Beta interferon (IFN-β) plays an essential role during the establishment of an antiviral state (8
). The transcriptional capacity of the IFN-β promoter is constitutively repressed in an adult normal cell and remains inactivated until an external stimulus such as virus infection triggers its activation. Activation of the transcriptional capacity of the IFN-β promoter is transient. It is turned on 4 to 6 h after virus infection, and it is turned off 10 to 12 h after it (10
). Regulation of the promoter transcriptional capacity requires specific binding of transcription factors as well as the orderly recruitment of chromatin-remodeling complexes on the promoter region (1
In the absence of virus infection, histone deacetylase activity maintains deacetylated lysine residues of histones H3 and H4 positioned on the IFN-β promoter region (22
). Shortly after virus infection, transcription factors and protein HMGI bind to the promoter on the nucleosome-free virus-responsive element region. The subsequent recruitment of histone acetyltransferases (HATs) CBP/p300 and GCN5/PCAF leads to the specific acetylation of certain lysine residues of histone H4 and H3, specially K8H4 and K14H3, which are essential for the recruitment of the RNA polymerase II holoenzyme complex and the SWI/SNF nucleosome-remodeling complex (2
). Finally, nucleosome remodeling allows the binding of TFIID to the TATA box, triggering initiation of transcription of the IFN-β gene.
We have recently published data indicating that transcription factor Yin Yang 1 (YY1) binds to the murine IFN-β (muIFN-β) promoter at two different sites and regulates promoter transcriptional capacity with a dual activator/repressor role (35
). The repressor role of YY1 appeared linked to its capacity to interact and recruit a histone deacetylase (HDAC) on the promoter region through its −90 site, while the exact molecular mechanisms governing the capacity of YY1 to activate the muIFN-β promoter remained to be elucidated.
YY1 is a ubiquitous, highly conserved zinc finger transcription factor (3
) that activates or represses several different eukaryotic genes, among which are the c-Myc, c-Fos, β-casein, α-actin, interleukin 3 and 5, and IFN-γ genes, as well as some viral promoters (15
). It binds to DNA through the recognition of a specific sequence containing a consensus (C/t/aCAT [uppercase letters represent preferred nucleotides; lowercase letters represent nucleotides tolerated to a lesser extent]) core motif (12
). Promoter context (20
), intracellular concentration (6
), and posttranslational modifications (37
) as well as the capacity of YY1 to interact with transcription factors and cofactors can influence the capacity of YY1 to act either as an activator or a repressor (7
). Particularly, the interaction of YY1 with HATs or HDACs can orient YY1 towards either an activator or a repressor role.
Using gel retardation and recombinant glutathione S
-transferase (GST)-YY1 protein we demonstrate in this work that the sequences surrounding the consensus YY1 DNA-binding core motifs present on the muIFN-β promoter strongly influence the binding of YY1 to these sites so that YY1 effectively binds only to the core motifs present at positions −122 and −90. Using the DNase I footprinting technique we have analyzed the capacity of YY1 for binding to its respective sites in the context of the entire promoter. We have observed that YY1 is able to bind both sites simultaneously protecting a region that extends beyond the −122 and −90 sites. Disruption of YY1 binding to either the −90 or the −122 site did not disrupt the binding of YY1 to the remaining intact site. In order to decipher the molecular mechanism governing the role of YY1 as activator of the transcriptional capacity of the murine IFN-β promoter, we have carried out chromatin immunoprecipitation (ChIP) assays on murine L929 cells and established cell lines containing either the wild-type muIFN-β promoter or the corresponding promoters mutated at either the −122 or the −90 YY1 site fused to a chloramphenicol acetyltransferase (CAT) reporter gene integrated in their genomes. The in vivo binding of CBP, AcK8H4, AcK14H3, IRF3, and YY1 to either the wild-type promoter or to the promoters mutated at the −122 (mut122) or −90 (mut90) site was analyzed before and at different times after virus infection. The results we have obtained here clearly indicated that the simultaneous presence of intact −90 and −122 sites was required to allow virus-induced CBP recruitment and K8H4/K14H3 acetylation on the muIFN-β promoter region as well as to reach virus-induced promoter transcriptional activation. The binding of YY1 to only one intact site, either −122 or −90, was not sufficient to allow CBP recruitment and K8H4/K14H3 acetylation. The binding of YY1 to the −90 site appeared constant, visible before as well as after infection, whereas the binding of YY1 to the −122 site was induced after infection so that simultaneous occupancy of both sites seemed possible only after infection. Contrary to the role of YY1 as activator of the muIFN-β promoter that, as demonstrated here, required that both the −122 and −90 sites be intact, only an intact −90 site has been previously described as required for the YY1-dependent repression of the promoter (35
We analyze here the essential role of YY1 during virus-induced CBP recruitment, K8H4/K14H3 acetylation, and muIFN-β promoter transcriptional activation and show that its role is predominant with respect to the main role generally attributed to IRF3 as a regulator of virus-induced CBP recruitment and IFN-β promoter activation. We also discuss here the possibility to regulate the orientation of YY1 either as a repressor or an activator by regulating the degree of occupancy of the respective YY1 binding sites present in a same promoter region.