Tanaka S, Ise W, Baba Y, Kurosaki T. The Role of TET Proteins in Biology. J Immunological Sci. (2021); 5(1): 1-5 Journal of Immunological Sciences

Mini review Article Open Access

The Role of TET Proteins in B Cell Biology Shinya Tanaka1*, Wataru Ise2, Yoshihiro Baba1, Tomohiro Kurosaki2,3# 1Division of and Biology, Medical Institute of Bioregulation, Kyushu University, Higashi-ku, Fukuoka, 812-0054, Japan 2Laboratory of Differentiation, WPI Immunology Frontier Research Center, Osaka University, Osaka, Suita, 565-0871,Japan 3Laboratory of Lymphocyte Differentiation, RIKEN Center for Integrative Medical Sciences, Yokohama, Kanagawa, 230-0045, Japan

Article Info ABSTRACT

Article Notes must be strictly controlled during cell differentiation Received: December 23, 2020 and function in mammalian systems. DNA plays an important Accepted: February 01, 2021 role in this process, and its pattern is shaped by balancing the activity of *Correspondence: methyltransferases and demethylases. Ten-eleven translocation (TET) was *Dr. Shinya Tanaka, Division of Immunology and Genome identified as a demethylase that catalyzes the oxidation reaction of the methyl Biology, Medical Institute of Bioregulation, Kyushu University, group of 5-methylcytosine (5mC), converting it to 5-hydroxymethylcytosine Higashi-ku, Fukuoka, 812-0054, Japan; TEL: (+81)-92-642-6839; (5hmC). Recently, indispensable roles of TET proteins in the regulation of Email: [email protected]. immune cells have been identified. Here, we review recent studies on the #Dr. Tomohiro Kurosaki, Laboratory of Lymphocyte biological consequences of dysregulation of TET proteins in the immune Differentiation, WPI Immunology Frontier Research Center, system, with a particular focus on B cell biology. Finally, we discuss future Osaka University, Osaka, Suita, 565-0871, Japan; TEL: (+81)-6- perspectives in this research field. 6879-4456; Email: [email protected].

© 2021 Tanaka S, Kurosaki T. This article is distributed under the terms of the Creative Commons Attribution 4.0 International Introduction License. The methylation pattern of eukaryotic DNA, which is critical Keywords: for appropriate cell differentiation and function, is dynamically B cell differentiation regulated by the activity of DNA methyltransferase and demethylases. Ten-eleven translocation DNA methylation Approximately ten years ago, Ten-eleven translocation 1 (TET1) was reported to act as a DNA demethylase in acute myeloid and lymphocytic leukemia1 and named after a t(10;11) (q22;q23) translocation. After this discovery, other TET family proteins TET2 and TET3 were

identified by sequence homology. These TET proteins2. In mammalianhave a CpG DNAcells, bindingthese TET motif proteins CXXC catalyzeat the N-terminus the oxidation (TET2 of 5-methylcytosine does not contain CXXC.) and a catalytic domain at the C-terminus

the(5mC), generation sequentially of an generating unmethylated 5-hydroxymethylcytosine . Recent publications (5hmC), have5-formylcytosine reported roles (5fC) of TETand proteins5-carboxylcytosine other than (5caC),their role resulting as tumor in suppressors3 in immune cells4-13. In this mini review, we discuss the role of TET proteins, especially in B cell biology. Role of TET Proteins in Early B Cell Development Tet2 and Tet3

andB defective cell-specific Ig kappa (κ) gene rearrangement, gene-deficient which mice is a using somatic an rearrangementMb1Cre driver showed process an of impaired Vκ and Jtransitionκ gene segments of Pro B toto Preproduce B cells a functional Igκ light chain gene, during early B cell development5, 6. An analysis at the molecular level revealed that TET2/TET3 double- κ locus transcripts and interferon regulatory factor (IRF)4/IRF8 expression5, 6, leading todeficiency speculation resulted that in TET-regulated decreased Ig IRF4/IRF8 might promote the expression of the germline transcripts, an activity that precedes

Page 1 of 5 Tanaka S, Ise W, Baba Y, Kurosaki T. The Role of TET Proteins in B Cell Biology. J Journal of Immunological Sciences Immunological Sci. (2021); 5(1): 1-5 and may be required for successful Ig light chain gene rearrangement. The Igκ region cis-regulatory elements, reduced at the TetE1, where BATF bound. In addition, TET2 3’ enhancer (3’Eκ) and distal 3’ enhancer (dEκ), contain andthat, BATFin the wereabsence physically of TET2/TET3, associated 5hmC in modification primary B cells was stimulated in vitro, suggesting that BATF and TET proteins development progresses5 coordinately induce AID expression11. CpG , which are demethylated as early B Tet2 cell As mentioned above, TET2 and TET3 play a critical role and Tet3 genes enhanced. DNA In Pro methylation B cells differentiated at the 3’Eκ, in differentiation11, 13, 14. We found that Tet2/ by co-culture with OP9 cells, disruption κof transcripts the Tet3 and Igκ germline transcripts, accompanied by impaired differentiation in vivo chromatinwhich in turnaccessibility reduced of the the amountIgκ locus. of Therefore, C DNA by the deletion observation caused of impaired defective -specific plasma cell differentiation plasma cell at 3’Eκ is a crucial epigenetic event for Igκ in vitro resulting from. This Tet2finding/Tet3 was further supported13 gene rearrangement. Although overexpression of IRF4 did closer examination, we could show that high level IRF4 not restore the defective Igκ rearrangement caused by Tet expression, which is required for plasma deficiency cell differentiation,. Upon was drastically diminished in Tet2/Tet3 κ cis- deficiency, knock down of the E-protein E2A and the ETS- cells, although low to medium level expression was still regulatory elements, with enhanced DNA methylation in -deficient B family protein PU.1 attenuated TET2-binding to Ig maintained. Mechanistically, DNA demethylation occurred at the 5’ region of the Irf4 gene during the transition from regulate Igκ rearrangement and expression during early B naive B cells to plasma cells. In contrast, in the absence of cellPre Bdevelopment cells, suggesting5. that E2A, PU.1 and TET coordinately TET2/TET3, this 5’ region was kept methylated. Therefore, Role of TET Proteins in Peripheral B Cell the epigenetic changes, including DNA demethylation Differentiation at these sites, may be required to induce high level IRF4 expression14. Notably, although IRF4 is necessary for early It has been reported that the TET proteins play a critical 16, 17 cell differentiation was observed in the Tet2/Tet plasma cells as well11, 13, 14. Tet2/Tet3 BGC cells. B cell This differentiation may be because, no the remarkable low to medium defect levelin GC ofB role in the differentiation of geminal center+ (GC) B and cells in vitro. 3-deficient A similar defect was observed in vivo after deficiency immunization did not 11,affect13 cell proliferation, but formation of IgG1 IRF4Role expressionof TET Proteins is sufficient in B forCell GC Tolerance B cell development. was rather increased in the absence of TET2/TET3 in B cells, even11. Thereby, though theTET2/TET3 number of were antigen-specific suggested to B cellsplay as a break of self-tolerance is a direct cause of autoimmune Self-tolerance is a vital biological event for homeostasis, which is an irreversible gene rearrangement process to an important role in class switch recombination (CSR), of self-reactive B cells, which is induced by discontinuous activationdisease. Peripheral due to lack B cell of tolerance help. is established In other words,by death B with this idea, the expression of activation-induced cell peripheral tolerance is established by an intrinsic cytidinegenerate deaminase different classes/isotypes (AID), which is anof .essential Consistentmutagenic suppressive mechanism of self-reactive B cell activation enzyme that catalyses the deamination of deoxycytidine and an extrinsic mechanism that prevents interaction of self-reactive B cells with self-reactive T cells. Although and gene conversion, was substantially reduced in several endogenous factors that dampen self-reactive B cell to deoxyuracil/ to initiate 11 CSR,. In fact, somatic we have hypermutation previously Tet2 Tet3 18, little is known about the Tet2/Tet3 deletion upon expression -deficient B cells latter mechanism. In our current study, we discovered one activation have been identified confirmed that acute of the mechanisms by which self-reactive T-B interaction is inhibited by TET2/TET3 to induce/maintain peripheral of a Tamoxifen-inducible Cre, decreasedTet2 AID/Tet3 expression, accompanied by impairment of IgG1 CSR (unpublished tolerance . B cells by enforced AID expression. On the other hand, the 19 expressiondata). IgG1 expressionlevel of µ and was γ 1restored germline in transcripts-deficient in Tet2/ In the above-cited publications5, 6, splenomegaly Tet3 Tet2/ Tet3 due -deficientto decreased B cellsAID expression. was equivalent Regarding to those the ofdetailed wild- and lymphadenopathy were evident in Mb1 CreTet2 x /Tet3 moleculartype B cells. mechanism, Therefore, theit defectivehas already IgG1 beenCSR wasreported solely conditional double knockfloxed outmice. mice We (hereafteralso found calledsimilar Tet lymphoid bDKO), that the factor basic leucine zipper ATF- tissuebut no abnormalitiesconspicuous defects in CD19 in early Cre-mediated B cell development. Like (BATF) can induce AID expression As discussed in our publication , this difference may be by directly binding to the 5’ region of the Aicda gene locus15. 19 and TetE2, in the 5’ region of the Aicda locus and found explained by the timing of Cre expression20, 21. Therefore, during early we Lio et al. identified TET2-binding regions, named TetE1 B cell development, since Mb1 Cre has been reported to be expressed earlier than CD19

Page 2 of 5 Tanaka S, Ise W, Baba Y, Kurosaki T. The Role of TET Proteins in B Cell Biology. J Journal of Immunological Sciences Immunological Sci. (2021); 5(1): 1-5 hypothesized that the tissue abnormalities might have demethylases, they also function as adapters, recruiting been caused by defective homeostasis of peripheral B cells, especially disruption of self-tolerance. In Tet bDKO suppression of gene expression in the hematopoietic mice, we found a constitutive activation of immune systemhistone through deacetylase deacetylation (HDAC)4, 8 that. Therefore, contributes we examined to the cells including B and T and myeloid cells, accompanied by autoimmune phenotypes: H3 acetylation caused by Tet2/Tet3 deletion in B cells at thechanges genome-wide in DNA methylation, level. Our meta-analysis HDAC binding, demonstrated and histone withproduction, these pathologies, cell infiltration Tet bDKO into mice non-lymphoid had renal damage tissues,, recruiter in B cells . On the other hand, an integrated suggestingand complement development deposition of a systemic in the lupus kidneys. erythematosus Consistent19 thatanalysis TET offunctioned epigenetic19 both data as aand demethylase gene expression and an HDAC data suggested that TET-dependent epigenetic changes development in the Tet bDKO mice, (SLE)-like disease. Regarding the mechanism underlying being a determinant of whether genes are transcribed or not.optimize At the (fine-tune) Cd86 transcription outcomes rather than Tetintroduction of a hen egg lysozyme (HEL)-specific+ T and BB cell and 3’ region of Cd86 intron 1, and this binding was TET- activationreceptor (MD4). Therefore, or B cell-specific antigen-mediated MHC class T-B II deletion interaction into gene locus, HDAC bound in the promoter was bDKO required 19mice forsuppressed the autoimmune the aberrant reaction, CD4 suggesting degree of acetylation of histone H3 in the promoter was that molecules which mediate T-B interaction may be enhanceddependent. Consistent with the reduced HDAC binding, the responsible for dysregulated T and B cell activation. As proteins negatively19 regulate the expression of the Cd86 . Collectively, these results suggest that the TET comprehensivemight be expected, gene the expression gene encoding analysis CD86, done an essential before andgene DNA by forming demethylation HDAC-dependent were observed suppressive in close chromatinproximity co-stimulatory molecule for T cells, was identified by a disease onset among up-regulated differentially expressed tostructures. the 3’ region Although of Cd86 both intron TET-dependent 1, it is currently HDAC unknown binding genes in the absence of TET2/TET3 in B cells. On the whether these two epigenetic events are functionally other hand, there was almost no change in gene coordinated. expression and in genes encoding other co-stimualtory In our current study, we elucidated a TET-dependent demonstrated that it was one of the responsible molecules inhibitory mechanism of T-B interaction that contributes to formolecules. induction Furthermore, of spontaneous a CD86 immune neutralizing cell activation experiment and autoimmune disease . 26, 27 19 peripheral B cell tolerance. Previous . Instudies addition, demonstrated a genome- in that CD86 expression was elevated in B cellsTET2 from and SLE TET3 and vivo, a transgenic model using HEL as a surrogate self- multipleas risk factor sclerosis genes (MS) in autoimmune patients diseases28, . Therefore, 22 antigenSince is it commonly is difficult used to identify to study self-reactive B cell tolerance B cells. In wide association study (GWAS) identified 29 a peripheral tolerance model, self-reactive B cells are described above may be utilized to control inthe humans mechanism as well. of suppression of CD86 expression upon HEL recognition, after which the activation is lost and thetemporarily cells are eliminated activated accompaniedby apoptotic cell by death CD8623 . inductionHowever, Conclusions and Future Perspectives Tet2/Tet3 As described, TET2 and TET3 are required not only for B cell early development but also differentiation -deficiency resulted in CD86 derepression and that the absence of Tet2/Tet3 may increase the risk of self- and homeostasis of mature peripheral B cells (Figure delayed elimination of HEL-specific B cells, suggesting reactive T cell activation, resulting in the development of 1). However, despite the recent accumulating studies, the role of TET proteins in B cell biology is not yet fully understood. First, it has not been fully investigated autoimmune disease. Our findings are consistent with 24 a in self-reactive B cells could activate self-reactive T cells . exactly in which stages of B cell biology TET proteins are previous report showing that enforced CD86 expression In addition, as only antigen stimulation without secondary necessary. In particular, little research has been conducted co-stimulation makes self-reactive T cells anergic25, we could interpret these results to indicate that TET proteins still largely unclear at a molecular level how TET2 and expressed in B cells actively contribute to induction of TET3to examine regulate a role the of above-mentioned TET in Bkey cells. processes Second, init isB cell biology. For instance, recent reports suggest a critical role of TET2/TET3 in development and/or homeostasis peripheral T cell tolerance by CD86 suppression. suggested to be one of the causes of the break in of marginal zone (MZ) B cells and B1 cells, because these peripheralBased ontolerance. these However, results, the dysregulated molecular mechanism CD86 was B cell subsets in spleen and peritoneal cavity almost been completely disappeared in the absence of TET2/TET3 in B cells6, . However, the molecular mechanism underlying by which CD86 expression was suppressed has 19 unknown. Although TET proteins were identified as DNA Page 3 of 5 Tanaka S, Ise W, Baba Y, Kurosaki T. The Role of TET Proteins in B Cell Biology. J Journal of Immunological Sciences Immunological Sci. (2021); 5(1): 1-5

Bone marrow TET2/TET3

Igκ, IRF4 HSCs Pre/pro B Pro B Pre B Immature B

Spleen Class TET2/TET3 Switched B Anergic TET2/TET3 B

Memory B FO B GC B Transi�onal LLPCs TET2/TET3 B MZ B TET2/TET3 SLPCs

Haematopoie�c stem cells (HSCs), Long-lived plasma cells (LLPCs), Short-lived plasma cells (SLPCs) Follicular (FO), Marginal zone (MZ), Germinal center (GC)

Figure 1: TET2/TET3 action points in B cell biology Ten-eleven translocation (TET)2 and TET3 regulate key steps in B cell development and tolerance. The expression of Igκ, interferon regulatory factor (IRF) 4 and activation-induced deaminase (AID) are regulated by the demethylation activity of TET2/TET3. In contrast, CD86 expression is repressed by TET-dependent HDAC recruitment, mediating B cell peripheral tolerance. this phenotype is largely unknown. Third is the issue of independent roles of TET family proteins. In the current and cancer could be addressed. studies, doubly Tet2/Tet3 knock out cells have been to establish effective treatments for inflammatory diseases utilized, because of the functional redundancy of TET2 Acknowledgement and TET3. Therefore, it has been unclear whether there This work was supported by research grants from the protein and if so, by what mechanism the selectivity is determinedare specific genesremains independently unclear. In addition, regulated the by eachrole TETand Ministry of Education, Culture, Sports, Science and Technology (MEXT) and AMED [Grant-in-Aid for Scientific Research (S) the . Regarding the more general question for T.K., AMED under grant no. 19gm6110004h0003 and aboutdownstream TET’s TET1molecular gene activities targets is in largely the regulation undefined inof Grant-in-Aid for Scientific Research (B) for Y.B and Grant-in- Conflict of Interest Statement gene transcription beyond understanding of role of TET Aid for Scientific Research (C) for S.T.] proteins in B cell biology, at least three questions should The authors declare no competing interests. be answered: How many TET-target genes are regulated in References manner? In what context are these two molecular activities 1. Tahiliani M, et al. either an HDAC-dependent or demethylation-dependent 5-hydroxymethylcytosine in mammalian DNA by MLL partner TET1. exerted? Do these two different TET activities coordinately Science 324, Conversion of 5-methylcytosine to regulate gene expression? A comprehensive understanding 2. et930-935 al. TETonic (2009). shift: biological roles of TET proteins in DNA of these issues may be obtained by addressing them with demethylation and transcription. Nat Rev Mol Cell Biol 14, 341-356 various different cell types. Finally, as these basic research (2013).Pastor W.A, issues progress, whether the regulation of immune cells by 3. et al. TET1 is a tumor suppressor of hematopoietic manipulation of TET activity can serve as a novel approach malignancy. Nat Immunol 16, 653-662 (2015). Cimmino L,

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