Transcription factor YY1 binds to the murine beta interferon promoter and regulates its transcriptional capacity with a dual activator/repressor role

Abstract

The induction of the beta interferon (IFN-beta) gene constitutes one of the first responses of the cell to virus infection. Its regulation is achieved through an intricate combination of virus-induced binding of transcription factors and local chromatin remodeling. In this work, we demonstrate that transcription factor YY1, known to interact with histone deacetylases (HDAC) and histone acetyltransferases, has a dual activator/repressor role during the regulation of the IFN-beta promoter activity. We show that YY1 specifically binds in vitro and in vivo to the murine IFN-beta promoter at positions -90 and -122. Overexpression of YY1 strongly repressed the transcriptional capacity of a stably integrated IFN-beta promoter fused to a chloramphenicol acetyltransferase reporter gene as well as the endogenous IFN activity of murine L929 cells via an HDAC activity. Stably integrated IFN-beta promoters mutated at the -90 site were no longer repressed by YY1, could no longer be activated by trichostatin A, displayed a retarded postinduction turn off, and a reduced virus-induced activity. Introduction of a mutation at the -122 site did not affect YY1-induced repression, but promoters with this mutation displayed a reduced virus-induced activity. Stably integrated full-length promoters (from position -330 to +20) mutated at both YY1-binding sites displayed extremely reduced promoter activities. We conclude that YY1 has a dual activator/repressor role on IFN-beta promoter activity depending on its binding site and time after infection.

FIG. 1.

Four potential YY1-binding sites are present in the muIFN-β promoter. The DNA sequence of the muIFN-β promoter region spanning from the TATA box to position −210 is shown (35). Positions of the VRE and NRDs are indicated. Arrows indicate the presence of the YY1 core motifs (5′ to 3′) of the four potential YY1-binding sites.

FIG. 2.

Protein YY1 forms protein-DNA complexes with sequences present in the muIFN-β promoter. (A) Nuclear extracts were incubated with the indicated unlabeled double-stranded DNA probes (whose sequences are listed in Table 1), submitted to a gel retardation assay, and transferred on a nitrocellulose membrane. The presence of protein YY1 in the retarded protein-DNA complexes was revealed with anti-YY1 H-414 raised against the full-length YY1 as the primary antibody. (B) Same experiment as described for panel A but with probe mut90 containing the −90 YY1-binding site mutated in its YY1 core motif and probe mut122 containing the −122 YY1-binding site mutated in its YY1 core motif. (C) Same experiment as described for panels A and B, except that in the last lane nuclear extracts were loaded in the presence of poly(dI-dC) but in the absence (−) of DNA.

FIG. 3.

Protein YY1 binds to the muIFN-β promoter at positions −90 and −122. (A) Nuclear extracts were incubated with labeled probes 122 and mut122 in the presence or absence of 2 μg of anti-YY1 monoclonal antibody (Ab) H-10X raised against the full-length YY1 protein. (B) Nuclear extracts were incubated with labeled probes 90 and mut90 in the presence or absence of 1 or 2 μg of anti-YY1 monoclonal antibody H-10X. (C) Competition experiments were carried out with nuclear extracts incubated with labeled probe 90 in the absence (−) or presence of a 150-fold excess (150X) of unlabeled probes 90, mut90, 122, mut122, 161, and 32. (D) Nuclear extracts were incubated with labeled probe 90 in the absence or presence of 50-, 100-, and 150-fold excesses of unlabeled probes 90 and 122. (E) Chromatin immunoprecipitation assay of genomic DNA from noninfected L929 wt330 cells. Increasing amounts of DNA immunoprecipitated (I.P.) with either anti-YY1 or anti-HMGI antibodies were amplified with primers specific for the integrated IFN-β promoter region.

FIG. 4.

YY1 down-regulates the transcriptional capacity of the muIFN-β promoter via histone deacetylation. (A) Murine L929 wt330 cells, carrying the stably integrated muIFN-β promoter (from position −330 to +20) fused upstream of a CAT reporter gene, were transiently transfected with 1 μg/well, final concentration, of YY1 expressing plasmid (pCMV-YY1) or the corresponding empty vector (pCMV). When indicated, TSA (100 ng/ml final concentration) was added to the medium 4 h after transfection. Cells were virus induced 48 h after transfection and collected 18 h after virus infection. TSA was removed from the medium after virus infection and thereafter until the collection of the cells. (B) Murine L929 wt330 cells were transiently transfected and virus infected as described for panel A with increasing amounts (62, 125, 250, 500, or 1,000 ng/well) of YY1 expressing a plasmid (pCMV-YY1) or the corresponding empty vector (pCMV). Repression fold corresponds to the CAT activity measured from pCMV-transfected cells divided by the CAT activity measured from pCMV-YY1 transfected cells. (C) Murine L929 wt110 cells, carrying the stably integrated muIFN-β promoter (from position −110 to +20) fused upstream of a CAT reporter gene, were transiently transfected and virus infected as described for panel A with 1 μg/well, final concentration, of YY1 expressing plasmid (pCMV-YY1) or the corresponding empty vector (pCMV).

FIG. 5.

An intact YY1 −90 site is required for YY1-induced repression and TSA-dependent activation of the IFN-β promoter activity. (A) Murine L929 wt330, mut122, and mut90 strains carrying, respectively, integrated wild-type or mutated muIFN-β promoters fused to a CAT reporter gene were transiently transfected with 250 ng/well, final concentration, of pCMV or pCMV-YY1 plasmid. Cells were virus induced 48 h after transfection and collected 18 h after infection. NDV, Newcastle disease virus. (B) Noninfected murine L929 wt330, mut122, and mut90 cells were treated or not with 100 ng of TSA/ml (final concentration) for 48 h before being collected. (C) Equivalent amounts of genomic DNA isolated from L929 wt330, mut122, and mut90 strains were immunoprecipitated (I.P.) with anti-YY1 antibodies. Immunoprecipitated DNA (1, 2, and 3 μl) was amplified with primers specific for the integrated IFN-β region. (D) Gel retardation assay of labeled wt330, mut122, and mut90 muIFN-β promoters (from −330 to +20) incubated with 75 ng of recombinant protein HMGI in the presence of 250 ng of sonicated, unlabeled salmon sperm DNA as a random, nonspecific competitor DNA.

FIG. 6.

Mutated promoters mut122 and mut90 display reduced virus-induced activities. Cells from the L929 wt330 strain (top), mut122 strain (middle), and mut90 strain (bottom) were virus infected and collected at 0, 2, 4, 6, 8, 10, and 18 h after infection. The corresponding absolute CAT activities (i − mi) were measured.

FIG. 7.

Promoter mut90 displays a retarded postinfection transcriptional turn off. Cells from the L929 wt330 (○), mut122 (▪), and mut90 (▴) strains were virus infected and collected at 0, 2, 4, 6, 8, 10, 24, 32, and 54 h after infection. Percentages of the corresponding absolute CAT activities (i − mi) were measured. We have considered the activity reached by each strain 10 h after infection to be 100%.

FIG. 8.

Mutation of both YY1-binding sites (mut122/90) strongly reduces the virus-induced transcriptional capacity of the IFN-β promoter. (A) Cells from the wild-type L929 wt330 strain or the double mutant L929 mut122/90 strain were mock transfected or transiently transfected with 250 ng/well, final concentration, of pCMV or pCMV-YY1 plasmid. Cells were virus induced 48 h after transfection and collected 18 h after infection. NDV, Newcastle disease virus. (B) Noninfected wild-type murine L929 wt330 cells and double mutant L929 mut122/90 cells were treated or not with 100 ng of TSA/ml (final concentration) for 48 h before being collected. TSA was removed from the medium after virus infection and thereafter until collection of the cells. (C) Cells from the double mutant L929 mut122/90 strain were virus infected and collected at 0, 2, 4, 6, 8, 10, and 18 h after infection. The corresponding absolute CAT activities (i − mi) were measured.

FIG. 9.

The dual activator/repressor role of YY1 could be related to its capacity to interact with HATs and HDACs. We propose here a model that attempts to explain the bifunctional role of YY1 during the regulation of the transcriptional capacity of the IFN-β promoter. Several points remain to be demonstrated, setting up directions for future work. Besides the data obtained during this work, the model we propose here relies on (i) previous results indicating that before virus infection HDAC participates in the establishment of the promoter constitutive repression state (30); (ii) the work of Agalioti et al. (1) which indicates that Gcn5 is recruited by the promoter 3 h after infection, peaks 6 h after infection, and is released from the promoter 9 h after infection, whereas CBP peaks between 9 and 12 h after infection, does not participate during promoter histone acetylation, and remains bound to the promoter 24 h after infection; and (iii) the work of Munshi et al. (25) that describes CBP as essential for the transcriptional turn off of the IFN-β promoter. We suppose that in noninfected cells YY1 is predominantly bound to its −90 site and participates in promoter repression through an HDAC activity that deacetylates (DeAc) histones positioned in the NRDII region (top panel). Shortly after infection, YY1, alongside virus-activated factors bound to the VRE, participates in the recruitment of Gcn5. Gcn5 induces histone acetylation (Ac) necessary for nucleosome sliding and promoter transcriptional activation (1, 21) as well as YY1 acetylation (Ac) that disrupts YY1-DNA interactions (39) and could therefore induce YY1 promoter unbinding (middle panel). After release of Gcn5 from the promoter, nonacetylated forms of YY1 bind the promoter at its strongest −90 site and participate in the promoter transcriptional turn off in association with CBP and HDAC. Acetylation (Ac) of YY1 by CBP stabilizes YY1-HDAC interactions (39) (bottom panel).