Assessing Interferon Regulatory Factor 4 Complex Formation: Differential Behavior of Homocomplexes Versus Heterocomplexes Induced by Mutations.

Interferon regulatory factor 4 (IRF4) is a crucial transcription factor that plays a vital role in lymphocyte development, including in the fate-determining steps in terminal differentiation. It is also implicated in the development of lymphoid tumors such as multiple myeloma and adult T-cell leukemia. IRF4 can form a homodimer and multiple heterocomplexes with other transcription factors such as purine-rich box1 and activator protein 1. Each protein complex binds to specific DNA sequences to regulate a distinct set of genes. However, the precise relationship among these complex formations remains unclear. Herein, we investigated the abilities of IRF4 proteins with functional mutations in the IRF-association domain and autoinhibitory region to form complexes using luciferase reporter assays. The assays allowed us to selectively assess the activity of each complex. Our results revealed that certain IRF-association domain mutants, previously known to have impaired heterocomplex formation, maintained or even enhanced homodimer activity. This discrepancy suggests that the mutated amino acid residues selectively influence homodimer activity. Conversely, a phosphomimetic serine mutation in the autoinhibitory region displayed strong activating effects in all complexes. Furthermore, we observed that partner proteins involved in heterocomplex formation could disrupt the activity of the homodimer, suggesting a potential competition between homocomplexes and heterocomplexes. Our findings provide new insights into the mechanistic function of IRF4.

DBS is activated by EPHB2/SRC signaling-mediated tyrosine phosphorylation in HEK293 cells.

It is well known that Rho family small GTPases (Rho GTPase) has a role of molecular switch in intracellular signal transduction. The switch cycle between GTP-bound and GDP-bound state of Rho GTPase regulates various cell responses such as gene transcription, cytoskeletal rearrangements, and vesicular trafficking. Rho GTPase-specific guanine nucleotide exchange factors (RhoGEFs) are regulated by various extracellular stimuli and activates Rho GTPase such as RhoA, Rac1, and Cdc42. The molecular mechanisms that regulate RhoGEFs are poorly understood. Our studies reveal that Dbl's big sister (DBS), a RhoGEF for Cdc42 and RhoA, is phosphorylated at least on tyrosine residues at 479, 660, 727, and 926 upon stimulation by SRC signaling and that the phosphorylation at Tyr-660 is particularly critical for the serum response factor (SRF)-dependent transcriptional activation of DBS by Ephrin type-B receptor 2 (EPHB2)/SRC signaling. In addition, our studies also reveal that the phosphorylation of Tyr-479 and Tyr-660 on DBS leads to the actin cytoskeletal reorganization by EPHB2/SRC signaling. These findings are thought to be useful for understanding pathological conditions related to DBS such as cancer and non-syndromic autism in future.

The interaction between PLEKHG2 and ABL1 suppresses cell growth via the NF-κB signaling pathway in HEK293 cells.

The Rho family small GTPases mediate cell responses through actin cytoskeletal rearrangement. We previously reported that PLEKHG2, a Rho-specific guanine nucleotide exchange factor, is regulated via interaction with several proteins. We found that PLEKHG2 interacted with non-receptor tyrosine kinase ABL1, but the cellular function remains unclear. Here, we show that the interaction between PLEKHG2 and ABL1 attenuated the PLEKHG2-induced serum response element-dependent gene transcription in a tyrosine phosphorylation-independent manner. PLEKHG2 and ABL1 were co-localized and accumulated within cells co-expressing PLEKHG2 and ABL1. The cellular fractionation analysis suggested that the accumulation involved actin cytoskeletal reorganization. We also revealed that the co-expression of PLEKHG2 with ABL1, but not BCR-ABL, suppressed cell growth and synergistically enhanced NF-κB-dependent gene transcription. The cell growth suppression was canceled by co-expression with IκBα, a member of the NF-κB inhibitor protein family. This study suggests that the interaction between PLEKHG2 and ABL1 suppresses cell growth through intracellular protein accumulation via the NF-κB signaling pathway.

The circulating immunoglobulins negatively impact on the parasite clearance in the liver of Leishmania donovani-infected mice via dampening ROS activity.

Visceral leishmaniasis, the most severe form of leishmaniasis, is caused by Leishmania donovani and L. infantum. Immunity to Leishmania infection has been shown to depend on the development of Th1 cells; however, the roles of B cells and antibodies during infection remain unclear. In the present study, we showed that AID and µs double-deficient mice (DKO), which have B cells but not circulating immunoglobulins (cIgs), became resistant to L. donovani infection, whereas µs or AID single-deficient mice did not. This resistance in DKO mice occurred in the liver from an early stage of the infection. The depletion of IFN-γ did not affect the rapid reduction of parasite burden, whereas NADPH oxidases was up-regulated in the livers of infected DKO mice. The inhibition of the reactive oxygen species pathway in vivo by apocynin, a NADPH oxidase inhibitor, resulted in a significant increase in the parasite burden in DKO mice. These results indicate that a circulating Ig deficiency induces a protective response against L. donovani infection by elevating IFN-γ-independent NADPH oxidase activity, and also that cIgs play a regulatory role in controlling L. donovani infection in mice.

M2-like macrophage polarization in high lactic acid-producing head and neck cancer.

Reprogramming of glucose metabolism in tumor cells is referred to as the Warburg effect and results in increased lactic acid secretion into the tumor microenvironment. We have previously shown that lactic acid has important roles as a pro-inflammatory and immunosuppressive mediator and promotes tumor progression. In this study, we examined the relationship between the lactic acid concentration and expression of LDHA and GLUT1, which are related to the Warburg effect, in human head and neck squamous cell carcinoma (HNSCC). Tumors expressing lower levels of LDHA and GLUT1 had a higher concentration of lactic acid than those with higher LDHA and GLUT1 expression. Lactic acid also suppressed the expression of LDHA and GLUT1 in vitro. We previously reported that lactic acid enhances expression of an M2 macrophage marker, ARG1, in murine macrophages. Therefore, we investigated the relationship between the lactic acid concentration and polarization of M2 macrophages in HNSCC by measuring the expression of M2 macrophage markers, CSF1R and CD163, normalized using a pan-macrophage marker, CD68. Tumors with lower levels of CD68 showed a higher concentration of lactic acid, whereas those with higher levels of CSF1R showed a significantly higher concentration of lactic acid. A similar tendency was observed for CD163. These results suggest that tumor-secreted lactic acid is linked to the reduction of macrophages in tumors and promotes induction of M2-like macrophage polarization in human HNSCC.

Four-and-a-half LIM Domains 1 (FHL1) Protein Interacts with the Rho Guanine Nucleotide Exchange Factor PLEKHG2/FLJ00018 and Regulates Cell Morphogenesis.

PLEKHG2/FLJ00018 is a Gßγ-dependent guanine nucleotide exchange factor for the small GTPases Rac and Cdc42 and has been shown to mediate the signaling pathways leading to actin cytoskeleton reorganization. Here we showed that the zinc finger domain-containing protein four-and-a-half LIM domains 1 (FHL1) acts as a novel interaction partner of PLEKHG2 by the yeast two-hybrid system. Among the isoforms of FHL1 (i.e. FHL1A, FHL1B, and FHL1C), FHL1A and FHL1B interacted with PLEKHG2. We found that there was an FHL1-binding region at amino acids 58-150 of PLEKHG2. The overexpression of FHL1A but not FHL1B enhanced the PLEKHG2-induced serum response element-dependent gene transcription. The co-expression of FHL1A and Gßγ synergistically enhanced the PLEKHG2-induced serum response element-dependent gene transcription. Increased transcription activity was decreased by FHL1A knock-out with the CRISPR/Cas9 system. Compared with PLEKHG2-expressing cells, the number and length of finger-like protrusions were increased in PLEKHG2-, Gßγ-, and FHL1A-expressing cells. Our results provide evidence that FHL1A interacts with PLEKHG2 and regulates cell morphological change through the activity of PLEKHG2.

In vivo analysis of Aicda gene regulation: a critical balance between upstream enhancers and intronic silencers governs appropriate expression.

The Aicda gene encodes activation-induced cytidine deaminase (AID). Aicda is strongly transcribed in activated B cells to diversify immunoglobulin genes, but expressed at low levels in various other cells in response to physiological or pathological stimuli. AID's mutagenic nature has been shown to be involved in tumor development. Here, we used a transgenic strategy with bacterial artificial chromosomes (BACs) to examine the in vivo functions of Aicda regulatory elements, which cluster in two regions: in the first intron (region 2), and approximately 8-kb upstream of the transcription start site (region 4). Deleting either of these regions completely abolished the expression of Aicda-BAC reporters, demonstrating these elements' critical roles. Furthermore, we found that selectively deleting two C/EBP-binding sites in region 4 inactivated the enhancer activity of the region despite the presence of intact NF-κB-, STAT6- and Smad-binding sites. On the other hand, selectively deleting E2F- and c-Myb-binding sites in region 2 increased the frequency of germinal-center B cells in which the Aicda promoter was active, indicating that E2F and c-Myb act as silencers in vivo. Interestingly, the silencer deletion did not cause ectopic activation of the Aicda promoter, indicating that Aicda activation requires enhancer-specific stimulation. In summary, precise regulation of the Aicda promoter appears to depend on a coordinated balance of activities between enhancer and silencer elements.

Identification of a Rho family specific guanine nucleotide exchange factor, FLJ00018, as a novel actin-binding protein.

FLJ00018/PLEKHG2 is a guanine nucleotide exchange factor for the Rho family small GTPases. FLJ00018 is directly activated by heterotrimeric G protein Gßγ subunits. Using two-hybrid screening, we have identified non-muscle cytosolic actin as a binding partner of FLJ00018. We found that there were two actin-binding regions in FLJ00018 at the N-terminal region (150-283 amino acids) and at the C-terminal region (465-1386 amino acids). The overexpression of non-muscle cytosolic actin attenuated the FLJ00018-induced serum response element-dependent gene transcription. These results suggest that non-muscle cytosolic actin may be a negative regulator of FLJ00018 through its interaction with the Dbl homology domain.

The AID dilemma: infection, or cancer?

Activation-induced cytidine deaminase (AID), which is both essential and sufficient for forming antibody memory, is also linked to tumorigenesis. AID is found in many B lymphomas, in myeloid leukemia, and in pathogen-induced tumors such as adult T cell leukemia. Although there is no solid evidence that AID causes human tumors, AID-transgenic and AID-deficient mouse models indicate that AID is both sufficient and required for tumorigenesis. Recently, AID's ability to cleave DNA has been shown to depend on topoisomerase 1 (Top1) and a histone H3K4 epigenetic mark. When the level of Top1 protein is decreased by AID activation, it induces irreversible cleavage in highly transcribed targets. This finding and others led to the idea that there is an evolutionary link between meiotic recombination and class switch recombination, which share H3K4 trimethyl, topoisomerase, the MRN complex, mismatch repair family proteins, and exonuclease 3. As Top1 has recently been shown to be involved in many transcription-associated genome instabilities, it is likely that AID took advantage of basic genome instability or diversification to evolve its mechanism for immune diversity. AID targets are therefore not highly specific to immunoglobulin genes and are relatively abundant, although they have strict requirements for transcription-induced H3K4 trimethyl modification and repetitive sequences prone to forming non-B structures. Inevitably, AID-dependent cleavage takes place in nonimmunoglobulin targets and eventually causes tumors. However, battles against infection are waged in the context of acute emergencies, while tumorigenesis is rather a chronic, long-term process. In the interest of survival, vertebrates must have evolved AID to prevent infection despite its long-term risk of causing tumorigenesis.

Imatinib mesylate directly impairs class switch recombination through down-regulation of AID: its potential efficacy as an AID suppressor.

Activation-induced cytidine deaminase (AID) is essential for class switch recombination and somatic hypermutation. Its deregulated expression acts as a genomic mutator that can contribute to the development of various malignancies. During treatment with imatinib mesylate (IM), patients with chronic myeloid leukemia often develop hypogammaglobulinemia, the mechanism of which has not yet been clarified. Here, we provide evidence that class switch recombination on B-cell activation is apparently inhibited by IM through down-regulation of AID. Furthermore, expression of E2A, a key transcription factor for AID induction, was markedly suppressed by IM. These results elucidate not only the underlying mechanism of IM-induced hypogammaglobulinemia but also its potential efficacy as an AID suppressor.

Histone chaperone Spt6 is required for class switch recombination but not somatic hypermutation.

Activation-induced cytidine deaminase (AID) is shown to be essential and sufficient to induce two genetic alterations in the Ig loci: class switch recombination (CSR) and somatic hypermutation (SHM). However, it is still unknown how a single-molecule AID differentially regulates CSR and SHM. Here we identified Spt6 as an AID-interacting protein by yeast two-hybrid screening and immunoprecipitation followed by mass spectrometry. Knockdown of Spt6 resulted in severe reduction of CSR in both the endogenous Ig locus in B cells and an artificial substrate in fibroblast cells. Conversely, knockdown of Spt6 did not reduce but slightly enhanced SHM in an artificial substrate in B cells, indicating that Spt6 is required for AID to induce CSR but not SHM. These results suggest that Spt6 is involved in differential regulation of CSR and SHM by AID.

Activation-induced cytidine deaminase expression in CD4+ T cells is associated with a unique IL-10-producing subset that increases with age.

Activation-induced cytidine deaminase (AID), produced by the Aicda gene, is essential for the immunoglobulin gene (Ig) alterations that form immune memory. Using a Cre-mediated genetic system, we unexpectedly found CD4(+) T cells that had expressed Aicda (exAID cells) as well as B cells. ExAID cells increased with age, reaching up to 25% of the CD4(+) and B220(+) cell populations. ExAID B cells remained IgM(+), suggesting that class-switched memory B cells do not accumulate in the spleen. In T cells, AID was expressed in a subset that produced IFN-γ and IL-10 but little IL-4 or IL-17, and showed no evidence of genetic mutation. Interestingly, the endogenous Aicda expression in T cells was enhanced in the absence of B cells, indicating that the process is independent from the germinal center reaction. These results suggest that in addition to its roles in B cells, AID may have previously unappreciated roles in T-cell function or tumorigenesis.

Preventing AID, a physiological mutator, from deleterious activation: regulation of the genomic instability that is associated with antibody diversity.

Activation-induced cytidine deaminase (AID) is essential and sufficient to accomplish class-switch recombination and somatic hypermutation, which are two genetic events required for the generation of antibody-mediated memory responses. However, AID can also introduce genomic instability, giving rise to chromosomal translocation and/or mutations in proto-oncogenes. It is therefore important for cells to suppress AID expression unless B lymphocytes are stimulated by pathogens. The mechanisms for avoiding the accidental activation of AID and thereby avoiding genomic instability can be classified into three types: (i) transcriptional regulation, (ii) post-transcriptional regulation and (iii) target specificity. This review summarizes the recently elucidated comprehensive transcriptional regulation mechanisms of the AID gene and the post-transcriptional regulation that may be critical for preventing excess AID activity. Finally, we discuss why AID targets not only Igs but also other proto-oncogenes. AID targets many genes but it is not totally promiscuous and the criteria that specify its targets are unclear. A recent finding that a non-B DNA structure forms upon a decrease in topoisomerase 1 expression may explain this paradoxical target specificity determination. Evolution has chosen AID as a mutator of Ig genes because of its efficient DNA cleavage activity, even though its presence increases the risk of genomic instability. This is probably because immediate protection against pathogens is more critical for species survival than complete protection from the slower acting consequences of genomic instability, such as tumor formation.

B cell-specific and stimulation-responsive enhancers derepress Aicda by overcoming the effects of silencers.

Activation-induced cytidine deaminase (AID) is essential for the generation of antibody memory but also targets oncogenes, among other genes. We investigated the transcriptional regulation of Aicda (which encodes AID) in class switch-inducible CH12F3-2 cells and found that Aicda regulation involved derepression by several layers of positive regulatory elements in addition to the 5' promoter region. The 5' upstream region contained functional motifs for the response to signaling by cytokines, the ligand for the costimulatory molecule CD40 or stimuli that activated the transcription factor NF-kappaB. The first intron contained functional binding elements for the ubiquitous silencers c-Myb and E2f and for the B cell-specific activator Pax5 and E-box-binding proteins. Our results show that Aicda is regulated by the balance between B cell-specific and stimulation-responsive elements and ubiquitous silencers.

AID-induced decrease in topoisomerase 1 induces DNA structural alteration and DNA cleavage for class switch recombination.

To initiate class switch recombination (CSR) activation-induced cytidine deaminase (AID) induces staggered nick cleavage in the S region, which lies 5' to each Ig constant region gene and is rich in palindromic sequences. Topoisomerase 1 (Top1) controls the supercoiling of DNA by nicking, rotating, and religating one strand of DNA. Curiously, Top1 reduction or AID overexpression causes the genomic instability. Here, we report that the inactivation of Top1 by its specific inhibitor camptothecin drastically blocked both the S region cleavage and CSR, indicating that Top1 is responsible for the S region cleavage in CSR. Surprisingly, AID expression suppressed Top1 mRNA translation and reduced its protein level. In addition, the decrease in the Top1 protein by RNA-mediated knockdown augmented the AID-dependent S region cleavage, as well as CSR. Furthermore, Top1 reduction altered DNA structure of the Smu region. Taken together, AID-induced Top1 reduction alters S region DNA structure probably to non-B form, on which Top1 can introduce nicks but cannot religate, resulting in S region cleavage.

Apex2 is required for efficient somatic hypermutation but not for class switch recombination of immunoglobulin genes.

The DNA cleavage step in both the class switch recombination (CSR) and somatic hypermutation (SHM) of Ig genes is initiated by activation-induced cytidine deaminase (AID). However, the detailed mechanisms of the DNA strand cleavage in SHM and CSR are still largely unknown. Recently, the apurinic/apyrimidinic endonucleases, Apex1 and Apex2, were reported to be involved in the DNA cleavage step of CSR. Here, we examined the role of Apex2 in SHM using Apex2-deficient mice and found that the Apex2 deficiency caused a drastic reduction in the frequency of SHM and the number of mutations per mutated clone without affecting the pattern of base substitution. These results suggest that Apex2 may play a critical role in SHM through its 3'-5' exonuclease activity. Unexpectedly, the efficiency of CSR was not reduced in Apex2-deficient B cells. In addition, Apex1 knockdown in CH12F3-2 B lymphoma cells did not affect the CSR frequency, suggesting that neither Apex1 nor Apex2 plays a major role in CSR.

Further evidence for involvement of a noncanonical function of uracil DNA glycosylase in class switch recombination.

Activation-induced cytidine deaminase (AID) introduces DNA cleavage in the Ig gene locus to initiate somatic hypermutation (SHM) and class switch recombination (CSR) in B cells. The DNA deamination model assumes that AID deaminates cytidine (C) on DNA and generates uridine (U), resulting in DNA cleavage after removal of U by uracil DNA glycosylase (UNG). Although UNG deficiency reduces CSR efficiency to one tenth, we reported that catalytically inactive mutants of UNG were fully proficient in CSR and that several mutants at noncatalytic sites lost CSR activity, indicating that enzymatic activity of UNG is not required for CSR. In this report we show that CSR activity by many UNG mutants critically depends on its N-terminal domain, irrespective of their enzymatic activities. Dissociation of the catalytic and CSR activity was also found in another UNG family member, SMUG1, and its mutants. We also show that Ugi, a specific peptide inhibitor of UNG, inhibits CSR without reducing DNA cleavage of the S (switch) region, confirming dispensability of UNG in DNA cleavage in CSR. It is therefore likely that UNG is involved in a repair step after DNA cleavage in CSR. Furthermore, requirement of the N terminus but not enzymatic activity of UNG mutants for CSR indicates that the UNG protein structure is critical. The present findings support our earlier proposal that CSR depends on a noncanonical function of the UNG protein (e.g., as a scaffold for repair enzymes) that might be required for the recombination reaction after DNA cleavage.

The C-terminal region of activation-induced cytidine deaminase is responsible for a recombination function other than DNA cleavage in class switch recombination.

Activation-induced cytidine deaminase (AID) is an essential factor for the class switch recombination (CSR) and somatic hypermutation (SHM) of Ig genes. CSR and SHM are initiated by AID-induced DNA breaks in the S and V regions, respectively. Because truncation or frame-shift mutations at the carboxyl (C)-terminus of AID abolishes CSR but not SHM, the C-terminal region of AID likely is required for the targeting of DNA breaks in the S region. To test this hypothesis, we determined the precise location and relative amounts of AID-induced DNA cleavage using an in situ DNA end-labeling method. We established CH12F3-2 cell transfectants expressing the estrogen receptor (ER) fused with wild-type (WT) AID or a deletion mutant lacking the C-terminal 16 aa, JP8Bdel. We found that AID-ER, but not JP8Bdel-ER, caused a CSR to IgA from the addition of 4-hydroxy tamoxifen. In contrast, both WT AID and JP8Bdel induced DNA breaks in both the V and S regions. In addition, JP8Bdel enhanced c-myc/IgH translocations. Our findings indicate that the C-terminal domain of AID is not required for S-region DNA breaks but is required for S-region recombination after DNA cleavage. Therefore, AID does not distinguish between the V and S regions for cleavage, but carries another function specific to CSR.

Molecular mechanism for generation of antibody memory.

Activation-induced cytidine deaminase (AID) is the essential enzyme inducing the DNA cleavage required for both somatic hypermutation and class switch recombination (CSR) of the immunoglobulin gene. We originally proposed the RNA-editing model for the mechanism of DNA cleavage by AID. We obtained evidence that fulfils three requirements for CSR by this model, namely (i) AID shuttling between nucleus and cytoplasm, (ii) de novo protein synthesis for CSR, and (iii) AID-RNA complex formation. The alternative hypothesis, designated as the DNA-deamination model, assumes that the in vitro DNA deamination activity of AID is representative of its physiological function in vivo. Furthermore, the resulting dU was removed by uracil DNA glycosylase (UNG) to generate a basic site, followed by phosphodiester bond cleavage by AP endonuclease. We critically examined each of these provisional steps. We identified a cluster of mutants (H48A, L49A, R50A and N51A) that had particularly higher CSR activities than expected from their DNA deamination activities. The most striking was the N51A mutant that had no ability to deaminate DNA in vitro but retained approximately 50 per cent of the wild-type level of CSR activity. We also provide further evidence that UNG plays a non-canonical role in CSR, namely in the repair step of the DNA breaks. Taking these results together, we favour the RNA-editing model for the function of AID in CSR.