Oncogenic IRFs Provide a Survival Advantage for Epstein-Barr Virus- or Human T-Cell Leukemia Virus Type 1-Transformed Cells through Induction of BIC Expression

Abstract

miR-155, processed from the B-cell integration cluster (BIC), is one of the few well-studied microRNAs (miRNAs) and is involved in both innate immunity and tumorigenesis. BIC/miR-155 is induced by distinct signaling pathways, but little is known about the underlying mechanisms. We have identified two conserved potential interferon (IFN) regulatory factor (IRF)-binding/interferon-stimulated response element motifs in the Bic gene promoter. Two oncogenic IRFs, IRF4 and -7, in addition to some other members of the family, bind to and significantly transactivate the Bic promoter. Correspondingly, the endogenous levels of IRF4 and -7 are correlated with that of the BIC transcript in Epstein-Barr virus (EBV)-transformed cells. However, RNA interference studies have shown that depletion of IRF4, rather than of IRF7, dramatically decreases the endogenous level of BIC by up to 70% in EBV- or human T-cell leukemia virus type 1 (HTLV1)-transformed cell lines and results in apoptosis and reduction of proliferation rates that are restored by transient expression of miR-155. Moreover, the endogenous levels of the miR-155 target, SHIP1, are consistently elevated in EBV- and HTLV1-transformed cell lines stably expressing shIRF4. In contrast, transient expression of IRF4 decreases the SHIP1 level in EBV-negative B cells. Furthermore, the level of IRF4 mRNA is significantly correlated with that of BIC in adult T-cell lymphoma/leukemia (ATLL) tumors. These results show that IRF4 plays an important role in the regulation of BIC in the context of EBV and HTLV1 infection. Our findings have identified Bic as the first miRNA-encoding gene for IRFs and provide evidence for a novel molecular mechanism underlying the IRF/BIC pathway in viral oncogenesis.

INTRODUCTION

MicroRNAs (miRNAs), like transcription factors, function as regulators of gene expression and are expressed in several eukaryotes as well as in some viruses, particularly in the family of herpesviruses (17). Study of miRNAs has exploded since their relatively recent discovery, and nearly 700 miRNA genes have been identified in humans to date (17). miRNAs have been shown to be key regulators of genes involved in innate immunity, cell growth, differentiation, tumorigenesis, and development acting at the posttranscriptional level (7, 11, 17, 20, 43, 45, 49). Different from small interfering RNA (siRNA), miRNAs inhibit the translation of select groups of mRNA transcripts containing imperfect annealing sequences in their 3′-untranslated regions (3′-UTRs) and less frequently through other regions of the transcript. Since miRNA profiles are different between normal and cancer cells, miRNA signatures can be used for diagnosis as well as prognosis of human malignancies (3).

miR-155 is an evolutionarily conserved miRNA which plays important roles in innate immunity (49, 72), and is the first oncogenic miRNA (oncomiR) shown to have increased expression in various types of cancers including lymphomas such as Hodgkin lymphoma and posttransplant lymphoproliferative disease (PTLD) (9, 28, 64, 70), breast cancer, leukemia, pancreatic cancer, and lung cancer (7, 15). miR-155 has also been shown to play a critical role in lymphocyte activation in vivo (53, 67). Accumulating evidence has revealed high levels of miR-155 in Epstein-Barr virus (EBV) latency 3, but not in latency 1 (4, 26, 28, 77), indicating that miR-155 expression is associated with EBV latency. The importance of miR-155 in cancers is underscored by the fact that at least two oncogenic herpesviruses, Kaposi's sarcoma-associated herpesvirus (KSHV) (19, 59) and Marek's disease virus (81), encode functional orthologs of miR-155. The KSHV ortholog miR-K12-11 also shares 100% seed sequence (first 8 nucleotides [nt]) homology with human miR-155 (59).

miR-155 is processed from a primary transcript, B-cell integration cluster (BIC), which can be processed via the intermediate precursor miR-155 (pre-miR-155) to the mature 22-nt miR-155 (63). BIC cDNAs from human, mouse, and chicken have 78% identity over 138 nucleotides. miR-155 preferentially targets SHIP1 (42), and also targets IKKε (19, 32, 69), FADD, RIP1 (69), P53BP1 (18), hypoxia-inducible factor 1 (HIF1), TAB2, PU.1, MyD88 (66), SMAD5 (50), FOXO3a (29), XAF1 (19), c-Myb (25), c-Maf (a transcription factor that promotes interleukin 4 [IL-4] production) (53), activation-induced cytidine deaminase (AID), and SOCS-1 (33), among others (12, 53, 68).

IRFs (interferon regulatory factors) are a family of transcription factors comprising 9 members in mammalian cells. The IRF family plays critical roles in multiple facets of host defense systems (22, 54). IRFs share significant homology within the conserved N-terminal DNA-binding domain (DBD), which consists of a signature tryptophan pentad (6). The consensus DNA for IRF recognition includes at least two GAAA repeats (21, 62), whereas other studies have implied that IRFs may have broader flexible binding capacities to the consensus sequence 5′-AANNGAAA-3′ (6, 10, 14, 47). The C termini of IRFs differ from one another, and this difference confers on each member distinct roles in regulation of tumorigenesis, cell growth, differentiation, and myeloid cell development (1, 41, 54, 62, 65). Among IRFs, IRF2, -4, and -7 have oncogenic potentials (41). IRF4 is overexpressed in EBV-transformed cells (73) and in multiple myeloma (24, 57), as well as in human T-cell leukemia virus type 1 (HTLV1)-infected cell lines and associated adult T-cell lymphoma/leukemia (ATLL) (51, 58, 61, 74). IRF7 has been shown to be associated with EBV latency and is overexpressed in a subset of EBV-positive and -negative lymphomas/leukemias (41).

BIC/miR-155 is induced by Toll-like receptor (TLR) signaling, tumor necrosis factor alpha (TNF-α), beta interferon (IFN-β), IFN-γ, EBV LMP1, LMP2A (8), and B-cell receptor (BCR) engagement. However, little is known about the mechanisms whereby miR-155 is regulated by these distinct signaling pathways (16). Many TLR signaling pathways activate IRFs (13, 30, 36, 52). EBV LMP1, a pleiotropic tumor necrosis factor receptor (TNFR) superfamily member (27), also activates IRF7 (23, 37, 60) and likely IRF4 (73). Type I IFNs (IFN-α/β) trigger signals through the IRF9-containing ISGF3 complex. Thus, specific IRFs are selectively involved in all these pathways. We therefore hypothesize that IRFs are involved in BIC induction by these signaling pathways.

In this study, we investigate whether the oncogenic IRFs, IRF4 and -7, that are overexpressed in EBV- and HTLV1-transformed cells (2, 34, 73) are able to regulate BIC expression in the context of EBV and HTLV1 infection. Our results show for the first time that oncogenic IRF4 plays an important role in induction of BIC in both EBV- and HTLV1-transformed cells and mediates cell proliferation at least in part via regulation of BIC. These novel findings have implications not only for viral oncogenesis but likely also for innate immune mechanisms governed by IRFs/BIC.

MATERIALS AND METHODS

Cell lines.

Sav I, Sav III, JiJoye, P3HR1, and IB4 are EBV-transformed B-cell lines. BJAB is an EBV-negative B-cell line. TLM01 is a Tax-positive HTLV1-transformed cell line. All B-cell lines were cultured in RPMI 1640 medium plus 10% fetal bovine serum (FBS) and antibiotics. 293 and HeLa cells were cultured with Dulbecco's modified Eagle's medium (DMEM) medium plus 10% FBS and antibiotics.

Plasmids, reagents, and antibodies.

Flag-tagged IRFs were cloned in the pCMV2-Flag vector. pGL3/BICp(−1494/+228)-Luc and pMSCV-miR-155 were kindly provided by Erik Flemington (77, 78). Mutants of pGL3/BICp(−1494/+228)-Luc were made by site-directed PCR. Ifnα4-Luc was described in our previous work (40). Anti-Flag M2 (Sigma), anti-LMP1 CS1-4 (Dako), anti-IRF4 H140 (Santa Cruz), anti-IRF7 H246 (Santa Cruz), anti-SHIP1 P290 (Cell Signaling), and anti-β-actin AC15 (Sigma) were used for Western blotting. Anti-IRF4 (Cell Signaling) and anti-IRF7 (Invitrogen) were purchased for chromatin immunoprecipitation (ChIP). The miR-155 inhibitor was purchased from Applied Biosystems.

Human ATLL sample collection.

Twenty-three primary ATLL tumor samples were chosen for this study (Table 1). The study was reviewed and approved by the institutional review boards of the University of Miami.

Table 1.

ATLL sample information

ATLL register	Type (as per Shimoyama criteria)	Sex	Age (yr)	Notes
ATLL1	Acute	M	47
ATLL2	Acute	M	37
ATLL12	Acute	F	66
ATLL13	Acute	F	68
ATLL17	Acute	F	27
ATLL20a	Acute	F	66
ATLL23	Acute	M	69
ATLL40	Acute	F	35
ATLL44	Acute	M	53	HIV+
ATLL47	Acute	M	33
ATLL54	Acute	F	44
ATLL81	Acute	F	43
ATLL7	Unfavorable chronic	F	77
BRA371	Chronic	M	21
ATLL30a	Lymphomatous	F	49
ATLL32	Lymphomatous	M	42	HIV+
Bra194	Lymphomatous	M	60
CTCL1	Cutaneous T-cell lymphoma	M	52
BRA367	Smoldering	M	35	Exclude. Pretreated/relapse

^aEach has a duplicate for this study.

RNA isolation and reverse transcriptase reactions.

Total RNA was isolated using the RNeasy minikit (Qiagen) according to the manufacturer's protocols. The eluted RNA was subjected to reverse transcriptase reactions, which were performed with the use of the GoScript reverse transcription (RT) kit (Promega) following the manufacturer's instructions.

Real-time quantitative PCR.

Quantitative PCR (qPCR) was performed with the use of SYBR green (Applied Biosystems) on an ABI 7300 real-time PCR system with SDS version 1.3.1. All reactions were run in duplicate. Mean cycle threshold (C_T) values were normalized to GAPDH (glyceraldehyde-3-phosphate dehydrogenase), yielding a normalized C_T (ΔC_T). The ΔΔC_T value was calculated by subtracting respective control from the ΔC_T, and expression level was then calculated by 2 raised to the power of the respective −ΔΔC_T value. Results are the average ± standard error (SE) of duplicates for each sample. Primers for qPCR include the following: BIC forward (5′-ACCAGAGACCTTACCTGTCACCTT-3′) and reverse (5′-GGCATAAAGAATTTAAACCACAGATTT-3′) (44); LMP1 forward (5′-CTTCAGAAGAGACCTTCTCT-3′) and reverse (5′-ACAATGCCTGTCCGTGCAAA-3′); and GAPDH forward (5′-ATGACATCAAGAAGGTGGTG-3′) and reverse (5′-CATACCAGGAAATGAGCTTG-3′).

Statistical analysis.

The tumor sample with the lowest BIC and IRF4 expression was set as the control. Three (14%) of the other 22 samples expressed lower levels of IRF7 than the control. For these 22 samples, Pearson correlation coefficients were computed to test the association between IRF4 and BIC, or between IRF7 and BIC. Log transformations of relative expression levels were used for descriptive statistics for IRF4 and -7. For BIC, the square roots of the log of relative expression levels were used.

Promoter-reporter assay.

Cells were transfected with expression plasmids as indicated together with IFNα4p-Luc and Renilla as the internal transfection control. Empty vector was used to equalize the total amounts of DNA in all transfections. Cells were collected 24 h after transfection. Luciferase activity was measured with equal amounts (10% of total for each sample) of protein lysates with the use of a dual luciferase assay kit (Promega). Results are the averages ± standard error (SE) of duplicates for each sample. Results obtained consistently from at least three independent experiments are shown. The ability of the vector controls to activate promoter was set to 1.

Chromosome immunoprecipitation assay.

A chromosome immunoprecipitation assay was performed with the use of ChIP-IT Express enzymatic kit (Active Motif) following the manufacturers' instructions. Briefly, 293T cells were transfected with Flag-IRFs using the CalPhos mammalian transfection kit (BD Clontech). Cells were harvested 48 h after transfection. For endogenous IRF proteins, IB4 cells, which express high levels of IRF4 and -7, were used. For cross-linking, cells were treated with 1% formaldehyde solution for 10 min, and the reaction was stopped by 125 mM glycine buffer. After lysis, nuclei were isolated with a Dounce homogenizer and digested in enzymatic shearing cocktail for 15 min. IP was performed overnight at 4°C with Flag antibody (Sigma) for 293T cells or rabbit monoclonal IRF4 (Cell Signaling) or IRF7 (Invitrogen) antibodies for IB4 cells. After washing with ChIP buffers 1 and 2, beads were eluted with buffer AM2. Cross-linking was reversed at 65°C, and samples were subjected to proteinase K digestion at 37°C for 1 h before PCR.

RNA interference (RNAi) and small hairpin RNA (shRNA) construction.

For shIRF4, oligonucleotides containing the target sequence (located at the 3′-UTR of the human IRF4 gene) or scramble control were synthesized and cloned into the inducible pTRIPz-Turbo-RFP vector (Thermo Scientific). Induction of shIRF4 was achieved by 1 μg/ml doxycycline (DOX). For shIRF7, a pool of four shIRF7 constructs (and control) in pGenClip (Promega) were purchased from SABiosciences. Knockdown efficiencies and specificities were verified by Western blotting. These shIRF7 and shIRF4 constructs did not knock down other IRFs. The one with the highest knockdown efficiency in the shIRF7 pool was chosen for this study.

Retrovirus-mediated transfection.

To produce retrovirus expressing shIRFs, 293T cells in 100-mm dishes were cotransfected with 7 μg of pVSV-G and pGAG-pol and 7 μg of either shIRFs or shControl by using CalPhos mammalian transfection kit (BD Biosciences). Retrovirus packing was performed following the manufacturer's instructions (Thermo Scientific). Supernatants containing retroviruses were collected 48 h later. For retroviral infections, cells were incubated with retroviruses in the presence of Polybrene (8 μg/ml). Infected cells were sorted by fluorescence-activated cell sorting (FACS) and/or were selected in puromycin (for shIRF7) or hygromycin (for shIRF4) for 2 weeks.

MTT proliferation assay.

The 3-(4,5-methylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) growth assay was used to measure the cell proliferation rate by using a CellTiter 96 nonradioactive cell proliferation assay kit (Promega) according to the manufacturer's recommendations. Basically, 5 × 10⁴ live cells were placed into 85 μl of RPMI medium plus 10% FBS in a 96-well plate. Retrovirus expressing green fluorescent protein (GFP) or miR-155 was incubated for 0 to 4 days. Dye solution (15 μl) was added to each well. The cells were incubated at 37°C for 4 h. Solubilization solution/stop mix (100 μl) was added, and cells were incubated for another hour. The absorbance at a wavelength of 562 nm was recorded using a 96-well plate reader. Results are the averages ± standard error (SE) of duplicates for each sample. Results obtained consistently from at least three independent experiments are shown. Proliferation rates at day 0 were set to 100%.

Apoptosis assay.

Apoptosis was assessed by measuring cleaved caspase activity with the use of a Caspase-Glo 3/7 assay kit (Promega) and by assessing DNA fragmentation. DNA was extracted from cells by using a DNA extraction kit (Qiagen), followed by proteinase K and RNase A digestion. DNA was purified by using an E.Z.N.A. cycle pure kit (Omega) before gel running.

RESULTS

Identification of IRF-binding element/interferon-stimulated response element (IRF-E/ISRE) in the Bic promoter.

The human Bic gene promoter was recently characterized by 5′ rapid amplification of cDNA ends (5-RACE), and the major start site of the transcript was found to be located at nucleotide 12596314 of human chromosome 21 (78). Consequently, the promoter fragment extending from −1494 to +228 relative to the start site was cloned, and the transcript of the Bic gene has been analyzed previously (78).

Using the combination of TRANSFAC, TESS, and Discover Studio Gene (Invitrogen) programs, we have identified two potential IRF-binding/interferon-stimulated response element motifs close to the start site of the human Bic promoter, along with several other potential important transcription factor-binding sites spanning −613/+228 (Fig. 1A, top). The core sequence for IRF binding 5′-AANNGAAA-3′ is conserved in both human and mouse Bic promoter (Fig. 1A, bottom).

Fig. 1.

Activation of and binding to the Bic promoter by IRFs. (A) Identification of potential transcription factor binding sites in the Bic promoter. The conserved core IRF-binding sequences are shown in underlined fonts. (B to D) HeLa cells in 12-well plates were transfected with 0.2 μg Flag-tagged IRF expression plasmids, 40 ng pGL3/BICp (−613/+228)-Luc (B), IFNα4p-Luc (C), or pGL3/BICp (−613/+228)-Luc mutants (D), and 10 ng Renilla. A dual luciferase assay was performed. Results are the averages ± standard error (SE) of duplicates. Representative results from at least three independent experiments are shown. The ability of the vector control to activate the promoter construct was set to 1. ISREm, the ISRE (GTTTCCTTTTC) was mutated to GccTCCTccTC; IRFEm, The IRFE (GAAAGGGAAAGG) was mutated to GAggGGGAggGG; DM, double mutant with ISREm and IRFEm. (E) 293T cells in 100-mm dishes were transfected with 5 μg of Flag-IRFs or empty vector. Cells were harvested 48 h after transfection. Cross-linking and ChIP assays were performed as described in Materials and Methods. The samples used for IP with IgG, beads, and nonspecific antibody (anti-β-actin) were prepared from Flag-IRF7-transfected cells. (F) IB4 cells (1 × 10⁷) were used for each ChIP. Anti-IRF4 (1: 25; Cell Signaling) and anti-IRF7 (2 μg; Invitrogen) and Protein A magnetic Dynalbeads (Invitrogen) were used for immunoprecipitation. Eluted DNA (3 μl from 100 μl total) was subjected for PCR (30 cycles). Inputs are 5% of DNA from 3% of total cell lysates. Primers used for PCR included the following: ISRE forward (5′-CCCCTCCAGCCGACTG-3′) and reverse (5′-AACACACGCCGTGTAC-3′), IRF-E forward (5′-CACGGCGTGTGTTGAGAG-3′) and reverse (5′-CTGCCTGTTCTTGGAACC-3′), and control (β-actin promoter) forward (5′-CCAACAAAGCACTGTGG-3′) and reverse (5′-GGGCGAAGGCAACGC-3′).

Regulation of Bic promoter activity by IRFs.

The pGL3/BICp(−1494/+228)-Luc construct was kindly provided by Erik Flemington (77, 78). To minimize the background activity, we have shortened the insert promoter fragment by digesting with the restriction enzyme KpnI to 640 bp (−613/+228), which contains the two potential IRF-E/ISRE and the potential IFN-γ-activated site (GAS) motifs (Fig. 1A, top).

As shown in Fig. 1B, promoter-reporter assay results show that several of the tested IRFs significantly activated the Bic promoter construct, pGL3/BICp(−613/+228)-Luc. Notably, the promoter may be preferably activated by the oncogenic IRFs, IRF4 and -7, and by IRF1 and -3, which play critical roles in induction of type I IFNs and IFN-stimulated genes (ISGs). As a positive control, we performed parallel experiments with an Ifna4 promoter construct, and the results show that the Bic and Ifna4 promoter constructs have similar activation patterns by the IRFs, although the Ifna4 promoter construct has a much greater ability to be activated by these IRFs (Fig. 1C). The results suggest that these IRFs may upregulate BIC expression in distinct biological contexts.

To check if ISRE and IRFE are responsible for the activation, we mutated each of these two sites or both and compared their abilities to be activated by IRFs with the wild-type promoter construct. Figure 1D shows that mutation in either of these two sites (ISREm or IRFEm) significantly reduces the promoter activity transactivated by an IRF7 mutant (IRF7m), which is a constitutively active IRF7 form with phosphomimetic substitutions on Ser477 and 479 (31). However, the double mutant (DM) with simultaneous mutation on both sites has much less activity stimulated by IRFs. Thus, we conclude that both of these two motifs are functional and should be responsive to BIC regulation by IRFs.

Binding of IRFs to Bic IRF-E/ISRE in vivo.

To check if IRFs can bind to the Bic ISRE/IRFE, we performed a ChIP assay in 293T cells with the use of ChIP-IT express enzymatic kit from Active Motif. The ChIP results (Fig. 1E) show that all the tested IRFs (IRF1, -2, -3, -4, -5, -7, and -9) can bind to the ISRE/IRFE. They may have different binding abilities, but each IRF has binding ability similar to those of both the ISRE and IRFE. Further, to check whether endogenous IRFs bind to the Bic promoter, we performed a ChIP assay using IB4 cells in which both IRF4 and -7 are expressed at remarkable levels. Anti-IRF4 and anti-IRF7 antibodies were used for this purpose. Figure 1F shows that endogenous IRF4 and -7 bind to the Bic ISRE and IRFE in vivo. Due to potential affinity differences of the used antibodies, the ChIP results shown here are not for quantification purposes and are not comparable to the ChIP results obtained from the Flag antibody (Fig. 1E).

Association of BIC transcript with IRFs in EBV latency.

In EBV latency 3, IRF4 and -7 are highly expressed and activated (37, 73), but in latency 1, both IRFs are expressed at very low levels, if at all. Thus, it is very interesting to investigate whether expression levels of these two IRFs and BIC are correlated in EBV latency programs.

Although a correlation between BIC transcript and mature miR-155 levels has been reported in many cases, the expression of mature miR-155 is likely regulated at both transcriptional and posttranscriptional levels, and the ratio of BIC transcript and mature miR-155 is not always constant (68). Therefore, for our purpose of transcriptional regulation of the Bic gene, we assayed BIC transcript rather than mature miR-155.

Sav I and Sav III are EBV-positive B-cell lines with type 1 and 3 latency programs, respectively. Western blotting results show that Sav III expresses a very high level of LMP1 and also high levels of IRF4, -7, and -2, but in Sav I cell line LMP1 and IRF4 were not detected and the levels of IRF7 and -2 are also much lower (Fig. 2A). These results are consistent with previous reports that both IRF4 (34, 73) and IRF7 (79) are induced by LMP1. In addition, IRF5 is also expressed at a higher level in type 3 latency than in type 1 latency (34, 39, 76). Correspondingly, BIC transcript was not detected by real-time PCR in Sav I but was detected at a high level in Sav III (Fig. 2B). We also performed testing in JiJoye and P3HR1 cell lines. P3HR1 was derived from JiJoye (type 3 latency) but has very low level of LMP1 due to the deletion of the EBNA2 gene. As shown in Fig. 2C, LMP1 in P3HR1 cells was not detected by Western blotting, but a low level of LMP1 was detected by real-time PCR (Fig. 2D). IRF4 and -7 were not detected in P3HR1 by Western blotting, but both IRF4 and -7 were detected at remarkable levels in JiJoye cells (Fig. 2C). These results confirm that both IRF4 (34, 73) and IRF7 (79) are induced by LMP1. Correspondingly, BIC transcript was not detected in P3HR1 cells with real-time PCR under our conditions, but BIC is expressed at a high level in JiJoye cells (Fig. 2D). These results indicate that BIC transcript is correlated with IRF4 and -7 in EBV latency and is likely induced by LMP1 at least in part through the contribution of IRF4 and -7.

Fig. 2.

Association of oncogenic IRFs with BIC in EBV latency. LMP1, IRF2, IRF4, and IRF7 were detected by Western blotting, and BIC was detected by real-time PCR. (A and B) Sav I and Sav III are EBV-positive cell lines with type 1 and 3 latency programs, respectively. (C and D) JiJoye and P3HR1 cell lines are EBV positive with type 3 latency. P3HR1 was derived from JiJoye but lacks the EBNA2 gene, which encodes the EBNA2 protein, a potent transactivator of the LMP1 promoter.

Transient expression of IRF4 in EBV-negative B cells increases BIC transcript.

Next, we transiently expressed IRF4 or -7 in EBV-negative BJAB cells to see whether BIC transcript could be elevated. As shown in Fig. 3A, BJAB cells transfected with control vector did not have a detectable IRF4 level and have a very low level of BIC, but transient expression of IRF4 increased the BIC transcript by 4.5-fold. However, transiently expressed IRF7 had little if any effect on BIC transcription (data not shown). These data suggest that IRF4, rather than IRF7, upregulates BIC expression in B cells.

Fig. 3.

Regulation of BIC by IRFs. (A) BJAB is an EBV-negative cell line. BJAB cells were transfected with IRF4-expressing construct. After 48 h, cells were collected for Western blotting and real-time PCR analyses. (B and C) JiJoye cells were infected with retrovirus expressing shIRF4 or shIRF7 (or shControls). Cells were sorted by FACS, and cell lines stably expressing shIRF4 or shIRF7 or shControls were established. Induction of shIRF4 was achieved by DOX. Knockdown of IRF4 and -7 and decrease of BIC are shown in the stable cell lines. (D) TLM01 is a HTLV1-positive cell line that expresses a high level of endogenous IRF4 (bottom panel). TLM01 cells were infected with shIRF4 (or Scramble control)-expressing retrovirus, and stable TLM01-shIRF4 and TLM01-Scramble cell lines were established. Knockdown of IRF4 and BIC levels are shown in the stable cell lines treated with 1 μg/ml DOX for 3 days. The BIC levels without DOX treatment are also shown.

Depletion of IRF4 decreases BIC transcript.

Further, we tried to knock down IRF4 or -7 expression in EBV-positive JiJoye cells. An shIRF4, which specifically targets the human IRF4 gene 3′-untranslated region, and a scrambled control sequence, were cloned in the inducible pTRIPZ-TurboRFP shRNA vector. Cells were infected with retrovirus expressing either shIRF4 or scramble shRNA. Infected cells were sorted by fluorescence-activated cell sorting (FACS), and stable cell lines were established. shIRF4 expression was induced by 1 μg/ml doxycycline (DOX). The knockdown efficiency was up to 90% by day 3, as verified by Western blotting (Fig. 3B, right). Expression of this shIRF4 did not affect the level of IRF7 (data not shown). Depletion of IRF4 resulted in an approximately 50% decrease in the endogenous BIC level (Fig. 3B). However, depletion of IRF7 resulted in only an approximately 20% decrease in the level of BIC (Fig. 3C).

Moreover, we performed siRNA study in the HTLV1-transformed cell line TLM01. HTLV1 is an oncogenic retrovirus and is associated with ATLL. TLM01 stable cell lines TLM01-shIRF4 and TLM01-Scramble were established as described above. Knockdown efficiency (up to 90%) was verified by Western blotting after induction by 1 μg/ml DOX for 3 days (Fig. 3D, bottom). A change in the IRF4 level was not observed before DOX treatment, as measured by real-time PCR (data not shown). Our results show that the endogenous level of IRF4 in HTLV1-infected cells is very high, consistent with the previous finding that IRF4 is overexpressed in HTLV1-infected cells (51). Depletion of IRF4 significantly decreased the BIC level by about 70% (Fig. 3D, top). Further, decreased BIC was restored by transient expression of IRF4 in TLM01-shIRF4 stable cells (data not shown).

Together, these results from RNA interference studies show that IRF4 plays an important role in regulating BIC transcription in the context of oncogenic virus infection. IRF7 makes only a minor if any contribution to BIC expression in these settings.

Regulation of the miR-155 target SHIP1 by IRFs in cancer cell lines.

miR-155 preferentially targets SHIP1 (42). Since IRF4 significantly regulates BIC transcription, we were interested to check if IRF4 can indirectly regulate SHIP1 expression. First, we compared the endogenous levels of BIC and SHIP1 in P3HR1 and JiJoye cell lines. As shown in Fig. 4A, in P3HR1 cells, which do not have detectable IRF4, the level of SHIP1 is higher than that in JiJoye cells, which express a high level of IRF4. Further, depletion of IRF4 in JiJoye cells consistently increased the endogenous level of SHIP1 (Fig. 4B). In EBV-negative BJAB cells, transient expression of IRF4 consistently decreased the level of SHIP1 (Fig. 4C). Similarly, in HTLV1-infected TLM01 cells, SHIP1 expression was elevated after depletion of IRF4 (Fig. 4D). These results indicate that IRF4 negatively modulates endogenous SHIP1 level through upregulation of BIC expression in EBV- or HTLV1-infected cells.

Fig. 4.

Regulation of the miR-155 target SHIP1 by IRF4. (A) The endogenous levels of IRF4 and BIC are reversely correlated in P3HR1 and JiJoye cells. (B) JiJoye cells were infected with retrovirus expressing shIRF4 or Scramble, and stable cell lines were established. Stable cells were treated with 1 μg/ml DOX for 3 days, and knockdown of IRF4 and BIC levels are shown. (C) EBV-negative BJAB cells were transfected with an IRF4-expressing construct or with vector control. SHIP1 is decreased in IRF4-transfected cells. (D) BIC levels are shown in TLM01-Scramble and TLM01-shIRF4 stable cell lines treated with 1 μg/ml DOX for 3 days.

Correlation of BIC with IRFs in ATLL tumors.

miR-155 has been implicated in many cancers; for example, it is highly expressed in pediatric Burkitt lymphoma (9, 35) and accumulates in other human B-cell lymphomas including ATLL (9). We were therefore interested to check the correlation of IRF4 and -7 with BIC in tumor samples. Twenty-three ATLL tumor samples were selected, and the information of these samples was detailed in Table 1. Total RNAs were extracted from these samples, and real-time PCR was performed for BIC, IRF4, -7, and GAPDH. Pearson correlation coefficient statistical results show that BIC was associated with IRF4 (R² = 0.388, P = 0.002) (Fig. 5A), but there was no significant association between BIC and IRF7 (R² = 0.009, P = 0.669) (Fig. 5B). Furthermore, there was no significant association between IRF4 and -7 (data not shown). These results are consistent with our conclusion that IRF4, rather than IRF7, upregulates BIC transcription in the context of oncogenic virus infection.

Fig. 5.

Correlation between IRFs and BIC in ATLL tumors. Twenty-three ATLL tumor samples were subjected to real-time PCR analysis for expression of IRF4, IRF7, and BIC. RNA extraction and real-time PCR are described in detail in Materials and Methods. Pearson correlation coefficient analysis was performed, and log scale scatter plots of BIC and IRF4 (A) and IRF7 (B) with fitted regression are shown.

BIC contributes to IRF4-mediated cell proliferation.

Since IRF4 upregulates BIC transcription, we reasoned that BIC contributes to IRF4-mediated cell proliferation. We therefore compared the cell proliferation rates between TLM01-Scramble and TLM01-shIRF4 stable cell lines, with MTT cell proliferation assay. Consistent with a previous report (73), the depletion of IRF4 results in dramatic reduction in cell proliferation (Fig. 6A). We then checked if transient expression of miR-155 can restore cell proliferation. Cells were infected with retroviruses expressing miR-155 or GFP, and cell proliferation rates were measured for 5 days. As shown in Fig. 6A, miR-155 partially restored the cell proliferation reduced by shIRF4, and by day 4, restored the rate from 45% to 70%.

Fig. 6.

BIC contributes to IRF4-mediated cell proliferation. (A) TLM01-Scramble and TLM01-shIRF4 stable cells were treated with 1 μg/ml DOX and infected with retrovirus expression GFP or miR-155. (B) BJAB-IRF4 and BJAB-GFP (control) stable cells were transfected with the miR-155 inhibitor (anti-miR-155) or scramble control with the use of Lipofectamine (Invitrogen). Cell proliferation was monitored by MTT proliferation assay. (C and D) IB4 cells in 6-well plates were infected with a combination of pSuper-shIRF4 (or Scramble control) retrovirus and pMSCV-miR-155 (or GFP control) retrovirus. Cell apoptosis was assessed on day 4 by DNA fragmentation assay (C) and on day 0 and 4 by measuring caspase-3/7 activity (D).

We also used miR-155 inhibitor to block miR-155 induced by IRF4 in BJAB cells. As shown in Fig. 6B, BJAB-IRF4 stable cells showed increased cell proliferation compared to BJAB-GFP stable cells; however, in the presence of the miR-155 inhibitor, the ability of IRF4 to promote cell proliferation was significantly decreased.

In the meanwhile, apoptosis was evaluated by caspase-3 activity. Both IRF4 (73) and miR-155 (46) have been reported to block caspase-3 activity. Knockdown of IRF4 in IB4 cells resulted in considerable increases in caspase-3 activity and DNA fragmentation characteristic of apoptosis, which was partially reversed by transiently expressed miR-155 (Fig. 6C and D).

Together, these results strongly suggest that miR-155 participates in IRF4-mediated cell proliferation.

DISCUSSION

In this study, we have identified Bic as the first non-protein-encoding gene transcriptionally targeted by IRFs. miR-155 is an important oncomiR that is implicated in various malignancies of B-cell or myeloid origin (7, 9, 15, 28, 70) and is overexpressed in many types of cancers (55, 68). It is also a novel crucial regulator of innate immunity (49, 72), hematopoiesis, and lymphocyte homeostasis and tolerance (12). Thus, the study of its regulation is of paramount importance. Recently, it is found that the induction of miR-155 by EBV LMP1 and by BCR engagement is through the activation of NF-κB (16) and AP1 (77, 78). A more recent study has shown that MYB induces miR-155 in chronic lymphocytic leukemia (71), and another report has shown that the transcription factor FOXP3 induces the high level of miR-155 in regulatory T cells (33). In addition, miR-155 induced by TLR signaling is through MyD88- or TRIF-dependent pathways, and that induced by IFN signaling requires an autocrine pathway involving TNF-α (44). Our results show that IRF4, which has oncogenic properties (56), induces BIC expression in the context of EBV and HTLV1 infection. Other IRFs that are important players in the production of type I IFNs, including at least IRF1, -3, and -7, also transactivate the miR-155 promoter construct, suggesting that they may play a role in induction of miR-155 in antiviral immunity.

IRF7, -4, and -2, three IRFs with oncogenic properties, are associated with EBV latency and may contribute to EBV oncogenesis. Overwhelming evidence also shows that IRF4 is overexpressed in HTLV1-infected cells and associated tumors (51, 58, 61, 74), suggesting that IRF4 may play a key role in HTLV1 tumorigenesis. Regulation of IRF4 expression in the context of HTLV1 infection is complicated. It is induced by HTLV1 in a Tax-independent manner in primary ATLL (61) but is induced by Tax in cell culture (51). IRF4 can also be induced by c-Rel or by other undefined cellular pathways in the absence of c-Rel (51). Nevertheless, the mechanisms of action of these IRFs in EBV or HTLV1 oncogenesis are not clear. As transcription factors, they may exert this function through transcriptional regulation of a set of genes involved in cellular growth and proliferation. However, only a very limited number of such genes have been identified so far. For example, we have shown that LMP1 is upregulated by IRF7 in the context of EBV infection (38). The positive regulatory circuit between IRF7 and LMP1 may potentiate the oncogenic effects of both factors and is negatively modulated by IRF5 (39), which in the context of EBV infection exists at least two natural variants, including the truncation mutant V12 (34) and the point mutant IRF5(A68P) (76). Both function as dominant negative mutants. As for IRF4, c-Myc, among other potential oncogenes, is a target for IRF4 in activated B cells and myeloma (57). Our present study has identified another important oncogene, Bic, for IRF4 in lymphoma/leukemia.

IRFs regulate many protein-encoding genes involved in immune responses, oncogenesis, and cell development and differentiation. For example, IRF7 is a multifunctional transcription factor which is the key regulator of type I IFNs and also regulates LMP1, transporter associated with antigen processing 2 (TAP2), and BamH I-A rightward transcript (BART) P1 promoter in EBV latency (5, 80), as well as potentially participates in many other cellular processes such as cell differentiation and autoimmune diseases (41). However, until now, no report has shown their regulation of a non-protein-encoding gene in any of these processes. This study uncovers the intersection between two major and quite diverse modes of regulation of these fundamental functions.

Although the oncogenic IRFs, as well as IRF5 (34, 39, 76), are associated with EBV latency, little is known about their functional interactions. We have shown IRF5 is physically associated with IRF7 and negatively regulates the IRF7/LMP1 regulatory circuit (39). Further investigation is needed to see if these oncogenic IRFs exhibit any cooperation in the regulation of BIC expression and viral oncogenesis.

miR-155 plays important roles in both immunity and cancer. The underlying mechanisms governed by miR-155 in these distinct settings may have overlaps, and therefore miR-155 may serve as a crucial link between immunity and cancer. In fact, the primary target of miR-155 in antiviral immunity, SHIP1 (42), is also a target for miR-155 in cancer (48, 75) and is shown here to be regulated indirectly by IRF4 via BIC (Fig. 4). A comprehensive comparison of miR-155 targetomes in immunity and cancer will be very helpful in understanding the interaction between immunity and cancer mediated by miR-155.

Our long-term goal is to provide evidence, for the first time, for a novel molecular mechanism underlying the interaction between the IRF4/miR-155 pathway and EBV/HTLV1 from aspects of oncogenesis as well as immunity in which both IRFs and miR-155 are important players. This is pivotal for understanding how the interaction between EBV/HTLV1 and the host immune system leads to viral oncogenesis and will provide the basis for the identification of molecular targets for therapeutic interventions.