walking the aid tightrope
TRANSCRIPT
nature immunology volume 11 number 2 february 2010 107
walking the AID tightropeMieun Lee-Theilen & Jayanta Chaudhuri
The mutator activation-induced cytidine deaminase is essential for immunoglobulin diversification but can be detrimental in other settings. A new comprehensive analysis investigates how its gene expression is regulated.
Mieun Lee-Theilen and Jayanta Chaudhuri are in
the Immunology Program, Memorial Sloan Kettering
Cancer Center, New York, New York, USA.
e-mail: [email protected]
efficient humoral immunity to foreign anti-gens requires that mature B lymphocytes
diversify their immunoglobulin genes through class-switch recombination (CSR) and somatic hypermutation (SHM)1. Activation-induced cytidine deaminase (AID), expressed mainly but not exclusively in activated mature B cells, has an essential role in the induction of both CSR and SHM1. Although a lack of AID activity leads to immunodeficiency syndromes, ectopic or higher AID expression results in malignant transformation of B cells and T cells as well as tumor development in various organs2. Despite the essential role of AID in immunity and the consequences of AID misexpression in cancer, transcriptional regulation of the gene encoding AID (Aicda) has not been examined in great detail. In this issue of Nature Immunology, Tran et al. provide evidence that the binding of ubiq-uitous proteins to silencer elements in the Aicda locus represses transcription in unactivated B lymphocytes and in non-B cells3. After activa-tion with cytokines that induce CSR, a battery of transcription factors associate with distinct elements of the Aicda locus to overcome the effect of silencers to induce AID expression. Thus, a fine balance between ubiquitous silenc-ers and cytokine-inducible activators regulates transcription of the gene encoding this potent mutator.
Antibodies or immunoglobulins produced by B lymphocytes are composed of immuno-globulin heavy and light polypeptide chains. The amino-terminal variable regions of immu-noglobulin heavy and light chains bind anti-gens, whereas the carboxy-terminal constant (C) region of the immunoglobulin heavy chain provides the effector function of the antibody molecule. The first antibody type produced by a B cell is immunoglobulin M (IgM), in which the C region of the immunoglobulin heavy chain is encoded by the Cµ gene seg-ments. During CSR, the Cµ region is replaced by one of a set of downstream C-region gene segments (Cγ, Cε or Cα) so that the B cell alters its expression of IgM to expression of IgG,
IgE or IgA, each with a distinct effector func-tion. CSR occurs between transcribed, repeti-tive DNA elements, called switch (S) regions, that precede each C-region gene. It is gener-ally believed that AID deaminates cytidines to uridines in transcribed S regions to initiate a cascade of reactions that induces the forma-tion of DNA double-strand breaks. Ligation of double-strand breaks between two distal S regions by components of the general DNA-repair pathways completes CSR4. During SHM, AID activity induces a very high rate of muta-tion of genes encoding the immunoglobulin heavy-chain and light-chain variable regions to generate immunoglobulins with greater antigen affinity4.
The immunoglobulin genes serve as primary and physiologically relevant AID targets, yet several non-immunoglobulin genes are fre-quently mutated by AID in normal B cells5. Non-immunoglobulin targets of AID include oncogenes, such as Myc, that are frequently translocated to the immunoglobulin loci in B cell malignancy6–8. Given the oncogenic poten-tial of AID, it is therefore not surprising that its activity in B cells is regulated at multiple levels, including microRNA-mediated degra-dation of its mRNA, active nuclear export and phosphorylation4. However, these post-tran-scriptional regulatory processes complement the fundamental regulatory mechanism of AID expression only at the level of transcription and by themselves cannot override the deleterious effects of ectopic AID expression. Indeed, the AID abundance that results from transgene overexpression or chronic infection has been associated with a greater incidence of tumors in many organs2. Thus, the most logical and effective step of ensuring stringent regulation of AID activity must occur at the level of tran-scription.
Published studies have identified several transcription factor–binding sites in Aicda. A promoter element (called ‘region 1’ in the article discussed here3) just upstream of the transcription start site contains binding sites for the transcription factors HoxC4, Sp1, Sp3, NF-κB and STAT6 (refs. 9–11; Fig. 1). However, the activity of this promoter has not been found to be lymphoid specific. The B cell–specific transcription factor Pax5 and the E-box transcriptional activator E47 have been
shown to induce AID expression through ele-ments residing in the first intron of Aicda10,12,13 (region 2; Fig. 1). Finally, a sequence located downstream of Aicda coding exons (region 3) has been reported to be required for AID expression in a bacterial artificial chromosome transgenic mouse system14 (Fig. 1). However, none of the studies reported above have dem-onstrated a mechanism by which Aicda expres-sion is actively repressed in nonactivated B cells and in non-B cells. Additionally, most of these studies analyzed each of the regions in isolation and thus do not provide any clues to how the elements influence each other.
To fully elucidate the role of the various cis elements in governing Aicda transcrip-tion, Tran et al. carry out a series of transient transfection assays in which they analyze the ability of various DNA elements in Aicda to drive luciferase expression in the CH12F3.2 B cell line, which has very low expression of AID. They find that stimulation with a bat-tery of activators and cytokines, composed of the ligand for the costimulatory molecule CD40, interleukin 4 and transforming growth factor-β (CIT), markedly induces AID expres-sion in these cells with concomitant CSR from IgM to IgA.
Tran et al. show that, consistent with pub-lished studies9–11, full-length region 1 stimu-lates luciferase expression four- to eightfold over that observed in the absence of the promoter. However, they find no additional induction of luciferase in response to CIT stimulation. As region 1 contains the transcrip-tion start site, the authors use a fragment of approximately 100 base pairs from this region that induces luciferase expression fivefold as the minimal promoter in subsequent experi-ments. The Pax5- and E-box-binding sites of region 2 stimulate the transcriptional activity of the minimal promoter10,12,13. However, the Pax5 and E-box fragments do not respond to CIT stimulation. Finally, region 3 does not show any activity in the reporter assays, which suggests that the region probably functions in the context of a chromatinized template. Therefore, none of these regions demonstrate any activity that could account for cytokine-dependent induction of Aicda expression.
In their quest to elucidate cytokine-induc-ible elements, Tran et al. turn their attention to
NEWS AND V IEWS©
201
0 N
atu
re A
mer
ica,
Inc.
All
rig
hts
res
erve
d.
STAT6, Smad3/4 and C/EBP after binding to region 4 that outweighs the suppressive activity of region 2 to allow high expression of AID. So how is Aicda transcription repressed in non-B cells? This again is probably achieved at mul-tiple levels. The E-box proteins that activate Aicda via binding to region 2 are B cell spe-cific. In addition, the basal activity of region 4 in non-B cells is only 50% of that in B cells. Finally, the silencer elements suppress basal Aicda transcription in non-B cells, consistent with the ubiquitous expression of the repres-sor proteins.
The report by Tran et al. raises several inter-esting questions about the nature of Aicda transcriptional regulation. First, what is the mechanism of AID transcriptional regulation by the various factors that potentially bind Aicda? In splenic B cells, mRNA for most of the region 4–binding factors, such as STAT-6 and NF-κB, are induced within 12 hours of cytokine stimulation but rapidly decrease thereafter to baseline. Yet AID expression does
an E2f-binding site; c-Myb-binding sites; and a 350–base pair CT-rich sequence. E2f and c-Myb have known repressor functions. A sequence similar to the 350–base pair CT-rich sequence has been shown to act as a suppres-sor element in keratinocytes. Point mutations in individual sites result in lower suppression activity, and combined disruption of all the binding sites completely eliminates suppressor activity. Therefore, these motifs function inde-pendently as suppressor elements to counteract the enhancer activities originating from region 2 and region 4.
The results reported by Tran et al. provide a detailed portrait of how AID expression is regu-lated in a multilayered fashion (Fig. 1). In non-activated B cells, the silencer activity of region 2 represses Aicda expression. In B lymphocytes, the E2A proteins and Pax5 provide activities that partially relieve the repression but are not sufficient for the high AID expression induced after cytokine stimulation. It is the cytokine-induced enhancer activity provided by NF-κB,
an element located upstream of the promoter (region 4). This region contains binding sites for several transcription factors, including NF-κB, STAT6, Smad3/4 and the enhancer-binding protein C/EBP (Fig. 1). In contrast to the other elements analyzed thus far, region 4 significantly induces luciferase expression in response to CIT stimulation. Additional analy-sis shows that interleukin 4, CD40 ligand and transforming growth factor-β independently activate Aicda transcription through STAT6, NF-κB and Smad3/4 sites, respectively, with each element providing an additive effect on AID expression. Finally, Tran et al. show that mRNA for NF-κB, STAT-6, Smad4 and C/EBP is rapidly induced in activated splenic B cells. Notably, cytokine stimulation of B cells induces binding of these proteins to region 4 DNA.
Tran et al. also make the striking observa-tion that region 2 strongly suppresses the basal activity of region 1 and even the cytokine-inducible activity of region 4. They find that three distinct sites confer silencing activity:
Stat6 NF-κB Smad3/4 C/EBP c-Myb Pax5 E-box E2f Sp HoxC4 1 2 AID
? ? TSS
?
IdId
Region 4
Additional elements thatrespond to other stimuli?
Region 1 Region 2 Region 3
CSR stimulation (CD40L, IL-4 and TGF-β)
Stat6 NF-κB Smad3/4 C/EBP c-Myb Pax5 E-box E2fSp
HoxC4-Oct
1 2AID
AIDAID
AID? ?TSS
?
Id
Id
Figure 1 A balance of silencers and activators regulates AID expression. Four distinct DNA elements (regions 1–4) regulate Aicda transcription. Region 1 contains the transcription start site (TSS). Regions 1, 2 and 4 have binding sites for many transcription factors, most of which are generally expressed. Pax5 and E47 are the only known B cell–specific proteins with binding sites in the Aicda locus. The activities of Pax5 and E47 are inhibited by Id proteins such as Id2 and Id3. Region 3 has been shown to be required for Aicda expression14, but the factors that regulate Aicda transcription through this region are unknown. In naive B cells and in non-B cells, silencer elements in region 2 bind the repressor proteins E2f and c-Myb to counter the activity of the transcriptional activators. Stimulation of B cells with cytokines that promote CSR induces activation signals through region 4 that are strong enough to overcome the effect of the repressor proteins at region 2, thereby enhancing Aicda transcription. Gray squares indicate exons 1 and 2 of Aicda; rectangles of various colors indicate DNA-binding elements; circles indicate proteins. CD40L, CD40 ligand; IL-4, interleukin 4; TGF-β, transforming growth factor-β.
108 volume 11 number 2 february 2010 nature immunology
NEWS AND V IEWS©
201
0 N
atu
re A
mer
ica,
Inc.
All
rig
hts
res
erve
d.
terial artificial chromosome transgenes and genetic knockout models, to elucidate the roles of DNA elements regulating Aicda expression in vivo. This exciting study also provides the impetus for screening for polymorphisms in Aicda that could potentially alter AID expres-sion and predispose people to either immuno-deficiency syndromes or cancer.
1. Muramatsu, M., Nagaoka, H., Shinkura, R., Begum, N.A. & Honjo, T. Adv. Immunol. 94, 1–36 (2007).
2. Okazaki, I.M., Kotani, A. & Honjo, T. Adv. Immunol. 94, 245–273 (2007).
3. Tran, T.H. et al. Nat. Immunol. 11, 148–154 (2010). 4. Delker, R.K., Fugmann, S.D. & Papavasiliou, F.N. Nat.
Immunol. 10, 1147–1153 (2009).5. Liu, M. & Schatz, D.G. Trends Immunol. 30, 173–181
(2009).6. Pasqualucci, L. et al. Nat. Genet. 40, 108–112
(2008).7. Robbiani, D.F. et al. Cell 135, 1028–1038 (2008).8. Wang, J.H. et al. Nature 460, 231–236 (2009).9. Dedeoglu, F., Horwitz, B., Chaudhuri, J., Alt, F.W. &
Geha, R.S. Int. Immunol. 16, 395–404 (2004).10. Yadav, A. et al. Mol. Immunol. 43, 529–541 (2006).11. Park, S.R. et al. Nat. Immunol. 10, 540–550 (2009).12. Sayegh, C.E., Quong, M.W., Agata, Y. & Murre, C. Nat.
Immunol. 4, 586–593 (2003).13. Gonda, H. et al. J. Exp. Med. 198, 1427–1437
(2003).14. Crouch, E.E. et al. J. Exp. Med. 204, 1145–1156
(2007).15. Petersen-Mahrt, S.K., Coker, H.A. & Pauklin, S. J. Mol.
Med. 87, 893–897 (2009).
line. It is also possible that additional positive and negative regulatory regions exist within or beyond the 100-kilobase region analyzed in this study.
Finally, although it was initially thought that AID expression is specific to activated B cell, it is now clear that AID is also expressed in several nonlymphoid cells, such as primary human hepatocellular carcinomas and helicobacter-infected gastric epithelial cells5. AID has also been detected in normal oocytes and embry-onic stem cells15. The physiological relevance of AID expression in these nonlymphoid cells is yet to be determined. But in the absence of any obvious role for AID in nonlymphoid cells, it is possible that its expression is an inadver-tent and often unfortunate consequence of an imbalance in the ubiquitous positive and negative regulatory factors that govern Aicda transcription. For example, NF-κB induced in helicobacter-infected cells as an innate immune response could provide the imbalance neces-sary for Aicda transcription.
In summary, Tran et al. present a compre-hensive map of inhibitory and activating fac-tors that regulate Aicda expression. It serves as the foundation for further studies, using bac-
not peak before approximately 60 hours after stimulation. Perhaps region 4 provides the initial burst of derepression but is dispensable for maintenance of the activated state. Rapid downregulation of the activating transcrip-tion factors could be a safeguard against a toxic buildup of AID. Likewise, how do the repressor proteins E2f and c-Myb regulate Aicda? E2f is a family of proteins that act as both transcrip-tional activators and repressors. Perhaps during cytokine stimulation an activating E2f protein transiently replaces the inhibitory E2f proteins at the binding sites in region 2 to promote derepression, followed by reestablishment of the repressed state.
Second, do the elements that respond to CIT stimulation in CH12 cells also function in vivo in the context of germinal centers where CSR and SHM occur? The elements that acti-vate Aicda in response to other CSR inducers, such as bacterial lipopolysaccharide, B cell–activation factor, tumor necrosis factor and interferon-γ are not known. It is likely that the influence of additional elements, such as the reported steroid hormone receptor–binding motifs in region 1 (ref. 15) or the region 3 ele-ment, could not be unmasked in the CH12 cell
Taming tissue-specific T cells: CTLA-4 reins in self-reactive T cellsAlison M Paterson & Arlene H Sharpe
CTLA-4 is a potent coinhibitory molecule that is critical for peripheral T cell tolerance. New data suggest that CTLA-4 exerts its critical immunoregulatory functions by controlling antigen-specific conventional T cells as well as regulatory T cells.
Alison M. Paterson and Arlene H. Sharpe are in the
Department of Pathology, Harvard Medical School,
Boston, Massachusetts, USA.
e-mail: [email protected]
It has been appreciated for some time that CTLA-4 (cytotoxic T lymphocyte antigen
4), a homolog of CD28, is an indispensable negative regulator of peripheral T cell func-tion1. The fatal lymphoproliferative pheno-type of Ctla4–/– mice revealed the critical negative regulatory function of CTLA-4 and provided the first evidence that costimulatory receptors could provide negative as well as positive second signals2,3. CTLA-4-deficient mice develop severe myocarditis and pancre-atitis and die within the first month of life. This pathology resembles an autoimmune
disease and gave impetus to studies investi-gating the role of CTLA-4 in T cell tolerance and autoimmunity4. Although the role of CTLA-4 in regulating peripheral T cell toler-ance has become firmly established, there are still many questions regarding how CTLA-4 exerts its key immunoregulatory functions. Among them is the unresolved question of whether or not T cells that infiltrate various tissues in the Ctla4–/– mice are autoreactive and tissue specific. In this issue of Nature Immunology, Ise et al. show for the first time that the hyperproliferative and destructive T cell populations in CTLA-4-deficient mice are not on autopilot but require specific sig-nals provided by autoantigens to cause tissue damage5. Their work points to an important role for CTLA-4 expression by effector T cells in restraining tissue-specific CD4+ T cells
from infiltrating, expanding their popula-tions and/or surviving in target organs and provides evidence that CTLA-4 can control the pathogenicity of self-reactive T cell at multiple levels.
Ise et al. analyze the T cell antigen recep-tor (TCR) repertoire of CTLA-4-deficient T cells by fixing the TCRβ chain of Ctla4–/– mice (DOβCtla4–/– mice). DOβCtla4–/– mice exhibit a pathology similar to but somewhat delayed relative to that of Ctla4–/– mice. The antigen-specific nature of pathogenic CTLA-4-deficient T cells is suggested by homing studies in which CD4+ T cells from the spleen or affected organs of DOβCtla4–/– mice are introduced into recipient mice deficient in recombination-activating gene 2. Although the transfer of splenic T cells recapitulates the multiorgan Ctla4–/– phenotype, T cells
nature immunology volume 11 number 2 february 2010 109
NEWS AND V IEWS©
201
0 N
atu
re A
mer
ica,
Inc.
All
rig
hts
res
erve
d.