Division of labor between IRF1 and IRF2 in regulating different stages of transcriptional activation in cellular antiviral activities
© Ren et al.; licensee BioMed Central. 2015
Received: 12 March 2015
Accepted: 27 March 2015
Published: 18 April 2015
Cellular antiviral activities are critically controlled by transcriptional activation of interferon-inducible genes, involving interferon regulatory factors (IRFs). Previous data suggested that IRF1 is an activator and IRF2 is a repressor, which functionally antagonize each other in transcriptional regulation. However, it is not clear how these two factors function to regulate cellular antiviral activities.
We show that IRF2 is critically required for the induction of the TLR3 and other interferon-inducible genes in a chromatin environment. While both IRF1 and IRF2 directly interact with the BAF chromatin remodeling complex, IRF2 is associated with the TLR3 promoter in the unstimulated state and IRF1 binding to the promoter is strongly induced by stimulation with interferon, suggesting that these two factors may function at different stages of gene induction in the recruitment of the BAF complex. IRF2 acts to maintain the basal level expression, an open chromatin structure, and active histone modification marks (H3K9, K14 acetylation and H3K4 tri-methylation) of the TLR3 promoter in the unstimulated state, while IRF1 serves to rapidly activate the promoter upon stimulation.
IRF1 and IRF2 of the IRF family of transcription factors play distinct roles in cellular response to viral infection. IRF2 binds to TLR3 and other IFN-inducible gene promoters and maintains an active chromatin structure in the unstimulated state, which is required for their induction, while IRF1 binding to these promoters activates their transcription upon viral infection. Thus, the division of labor between the IRF transcription factor family members plays a pivotal role in coordinating the transcriptional activation in the cellular antiviral response.
KeywordsIRF1 IRF2 BRG1 ChIP Transcription Antiviral activities
Genome analyses indicate that human genome contains only about twice the number of genes than the simple nematode worm, C. elegans, has [1-3]. It has been hypothesized that the more complexity of human compare to the worm is caused by the diversity of transcriptional regulatory DNA elements and transcription cofactor complexes . Gene duplication in human genome results in the existence of multiple family members of transcription factors, which usually have highly conserved DNA-binding domains and recognize the same DNA sequence. How do the different family members contribute to the complexity and accuracy of their target genes regulation? To provide insights to this question, we decided to study the transcriptional regulation of toll-like receptor 3 (TLR3) by the interferon regulatory factors IRF1 and IRF2 . IRF1 and IRF2 belong to the nine-member IRF family with highly homologous N-terminal DNA-binding domains [6-8]. Previous studies suggested that IRF1 is an activator and IRF2 is a repressor of transcription; and they function antagonistically by recognizing the same DNA motif .
TLR3 is a critical regulator of cellular antiviral activities . It recognizes double-stranded (ds) RNA, which is an intermediate of viral replication, and transmits signals to induce the key cytokines of the cellular antiviral system, the type I interferons IFN-α/β . The expression of IFN-α/β is activated by NF-κB and TLR3. IFN-α/β signal through their cell surface receptors to activate and translocate the trimeric ISGF3 complex consisting p48, STAT1, and STAT2 to the nucleus, which is required for the activation of hundreds of target genes . Both the basal and induced levels of expression of the interferon-inducible genes are critical for the innate and activated cellular antiviral activities . Thus understanding the transcriptional regulation of TLR3 will provide insights to the cellular antiviral activities. In this study we present data to show the differential function of IRF1 and IRF2: IRF2 is associated with TLR3 and other IFN-inducible gene promoters in unstimulated states and potentiates their induction in response to viral infection by maintaining an active chromatin structure while IRF1 activates transcription of these genes in response to viral infection.
Results and discussion
To identify the DNA elements that mediate the BAF complex activity, 5′-deletion analysis of the TLR3 promoter was performed. The 509 bp DNA fragment of promoter was responsive to IFN-α stimulation and to the BRG1 expression (Figure 1C). Both together can activated the promoter further. Deletion to −251 reduced the activity of the promoter. However, the responsiveness to IFN-α and BRG1 remained, even though an apparent ISRE was deleted. Deletion to −11, which removed an IRF-E sequence, completely abolished the activity of the promoter. These data suggest that the Sp1 binding site contributes to and the IRF-E plays an essential role in mediating the activity of the BAF complex. Consistent with this, the point mutation of the Sp1 binding site decreased the promoter activity, and point mutation of the IRF-E site completely abolished the activity of the promoter (Figure 1D). These results are consistent with the observation that Sp1 stabilizes the BAF complex binding to target promoters .
Why do the cells take the trouble to make two proteins binding to the same site? We hypothesize that the TLR3 gene needs to be expressed at low level in the absence of viral infection and needs to be rapidly induced to high level in the presence of viral infection. Furthermore, the chromatin structure at the promoter should be prepared for rapid activation in response to viral infection. Based on the in vivo binding and knock-down results (Figure 2), we hypothesize that IRF2 may be the factor to prepare the chromatin structure and direct low level expression, and IRF1 may be the factor to direct highly induced expression.
Activation of the TLR3 gene by viral infection or polyI/polyC leads to the induction of the IFN-β gene, which is mediated by NF-κB and TLR3 . Our data that IRF2 is required for the basal and induced levels of TLR3 expression suggest that knocking down IRF2 may cripple the cellular response to polyI/polyC. To confirm this, HeLa cells, which were either transfected with a control vector or siIRF2, were treated with polyI/polyC. The induced expression of the IFN-β gene was abolished by knocking down IRF2 (Figure 3E), suggesting that IRF2 is a key molecule that controls the cellular antiviral activities.
Next we investigated whether IRF2 binding impacts histone modifications. Histone H3 is subject to extensive modifications including acetylation and methylation, which is correlation with transcriptional activation or repression . To test whether IRF2 facilitates histone modifications at the TLR3 promoter, we measured H3K4me3 and H3K9/K14ac using ChIP assays. Our data revealed that these modifications at the promoter region were dramatically decreased by knocking down IRF2, while no significant changes were detected at the exon II region of the TLR3 gene (Figure 4B, C). These data confirmed our hypothesis that IRF2 is essential for keeping an open chromatin structure at the TLR3 promoter.
IRF1/2 are known to recognize ISRE , which mediates the induction of IFN-α target genes by the ISGF3 complex. Since the BAF complex regulates most of the IFN-α target genes , IRF2 may serve as a general recruiter of the BAF complex to the IFN-α target genes. To test this idea, we analyzed IRF1 and IRF2 binding at several IFN-α inducible genes by ChIP assays. As shown in Figure 5B and C, both IRF1 and IRF2 bound to the promoters of TLR3, IFITM1, IFITM3, and STAT2. Interestingly, IRF2 disappeared from all of these promoters after IFN-α treatment. We assume that IRF2 was replaced by the ISGF3 complex at the IFITM1, IFITM3, and STAT2 promoters as suggested by the ChIP data summarized in Figure 5D. Similarly to the induction of TLR3, knocking down of IRF2 also significantly inhibited the expression of IFITM1, IFITM3, and STAT2 (Figure 5E). Therefore, these data define a general role of IRF2 to potentiate the induction of the IFN-α target genes in response to viral infection.
Recent studies suggested that epigenetic mechanisms play roles in controlling the function of IRF1 and IRF2 . In particular, histone acetyltransferases can directly enhance their transcriptional activity by modifying nucleosome or IRFs themselves [24,25]. Our previous studies suggested that the ATP-remodeling BAF complexes are required for maintaining the basal and induced expression of IFN-inducible genes . However, it is not clear what are the roles of the constitutively expressed IRFs in this process. Although IRF2 has been considered a transcriptional repressor and an antagonist of IRF1 [5,26], it has been difficult to explain certain phenotypes in mice associated with disruption of IRF1 and IRF2 [27,28]. In this study, our data argue against the notion that IRF2 is a repressor and thus antagonizes the activity of IRF1. Instead, we demonstrate that IRF1 is a strong transcription activator and IRF2 is a weak activator, which act at different stages of TLR3 activation. IRF2 serves two roles at the TLR3 promoter: 1) to recruits the BAF complex to prepare an open chromatin structure for rapid activation upon viral infection; 2) to maintain a basal level expression of the gene for the innate antiviral activity. IRF1 serves to rapidly activate the promoter by replacing IRF2 upon induction. However, if the chromatin structure at the promoter was not prepared, induction completely failed. The functional difference between IRF1 and IRF2 may arise from two mechanisms. One is the control of their expression: IRF2 is constitutively expressed while IRF1 is inducible by viral infection or interferon treatment, which results in different binding patterns during different stages of cellular antiviral activity. The other may reflect the differences of their activation domains: although IRF1’s activation domain can potently activate transcription, our data argue that only the activation domain of IRF2 can act as a pioneering factor to prepare chromatin for rapid transcription induction in response to stimulation. Therefore, our data in this report demonstrate that different members in a transcription factor family are made to meet different requirements for elaborate transcriptional regulation. The vertebrate organisms gain an extra level of transcriptional control by duplication of gene families.
Materials and methods
Constructs and antibodies
pREP4-TLR3pr-luc, and pGL3-TLR3pr-luc were constructed by cloning the PCR-amplified 509 bp fragment of the TLR3 promoter (from −509 to +1) into appropriate restriction sites in the pREP4-luc  and pGL3 vectors. The promoter deletion reporter constructs were generated by cloning the corresponding PCR fragments in the pREP4-luc vector. The reporter constructs with point mutations of the Sp1 and IRF-E motifs were generated from the 509-bp promoter construct using a Stratagene mutagenesis kit. The shRNA constructs for IRF1 and IRF2 were generated by inserting the corresponding cDNA sequences into the pREP4-puro vector. pREP4-U6-shBAF47 construct was described previously .
The oligonucleotides for Sp1 and IRF-E site mutations are listed below:
The Oligonucleotides used for shRNA constructs are listed below:
Antibodies used in this study are from the following sources
Anti-IRF1(sc-497X; Santa Cruz), anti-IRF2(sc-498X; Santa Cruz), anti-TLR3(H-125; Santa Cruz), anti-BRG1(sc-17796X; Santa Cruz), anti-hBRM(sc-6450; Santa Cruz), anti-BAF47(home made see paper), anti-p48(sc-496; Santa Cruz), anti-Stat1(06–502; upstate), anti-Stat2(sc-346-G; Santa Cruz), anti-PloII (sc-899X; Santa Cruz), anti-CBP(ab10489; abcam), anti-H3K4me3(17–614; Millipore), anti-H3k9ac/14 ac (ab4441;abcam), anti-H3(ab1791-100; abcam).
Cell culture and transfection
SW-13 cells and HeLa cells were maintained in DMEM supplemented with 10% fatal calf serum and 1% penicillin-streptomycin. The THP-1 cells were maintained in ATCC-formulated RPMI-1640 Medium (Catalog No. 30–2001) supplemented with 10% fatal calf serum and 1% penicillin-streptomycin. Transfections of SW-13, HeLa cells, and THP-1 cells were performed using Superfect (Qiagen) as instructed by manufacture. The cells were selected in 1 μg/ml puromycin for 48 hours before stimulation with 500 Units/ml IFN-α for 12 hours for chromatin immunoprecipitation and TLR3 gene expression analysis.
TaqMan® Gene Expression Assays for TLR3 and IFN-β gene were ordered from ABI (Cat. # Hs01551078_m1; Cat. # Hs01077958_s1). RT-PCR analyses were performed as described previously , by using total RNAs isolated from HeLa, SW-13, and THP-1 cells.
Forward primer: 5′-ACAGTGTGGCGATTCCTCAAG-3′, Reverse primer: 5′-GTAATGGGATTGCTGGGTCAA-3′, Probe: 5′-ATCTAGAACCAGAAATACTG-3′ were used.
This work was supported by Division of Intramural Research, National Heart, Lung and Blood Institute, National Institutes of Health. G.R. was supported by a Ph.D. Student Fellowship from the China State Scholarship Fund (2012–2014).
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