpression

(A) Luciferase assay in U-87 MG glioblastoma cells using reporter plasmid containing the BAG3 promoter (−831 to +306) DNA sequence (left bars) or Id-1 promoter (−2114/+95) sequence (right bars) upstream of the luciferase gene. Cells were co-transfected with empty vector or BAG3 full-length cDNA plus TK-Renilla luciferase as normalization control. Twenty-four hours after transfection, cells were harvested and firefly-luciferase and constitutively active thymidine kinase-Renilla luciferase activities were assayed. The data are expressed as fold effect from three independent experiments where luciferase activity was normalized with Renilla expression. (B) BAG3 cDNA dose-dependent response of the BAG3 promoter −831 to +306. Increasing amounts of BAG3 full-length cDNA were co-transfected with BAG3 promoter-luciferase in T98G cells as depicted. Firefly-Renilla luciferase assay was carried out and analyzed as in Panel A. BAG3 full-length protein overexpression was evaluated by Western blot ; α-tubulin was used as loading control. (C) Luciferase assay in primary human fetal astrocytes. Primary human fetal astrocytes were transfected with BAG3 promoter DNA plasmid and luciferase assay was performed according to the method described in Panel A.

Figure 2. Identification of BAG3 protein region required for BAG3 promoter activation

(A) Schematic representation of BAG3 protein domains. (B) T98G cells were co-transfected with the BAG3 promoter sequence −831 to +306. Firefly-luciferase plus empty vector (lane 1) BAG3 FL (lane 2) DC 1-420 (lane 3) or C-term 421-576 (lane 4). Luciferase assay was carried out 24 h after transfection as described above. Ten micrograms of total protein lysates was analyzed by Western blot and hybridized with a polyclonal anti-His antibody raised against the 6X Histidine tag present at the C terminus of recombinant protein.(C) T98G cells were transfected as in Fig. 2B. Total RNA was purified and 12 μg were analyzed by Northern blot. The filter was hybridized with firefly-luciferase probe. 18S ribosomal RNA was used as loading normalization control.

Figure 3. BAG3-BAG domain activates BAG3 promoter

(A) Luciferase assay in T98G cells cotransfected with BAG3 promoter −831/+306 + empty vector (lane 1) or 1X (lane 2) 2X (lane 3) and 3X of C-terminus (421-576) tandem repeats (lane 4). Overexpression of transfected cDNAs was assayed in the same lysates by imunoblotting with anti-His polyclonal antibody. (B) Luciferase assay in T98G glioblastoma cells co-transfected with equal amounts of the BAG3 promoter (−831/+306) and empty vector (left), BAG3 FL cDNA (middle) or BAG5 cDNA (right) plasmids. Fold activation was evaluated from three independent experiments, after normalization with Renilla activity, considering empty vector as basal luciferase level.

Figure 4. Identification of BAG3 promoter region responsive to BAG3 full-length protein

(A) Promoter-reporter activity of sequential deletions of the BAG3 promoter. At left are the various regions cloned into the pGL3 basic plasmid. The numbers indicate the positions relative to the transcription start site at +1. Fold activation was determined from three independent experiments, after normalization by Renilla activity, by comparing BAG3 FL with empty vector transfected cells. (B) T98G cells were infected at 20 m.o.i. with Adenovirus-Null (lanes 1 and 3) or Adenovirus-expressing BAG3-targeting siRNA (lanes 2 and 4) for 24 h. Cells were then transfected with equal amounts of BAG3 promoter-Luciferase −831/+306 (lanes 1 and 2) or −831/+1 (lanes 3 and 4) in both infected conditions. Twenty-four hours post-transfection cells were harvested and analyzed by luciferase assay as described above. Luciferase activity was present in arbitrary units of 1 to 20. Ten micrograms of whole cell lysate were checked by Western blot for BAG3 expression.

Figure 5. Induction of endogenous BAG3 by C-terminal BAG in T98G cells

(A) Time course of C-terminus 1x overexpression in T98G cells. After transfection, cells were harvested at indicated time points. Forty micrograms of lysate was subjected to Western blot and probed with rabbit polyclonal antibody raised against the full-length BAG3 protein. (B) Time course of C-terminus 2X overexpression in T98G cells. The procedure was performed as in Panel A. (C) qRT-PCR analysis of RNA in the same samples as in Panel B. The BAG3 transcript was measured and β-Actin mRNA was used as the normalization control

Figure 6. Subcellular localization of BAG3

(A) Untransfected cells and cells (U-87 MG and T98G) transfected with BAG3 FL were harvested after 24 h and nuclear-cytoplasm fractionated as described in Materials and Methods. Five and 15 μg of nuclear and cytoplasmic fractions, respectively, were evaluated by Western blot and probed with a rabbit polyclonal antibody against BAG3. The position of BAG3 is shown by an arrow. An asterisk points to a non-specific band. The purity of fractionation was checked by hybridization with anti-Lamin A/C and anti-α-tubulin antibodies. (B) U-87 MG cells were transfected as in A, hybridized with polyclonal BAG3 antibody and FITC-labeled secondary anti-rabbit, and microscopically analyzed.