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. 2000 Nov;20(22):8489-98.
doi: 10.1128/MCB.20.22.8489-8498.2000.

Acetylation by PCAF enhances CIITA nuclear accumulation and transactivation of major histocompatibility complex class II genes

C Spilianakis  1 , J Papamatheakis, A Kretsovali
Affiliations

Acetylation by PCAF enhances CIITA nuclear accumulation and transactivation of major histocompatibility complex class II genes

C Spilianakis et al. Mol Cell Biol. 2000 Nov.

Abstract

The class II transactivator (CIITA), the master regulator of the tissue-specific and interferon gamma-inducible expression of major histocompatibility complex class II genes, synergizes with the histone acetylase coactivator CBP to activate gene transcription. Here we demonstrate that in addition to CBP, PCAF binds to CIITA both in vivo and in vitro and enhances CIITA-dependent transcriptional activation of class II promoters. Accordingly, E1A mutants defective for PCAF or CBP interaction show reduced ability in suppressing CIITA activity. Interestingly, CBP and PCAF acetylate CIITA at lysine residues within a nuclear localization signal. We show that CIITA is shuttling between the nucleus and cytoplasm. The shuttling behavior and activity of the protein are regulated by acetylation: overexpression of PCAF or inhibition of cellular deacetylases by trichostatin A increases the nuclear accumulation of CIITA in a manner determined by the presence of the acetylation target lysines. Furthermore, mutagenesis of the acetylated residues reduces the transactivation ability of CIITA. These results support a novel function for acetylation, i.e., to regulate gene expression by stimulating the nuclear accumulation of an activator.

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Figures

FIG. 1
FIG. 1
The activation domain of CIITA binds to both CBP and PCAF. (A) GST fusions of different parts of the CIITA protein used for interaction with PCAF and CBP are shown schematically. (B) In vitro-translated and 35S-labeled PCAF or two regions of CBP (CBP 1–1098 and CBP 1620–1877) were used in a GST pull-down assay with equal amounts (1 μg) of GST alone (lanes 2, 10, and 16) or fused with the indicated fragments of CIITA. Input was 5% in lane 1, 30% in lane 15, and 20% in lane 21.
FIG. 2
FIG. 2
Regions of PCAF that interact with CIITA. (A) Scheme of the GST-PCAF proteins used for interaction with CIITA. Br., bromodomain. (B) In vitro 35S-labeled CIITA was used in a GST pull-down assay with equal amounts of GST alone (lane 2) or GST fused to the indicated regions of PCAF (lanes 3 to 12).
FIG. 3
FIG. 3
In vivo interaction between CIITA and PCAF. Whole-cell extracts from COS-1 cells transfected with the indicated plasmids (5 μg each) were immunoblotted (WB) with anti-HA antibody (α-HA) before (lanes 1 to 5) or after (lanes 1' to 5') immunoprecipitation (IP) with anti-Flag M2 agarose. In lanes 1 to 5 inputs are 10% of the extract used in immunoprecipitation. Equivalent amounts of inputs were also checked for expression of PCAF derivatives using anti-Flag antibody (α-Flag).
FIG. 4
FIG. 4
PCAF coactivates MHC class II gene expression. (A) Plasmids encoding PCAF or vector control (0.5 to 1 μg) were cotransfected with 1 μg of an MHC class II promoter-CAT reporter plasmid into HeLa cells. Uninduced and IFN-γ (50 U/ml)-induced activities were assayed 24 h after IFN-γ addition. The CAT activity of vector transfected-uninduced cells was set to 1. Bars represent the standard error of the mean (SEM) from four experiments. (B) HeLa cells were cotransfected with a class II-luciferase reporter (1 μg) a CIITA-expressing plasmid (20 ng), and 0.5 to 1 μg of plasmids expressing wild-type PCAF, a deletion lacking its first 352 amino acids (PCAF-ΔN), a mutant that has no HAT activity (PCAF-HAT−) a mutant containing only the first 511 amino acids (PCAF 1–511), and a mutant that lacks amino acids 653 to 736 (PCAF-Δ ADA). Luc activity was measured 24 h after transfection. The activity of the class II-Luc reporter in the presence of CIITA protein was set to 1 and represents an induction range between 7- and 15-fold. Bars represent SEM from four experiments. The expression levels of the PCAF molecules were analyzed by Western blotting (WB) using an anti-Flag antibody (α-Flag). (C) HeLa cells were transfected with 1 μg of class II-CAT plasmid along with vector control or increasing amounts of plasmids expressing the indicated E1A products. Results are means of three experiments, with standard deviation less than 25% of the mean value. IFN-γ induction ranged between 15- and 45-fold. (D) HeLa cells were cotransfected with 1 μg of class II-CAT and 100 ng of a CIITA-expressing plasmid and vector control plasmid or increasing amounts of plasmids expressing the indicated E1A derivatives. Results are expressed as a percentage of the vector control activation by CIITA and are averages of three experiments, with standard deviation less than 30% of the mean value. Activation by CIITA ranged between 75- and 120-fold.
FIG. 5
FIG. 5
CIITA is acetylated in vitro and in vivo. (A) GST-CIITA fusion proteins that were assayed for in vitro acetylation. The aligned amino acid sequence of the human and murine CIITA region spanning amino acids 141 to 159 is shown, and critical lysine residues are underlined. mK1 and mK2 designate mutations of lysine pairs K141,144 and K156,159, respectively. (B) PCAF acetylation assays. Portions (1 to 2 μg) of the indicated CIITA fragments were subjected to in vitro acetylation using 500 ng of GST-PCAF (lanes 1 to 13) or PCAF immunoprecipitated on anti-Flag M2 agarose from COS-1 cells (lanes 14 and 15). ∗, PCAF autoacetylation. WT, wild type. (C) CBP immunoprecipitation-acetylation assay. CBP was immunoprecipitated (IP) from COS-1 cell extracts with anti-CBP antibodies and used for acetylation of the indicated CIITA fragments. ∗, CBP autoacetylation. (D) In vivo acetylation. COS-1 cells transfected with the indicated plasmids were labeled with [3H]acetate for 1 h. The resulting whole-cell extracts were immunoprecipitated with anti-GFP monoclonal antibody, subjected to SDS-PAGE (10% polyacrylamide), and subjected to autoradiography. Identical nonradiolabeled samples were immunoblotted (W.B.) with anti-GFP antibody (α-GFP).
FIG. 6
FIG. 6
An NLS is contained between amino acids 141 and 159 of CIITA. GFP fusions of the indicated regions of CIITA were transfected in HeLa cells. Green fluorescence was observed 24 h later.
FIG. 7
FIG. 7
CIITA exits from the nucleus via CRM-1. The nucleocytoplasmic distribution of wild-type and mutant GFP-CIITA fusion proteins in HeLa cells is shown. LMB indicates a 2-h treatment with 20 nM LMB.
FIG. 8
FIG. 8
Inhibition of deacetylases by TSA enhances nuclear accumulation of CIITA. HeLa cells were transfected with GFP-CIITA or GFP-p65 expression plasmids and further cultivated in the absence (Control) or presence of 1 μM TSA (+TSA). The picture shows representative fields of fluorescent cells 24 h after the addition of TSA.
FIG. 9
FIG. 9
PCAF promotes nuclear localization of CIITA. (A) Cells were transfected with 1 μg of GFP-CIITA fusion plasmid and 3 μg of Flag-PCAF or Flag-PCAF-HAT− expressing plasmids or empty vector alone. The cells were analyzed 20 to 24 h later. (B) Coexpression of PCAF produces high levels of nuclear CIITA. Cells transfected with GFP-CIITA and Flag-PCAF were stained with an anti-Flag antibody followed by rhodamine-conjugated secondary antibody and analyzed for either GFP fluorescence (a) or rhodamine stain (b). (C) Quantitative analysis of the above results. The percentage of cells expressing predominantly or fully nuclear CIITA (N > C) in the presence of vector or the indicated PCAF expression plasmids is shown. Results presented were derived from the analysis of more than 300 cells 24 h after transfection and are averages from four independent experiments.
FIG. 10
FIG. 10
Transactivation by CIITA acetylation mutants. COS-1 cells were cotransfected with 1 μg of Eα class II-Luc reporter and 25 ng of the indicated wild-type (wt) or mutant CIITA expression plasmids. Luciferase activity was measured 24 h posttransfection. The results are presented as the percentage of promoter activation obtained by the wild-type CIITA, which is set to 100%, and they are average values of four experiments. The solid and open bars indicate the activities of CIITA derivatives in the absence or presence of the SV40 NLS, respectively. The expression pattern of CIITA proteins was analyzed by immunoblotting (WB) with an anti-HA antibody.
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