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Epigenetic Regulation of Kcna3-Encoding Kv1.3 Potassium Channel by Cereblon Contributes to Regulation of CD4+ T-Cell Activation

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Epigenetic Regulation of Kcna3-Encoding Kv1.3 Potassium Channel by Cereblon Contributes to Regulation of CD4+ T-Cell Activation Epigenetic regulation of Kcna3-encoding Kv1.3 potassium channel by cereblon contributes to + regulation of CD4 T-cell activation Jung-Ah Kanga,1, Sang-Heon Parka,1, Sang Phil Jeonga, Min-Hee Hana, Cho-Rong Leea, Kwang Min Leea, Namhee Kima, Mi-Ryoung Songa, Murim Choib, Michael Yec, Guhung Jungd, Won-Woo Leee, Soo Hyun Eoma, Chul-Seung Parka, and Sung-Gyoo Parka,f,2 aSchool of Life Sciences, Gwangju Institute of Science and Technology, Gwangju 61005, Republic of Korea; bDepartment of Biomedical Sciences, College of Medicine, Seoul National University, Seoul 03080, Republic of Korea; cSchool of Liberal Arts and Sciences, Gwangju Institute of Science and Technology, Gwangju 61005, Republic of Korea; dDepartment of Biological Sciences, Seoul National University, Seoul 08826, Republic of Korea; eDepartment of Microbiology and Immunology, College of Medicine, Seoul National University, Seoul 03080, Republic of Korea; and fCell Logistics Research Center, Gwangju Institute of Science and Technology, Gwangju 61005, Republic of Korea Edited by Kenneth M. Murphy, Washington University, St. Louis, MO, and approved June 14, 2016 (received for review February 2, 2015) The role of cereblon (CRBN) in T cells is not well understood. We binding of CRBN to Kcna3 leads to epigenetic modification of generated mice with a deletion in Crbn and found cereblon to be the Kcna3 locus and reduces the expression of Kv1.3. Triggering an important antagonist of T-cell activation. In mice lacking CRBN, of TCR signaling in CRBN-deficient T cells results in (i) in- + CD4 T cells show increased activation and IL-2 production on creased potassium and calcium flux, (ii) increased activation of T-cell receptor stimulation, ultimately resulting in increased potas- transcription factor NF-AT, and (iii) increased production of sium flux and calcium-mediated signaling. CRBN restricts T-cell ac- IL-2. Our data identify CRBN as an important regulator of T-cell tivation via epigenetic modification of Kcna3, which encodes the activation, restricting calcium influx via epigenetic repression Kv1.3 potassium channel required for robust calcium influx in T of Kcna3. cells. CRBN binds directly to conserved DNA elements adjacent to Kcna3 via a previously uncharacterized DNA-binding motif. Conse- Results quently, in the absence of CRBN, the expression of Kv1.3 is dere- CRBN Deficiency Does Not Affect T-Cell Development, but Does pressed, resulting in increased Kv1.3 expression, potassium flux, and + Increase T-Cell Activation. Analysis of the relative levels of CRBN CD4 T-cell hyperactivation. In addition, experimental autoimmune – Crbn transcripts in multiple tissue types using the Novartis BioGPS encephalomyelitis in T-cell specific -deficient mice was exacer- expression array database (biogps.org) (9) suggested that the ex- bated by increased T-cell activation via Kv1.3. Thus, CRBN limits + pression of CRBN mRNA is higher in lymphoid cells, including CD4 T-cell activation via epigenetic regulation of Kv1.3 expression. + CD4 T cells, than in other cell types (Fig. S1A). Thus, we used Crbn gene-targeted mice to examine the effect of CRBN defi- T-cell activation | CRBN | Kv1.3 | calcium flux | potassium flux ciency in T-cell development and activation. First, we confirmed + the loss of CRBN protein from CD4 T cells isolated from Crbn- − − ecent studies have identified cereblon (CRBN) as a thalidomide- deficient (Crbn / ) mice (Fig. 1A). There were no obvious changes binding protein (1, 2). CRBN is expressed in the cytoplasm, R in thymic T-cell development (Fig. 1B). In addition, peripheral nucleus, and peripheral membranes of a wide variety of cells and − − T-cell and B-cell populations in 6- to 8-wk-old Crbn / mice were tissues (1, 3–5). CRBN forms an E3 ubiquitin ligase complex with + − no different from those in 6- to 8-wk-old littermate control (Crbn / ) damaged DNA-binding protein (DDB) 1 and Cul4A, and this + + mice (Fig. 1 C and D and Fig. S2A). Moreover, the Foxp3 CD4 complex is involved in limb outgrowth and controls fibroblast −/− INFLAMMATION Crbn IMMUNOLOGY AND growth factor 8 expression in zebrafish and chicks (1). When T-cell population in the spleens of mice was similar to that thalidomide binds to CRBN, it inhibits formation of the E3 ubiquitin ligase complex, which in turn reduces the activity of the Significance ubiquitin ligase and results in defective limb development (1). Recent work suggests that immunomodulatory drugs (IMiDs) In resting T cells, CRBN normally represses expression of the stimulate increased IL-2 production in T cells by binding to CRBN. Kv1.3 potassium channel by regulating histone modifications This binding alters the substrate specificity of CRBN-containing to prevent hyperactivation of T cells. It does this by controlling ubiquitin ligase complexes (6, 7) even though IMiDs reduce the recruitment of EZH1 to the potassium channel region of overall activity of E3 ubiquitin ligase (1). As a result, binding the Kcna3 gene (or locus). However, lack of CRBN causes up- promotes abnormal degradation of the Ikaros zinc finger proteins 1 regulation of Kv1.3 expression, which in turn increases potas- and 3 (IKZF1 and IKZF3), which in turn increases IL-2 production sium flux, thereby triggering increased calcium flux during in T cells (6–8). However, although the CRBN-mediated effects of T-cell activation. IMiDs are known, the physiological role of CRBN in T cells remains unclear. Author contributions: S.-G.P. conceived of the study; J.-A.K., S.-H.P., M.-R.S., M.C., G.J., Crbn W.-W.L., and S.-G.P. designed research; J.-A.K., S.-H.P., S.P.J., M.-H.H., C.-R.L., N.K., and We deleted the gene from murine T cells to examine the S.-G.P. performed research; K.M.L., S.H.E., and C.-S.P. contributed new reagents/analytic physiological role of CRBN during T-cell activation, with the aim tools; J.-A.K., S.-H.P., S.P.J., M.-H.H., C.-R.L., N.K., M.Y., and S.-G.P. analyzed data; and J.-A.K., of gaining new insight into the regulation of potassium flux M.Y., and S.-G.P. wrote the paper. during T-cell signaling. Deletion of Crbn from T cells led to IL-2 The authors declare no conflict of interest. + production and differentiation of CD4 T cells into Th17 ef- This article is a PNAS Direct Submission. fector cells, as well as worsening of the phenotype associated Freely available online through the PNAS open access option. with experimental autoimmune encephalitis (EAE). CRBN re- 1J.-A.K. and S.-H.P. contributed equally to this work. presses T-cell activation by binding to the chromosomal regions 2To whom correspondence should be addressed. Email: [email protected]. Kcna3 adjacent to the locus, a gene encoding the Kv1.3 potas- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. sium channel, which participates in calcium influx in T cells. The 1073/pnas.1502166113/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1502166113 PNAS | August 2, 2016 | vol. 113 | no. 31 | 8771–8776 Downloaded by guest on September 30, 2021 Fig. 1. CRBN deficiency does not affect T-cell development, but does increase T-cell activation. (A) Loss of CRBN protein expression in CD4+ T cells from Crbn−/− mice was confirmed by immunoblot analysis with an anti-CRBN antibody. (B) T-cell development in the thymus was analyzed by flow cytometry. (C and D) Lymphocytes in + − − + − the spleen (C) and lymph nodes (D) were analyzed by flow cytometry. (E)CD4 Tn and Tem cells from Crbn / mice and Crbn / mice were stimulated with anti-CD3 + and anti-CD28 antibodies for the indicated times, after which cell surface expression of CD25 was analyzed by flow cytometry. (F) Proliferation of CD4 Tn and Tem + cells stimulated with anti-CD3 and anti-CD28 antibodies was measured by [3H]thymidine incorporation. (G–J) IL-2 secretion and IL-2 mRNA expression in CD4 Tn and Tem cells stimulated by either anti-CD3 or anti-CD28 antibodies (G and H) or PMA and ionomycin (I and J) were analyzed by ELISA and quantitative RT-PCR. Data are representative of two (A), four (B–D), or three (E–J) independent experiments. Results are expressed as the mean ± SD. *P < 0.05; **P < 0.01, unpaired two-tailed Student’s t test. + − + in Crbn / mice, suggesting that CRBN does not affect the selection ERK and JNK was unaffected in CRBN-deficient CD4 Tn and + of T cells during development (Fig. S2B). However, when CRBN- Tem cells (Fig. 2A). The NF-κB pathway in CD4 T cells also + + deficient CD4 T cells, including CD4 naïve T (Tn) cells and ef- was unaffected, because IκBα degradation (Fig. 2A) and NF-κB − − fector memory T (Tem) cells, from Crbn / mice were stimulated DNA-binding activity (Fig. 2B) were unchanged in CRBN- + + with anti-CD3 and anti-CD28 antibodies, both CD4 Tn and Tem deficient CD4 Tn and Tem cells. However, we found it interesting cells expressed more CD25 on the cell surface compared with the that NF-AT DNA binding (Fig. 2 B and C) and nuclear local- + +/− + control CD4 Tn and Tem cells from their Crbn littermates (Fig. ization (Fig. 2D) in CRBN-deficient CD4 Tn and Tem cells 1E). In contrast to T cells, on B cells a loss of CRBN did not affect were increased after activation, suggesting that loss of CRBN + the activation-induced cell surface expression of CD69 and CD86 in CD4 T cells affects primarily the NF-AT activation signaling C (Fig. S2 ). pathway. Along with the expression of activation markers on the cell + To identify the genes responsible for the increased activation – – + surface, anti-CD3 and anti-CD28 stimulated CD4 Tn and of NF-AT observed in CRBN-deficient CD4 T cells, we ex- Tem cells from CRBN-deficient mice were more proliferative + F + amined gene expression patterns in CD4 Tn cells from CRBN- (Fig.
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