Selective potentiation of Stat-dependent gene expression by collaborator of Stat6 (CoaSt6), a transcriptional cofactor

Abstract

The molecular mechanisms by which transcription is selectively activated and precisely controlled by signal transducer and activator of transcription (Stat) factors represent a central issue in cytokine-mediated cellular responses. Stat6 mediates responses to IL-4 and antagonizes Stat1 activated by IFN-gamma. We have discovered that Stat6 binds to collaborator of Stat6 (CoaSt6), a protein that lacks conventional coactivator motifs but contains three iterations of a domain found in the variant histone macroH2A. Although macroH2A participates in transcriptional silencing, the macro domains of CoaSt6 increased IL-4-induced gene expression. Moreover, CoaSt6 amplified Stat6-mediated but not IFN-gamma-induced gene expression, providing evidence of a selective coregulator of Stat-mediated gene transcription.

Conflict of interest statement

Conflict of interest statement: No conflicts declared.

Figures

Fig. 1.

Cloning and association of Stat6 and CoaSt6. (a) Diagram of the CoaSt6 cDNA. One partial cDNA repeatedly isolated in a two-hybrid screen by using full-length Stat6 as a bait and a mouse splenic cDNA library as target was designated as “clone 35,” which encoded an ORF of 1,806 nucleotides followed by a 3′ untranslated region of ≈2 kb (not shown). The full-length cDNA matched to the complete mouse genomic sequence at 35,560–35,605 K on chromosome 16. blast comparisons with the mouse and human genome sequence databases revealed homologies to macro (also called his-macro) domains (cross-hatched) of the BAL gene. ∗, Position of a mouse-specific portion of the predicted sequence used for preparation of anti-peptide antisera. (b) 293 cells were transiently transfected with expression constructs either lacking insert or encoding the indicated cDNA. After treatment of the transfected cells with or without IL-4, cellular lysates were subjected to IP and immunoblot analysis with the indicated antibodies. Total lysates were also probed with anti-Stat6 and anti-CoaSt6 without prior IP. (c) Lysates (cytoplasmic-C or nuclear-N fraction) from M12 B lymphoma cells treated with IL-4 (or not) were probed with anti-Stat6 and anti-CoaSt6 as indicated, whereas larger equal portions were subjected to IP with anti-Stat6 or an isotype-matched control Ig, followed by Western blot analysis of the precipitated proteins using anti-CoaSt6 or -Stat6 antibodies as indicated. (d) Cytoplasmic (C) or nuclear (N) extracts were made from IL-4-treated splenocytes isolated from the WT or Stat6-deficient (Stat6−/−) mice. The extracts were subjected to immunoprecipitation with anti-Stat6 and probed with the indicated antibodies.

Fig. 2.

CoaSt6 potentiates IL-4-induced, Stat6-mediated transcriptional activation. (a) Expression vector with or without CoaSt6 was cotransfected into HepG2 cells along with the Stat6 reporter and either empty vector (pcDNA3), Stat6 cDNA, or pcDNA3 encoding Stat6ΔC. The magnitude of IL-4-induced expression (mean ± SEM from three independent experiments), normalized to a separate reporter plasmid (pCMV-β-gal), is shown. (b) Increasing amounts of a CoaSt6-containing expression plasmid (pcDNA3) were transfected into HepG2 cells along with a reporter plasmid responsive to Stat6 and IL-4. Shown is the mean (± SEM) magnitude of transcriptional induction by IL-4, normalized as in a (mean of three independent experiments). (c) Jurkat cells were transfected with a Stat6 reporter and the indicated expression plasmids. The mean (± SEM) of IL-4-mediated fold induction of the reporter from three independent experiments is plotted. (d) A reporter containing three copies of an isolated Stat6-binding site were transfected into HepG2 cells along with the indicated expression plasmids, and the IL-4-dependent promoter activity was determined. Shown are mean values ± SEM from three independent experiments. (e) Failure of CoaSt6 to enhance Stat1-mediated IFN-γ inducibility of the IRF-1 promoter. A Stat1-dependent, IFN-γ-responsive reporter (driven by the 1.3-kb IRF-1 promoter linked to luciferase) was transfected into HepG2 cells along with either empty expression vector or the same vector encoding CoaSt6. Shown are the mean (± SEM) measurements of IFN-γ inducibility (three independent experiments). (f) Lack of interaction between Stat1 and CoaSt6. Co-IP experiments were performed by using extracts of the 293 cells transfected with plasmids encoding Stat1 and FLAG-tagged CoaSt6. Anti-FLAG IP were probed with the indicated antibodies. Whole-cell extracts were probed with anti-Stat1 (Lower).

Fig. 3.

CoaSt61216–1817 potentiates IL-4-induced transcription of the endogenous Stat6-dependent CD23 gene. (a) Expression plasmids encoding CoaSt6, or CoaSt61216–1817, and Stat6 were cotransfected into HepG2 cells along with a Stat6-responsive reporter. (Inset) Data from transfections in which the Stat6 expression plasmid was not included. The mean (± SEM) IL-4 induction values are plotted (three independent experiments). (b) Stat6−/− B cells were doubly transduced with two retroviruses, one with the GFP marker alone or containing GFP and Stat6 cDNAs. The other retrovirus encoded Thy1.1 with or without CoaSt61216–1817. Retrovirally infected B cells were treated with IL-4, and the CD23 expression on B220 positive cells expressing GFP and Thy1.1 was monitored by FACS. The number below each panel is the mean fluorescence intensity (MFI) indicating CD23 expression levels, and the value within each box represents the percentage of cells hyperexpressing CD23. Shown is a representative data set from one of three experiments with similar results. (c) Overexpression of CoaSt6 in B cells. A panel of stably transfected M12 B cells was generated along with empty vector-transfected cells. Anti-CoaSt6 immunoblots from four representative clones containing a full-length CoaSt6 cDNA (C6-1 to C6-4) are shown in comparison with the parental cells (P) and three neo-selected clones transfected with empty vector (E-1 to E-3). (d) Enhanced IL-4-induced CD23 expression on transfected B cells. Each of the above cell lines was treated with IL-4 and analyzed by flow cytometry for CD23 expression. Shown are profiles from the cells characterized in c. (e) IFN-γ induction of IRF-1 unaffected by CoaSt6. RNAs prepared from the same of CoaSt6-transfected M12 B cells and controls were analyzed in Northern blots probed with IRF-1 cDNA. Quantitation by phosphorimaging revealed no significant difference in IRF-1 expression between control and CoaSt6-transfected cells.

Fig. 4.

Knockdown of CoaSt6 expression decreases IL-4 induction of CD23 but not IFN-γ-mediated up-regulation of IRF-1. (a) The indicated CoaSt6 variants were transfected into 293T cells along with plasmids containing Shs either targeting CoaSt6 or LacZ. Cell extracts from these transfectants were then blotted with anti-CoaSt6. (b) M12 cells were stably transfected with plasmids containing Shs targeting LacZ, N-terminal (C6 Sh N), middle (C6 Sh M), and C-terminal (C6 Sh C) portions of CoaSt6. Western blots of CoaSt6 expression in two clones from each transfection were quantitated by a linear fluorescence energy detector, and the relative expression as compared with controls was calculated. (c) The CD23 expression profile with and without IL-4 treatment for the indicated M12 lines was determined by flow cytometry. Shaded histograms represent the CD23 expression profile on untreated cells whereas the bold line indicates that from cells treated with IL-4. (d) Knockdown of CoaSt6 expression by the N-terminal hairpin recloned into the pSIREN retrovector was evaluated as in a. (e) WT B-lymphoblasts were infected with the indicated retrovectors (n.s. = nonspecific hairpin), and the IL-4-dependent CD23 expression was evaluated as in Fig. 3b. Shown is a representative data set from four independent experiments. (f) Total RNAs isolated from the same cell lines, left untreated or treated with IFN-γ, were probed with cDNAs corresponding to IRF-1 and GAPDH. Quantification and normalization of the signals by phosphorimaging revealed no significant difference in IRF-1 expression between control and cells transfected with the Sh targeting CoaSt6.

Fig. 5.

MacroH2A-like domains of CoaSt6 amplify Stat6-mediated gene expression induced by IL-4. (a) Epitope-tagged segments of CoaSt6 were transfected into HepG2 cells along with a Stat6-dependent reporter and pcDNA3-Stat6. Shown are the mean (± SEM) induction by IL-4 (calculated after normalizing for transfection efficiency) from four independent experiments. (b) Bi-cistronic retrovectors containing cDNAs encoding the indicated CoaSt6 variants and Thy1.1 were used to infect LPS lymphoblasts from Stat6−/− mice along with retrovirus encoding Stat6 and GFP. Levels of CD23 expression induced by IL-4 were measured by flow cytometry of infected B cells expressing both Thy1.1 and GFP or expressing them singly. The numbers within and below each FACS panel are as in Fig. 3b.