And-1 is required for the stability of histone acetyltransferase Gcn5

Abstract

Histone acetyltransferases (HATs) have a central role in the modification of chromatin as well as in the pathogenesis of a broad set of diseases including cancers. Gcn5 is the first identified transcription-related HAT that has been implicated in the regulation of diverse cellular functions. However, how Gcn5 proteins are regulated remains largely unknown. Here we show that acidic nucleoplasmic DNA-binding protein (And-1, a high mobility group domain-containing protein) has remarkable capability to regulate the stability of Gcn5 proteins and thereby histone H3 acetylation. We find that And-1 forms a complex with both histone H3 and Gcn5. Downregulation of And-1 results in Gcn5 degradation, leading to the reduction of H3K9 and H3K56 acetylation. And-1 overexpression stabilizes Gcn5 through protein-protein interactions in vivo. Furthermore, And-1 expression is increased in cancer cells in a manner correlating with increased Gcn5 and H3K9Ac and H3K56Ac. Thus, our data reveal not only a functional link between Gcn5 and And-1 that is essential for Gcn5 protein stability and histone H3 acetylation, but also a potential role of And-1 in cancer.

Figure 1. And-1 is complexed to histone H3 and Gcn5

(A) Schematic of the And-1 protein domains and deletions used for protein interactions in Fig. 1. (B) And-1 interacts with histone H3 in vivo. 293T cells transfected with indicated Flag-And-1 mutants were lysed and immunoprecipitated with anti-Flag, resolved on SDS-PAGE, and immunoblotted with indicated antibodies. (C) And-1 interacts with histone H3 in vitro. Flag tagged full length And-1 as well as its mutants was expressed in 293T cells and immunopurified Flag-And-1 was mixed with histone core particles. After incubation and extensive washing, Flag-And-1 immunoprecipitates were resolved on SDS-PAGE, and immunoblotted with indicated antibodies. (D) Gcn5 interacts with And-1. HCT116 cell lysates immunoprecipitated with IgG, anti-And-1, or anti-Gcn5 and immunoblotted with anti-And-1 or anti-Gcn5 as indicated. (E) Gcn5 interacts with full length And-1. 293T cells transfected with Flag-And-1 and treated as in (B). (F) Gcn5 interacts with And-1 in vitro. Immunopurified Gst-And-1 and Flag-Gcn5 from 293T cells were mixed and incubated for one hour. After washing, Gst-And-1 precipitates were then resolved on SDS-PAGE, and immunoblotted with indicated antibodies.

Figure 2. Cell cycle localization of And-1 and Gcn5

(A) Cell cycle immunofluoresence image of HCT116 cells showing that the majority of Gcn5 co-localizes with And-1 in the cell cycle except in telophase during which And-1 re-associates with chromatin earlier than Gcn5. (B) Cell cycle analysis of Gcn5, acetylation of histone H3 and And-1 proteins. HCT116 cells were synchronized at S phase by a double thymidine procedure and subsequently released into the cell cycle. Samples were taken at indicated time points after release and subjected to western blot analyses of whole cell extracts and probed with indicated antibodies. Asy, Asynchronous cells. (C) FACS analysis of cells harvested as in (B).

Figure 3. And-1 depletion reduces Gcn5 expression

(A) And-1 downregulation decreased endogenous Gcn5 expression. HCT116 cells transfected with siRNA control Gl2 or two independent siRNAs were harvested, resolved on SDS-PAGE, and immunoblotted with indicated antibodies. (B) And-1 downregulation decreased exogenous Gcn5 protein levels. Flag-Gcn5 was co-transfected with siRNA control Gl2 or And-1 in 293T cells. Cells were harvested 48 hours after transfection, resolved on SDS-PAGE, and immunoblotted with indicated antibodies. (C) Downregulation of And-1 does not affect expression of other components of hSAGA and ATAC. HCT116 cells treated as in (A) were harvested and immunoblotted with indicated antibodies. (D) Decreased Gcn5 proteins can be rescued by expression of And-1 from cDNA. U2OS cells were transfected with Gl2 siRNA or And-1 siRNA targeting its 3’UTR mRNA sequence alone, or co-transfected with vector plasmid or And-1 plasmid. Cells were harvested 48 hours after transfection and western blotted for indicated proteins.

Figure 4. And-1 specifically stabilizes Gcn5 proteins

(A) And-1 depletion does not affect Gcn5 mRNA. mRNA level of Gcn5 tested by real-time RT-PCR in HCT116 cells transfected with siRNAs against Gl2 or And-1. Experiments were performed two times. mRNA level data were normalized to GAPDH, and Gl2 transfected group values were set as 1. Error bars represent SD from the mean. (B) Cell cycle immunofluoresence image of HCT116 cells transfected with siRNA And-1 showing Gcn5 proteins are decreased in the absence of And-1 during cell cycle. (C) Gcn5 protein half-life is reduced in And-1 depleted cells. HCT116 cells transfected with siRNA Gl2 or And-1 were treated with cycloheximide at 40 hours after transfection. Cells were harvested at indicated time points, and immunoblotted for indicated proteins.

Figure 5. And-1 overexpression stabilizes Gcn5

(A) And-1 overexpression increases endogenous Gcn5 protein levels. U2OS cells co-transfected with Flag-And-1 or vector were harvested 48 hours after transfection. Cells were resolved on SDS-PAGE, and immunoblotted with indicated antibodies. (B) And-1 overexpression increases exogenous Gcn5 protein levels. U2OS cells were co-expressed with Flag-Gcn5 and titrated amounts of vector or Flag-And1 plasmids as indicated. Cells were harvested 48 hours after transfection and treated as in (A). (C) Full length Flag-And-1 stabilizes Gcn5 proteins. And-1 and its mutants were transfected together with Flag-Gcn5 plasmids in U2OS cells. Cells were harvested 48 hours after transfection and treated as in (A).

Figure 6. Acetylation of H3K9 and H3K56 is decreased in And-1 downregulated cells

(A) And-1 depletion reduces the acetylation of H3K9 and H3K56. HCT116 cells transfected with indicated siRNAs were harvested, resolved on SDS-PAGE, and immunoblotted with indicated antibodies. (B) The acetylation of H3K9 at promoters of Cyclin B1 is reduced in And-1 depleted cells. HCT116 cells were transfected with indicated siRNAs before analyses by ChIP. The PCR-amplified products were analyzed by agarose gel (middle). The amount of DNA was quantitated by Scion imaging software (top). The amount of DNA in Gl2 siRNA transfected cells was taken as 100%. Schematic representation of Cyclin B1 genes showing positions analyzed by ChIP (bottom). (C) The acetylation of H3K9 at promoters of Cdk1 is reduced in And-1 depleted cells. Assay was performed as in (B).

Figure 7. And-1 expression in tumors and tumorigenic cell lines

(A) And-1 is more highly expressed in colorectal carcinomas compared to paired normal colon tissue. A representative fluorescent immunohistochemical image is shown. (B) Plots of And-1 expression in five paired colon tumor and normal tissues from individual patients. And-1 expression was increased in 4 of the 5 patients examined. Overall expression of And-1 was significantly higher in tumors compared to paired normal tissues by paired t-test (p < 0.05). (C) And-1 expression is increased in tumorigenic cell lines compared to normal cells. A375 (melanoma) and D-1 (normal skin fibroblast) cells were harvested for immunoblotting for indicated proteins.