Lysine Acetylation Regulates Bruton's Tyrosine Kinase in B Cell Activation Zhijian Liu, Antonello Mai and Jian Sun This information is current as J Immunol 2010; 184:244-254; Prepublished online 30 of September 27, 2021. November 2009; doi: 10.4049/jimmunol.0902324 http://www.jimmunol.org/content/184/1/244 Downloaded from Supplementary http://www.jimmunol.org/content/suppl/2009/12/11/jimmunol.090232 Material 4.DC1 References This article cites 44 articles, 14 of which you can access for free at: http://www.jimmunol.org/content/184/1/244.full#ref-list-1 http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! 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The Journal of Immunology Lysine Acetylation Regulates Bruton’s Tyrosine Kinase in B Cell Activation Zhijian Liu,*,†,‡ Antonello Mai,‡,x and Jian Sun*,{ Bruton’s tyrosine kinase (Btk) is essential for BCR signal transduction and has diverse functions in B cells. Although Btk has been extensively studied, the role of lysine acetylation in Btk regulation has not been reported. In this study, we show that BCR cross- linking induces histone lysine acetylation at the Btk promoter, correlating with marked recruitment of histone acetyltransferase E1A-associated 300-kDa protein (p300) to the locus. These effects enhance Btk promoter activity and increase the expression of Btk mRNA and protein. Consistent with these results, activated B cells display increased p300 expression and total histone acetyl- transferase activity in vitro and in vivo, resulting in global histone acetylation. Interestingly, we found that BCR signaling induces Btk lysine acetylation mediated by p300. Moreover, lysine acetylation of Btk promotes its phosphorylation. Together, our results indicate a novel regulatory mechanism for Btk transcription and reveal a previously unrecognized posttranslational modification of Downloaded from the Btk protein and its association with phosphorylation in B cell activation. The Journal of Immunology, 2010, 184: 244–254. ruton’s tyrosine kinase (Btk) is a member of the nonreceptor is enhanced in response to BCR cross-linking. Several transcription tyrosine kinase Tec family and is expressed in many hema- factors, including Sp1, Sp3, Spi-B, PU.1, Bright, OBF-1, and NF-kB, B topoietic lineages, but not in T cells (1). It is well known that have been identified as regulators of Btk transcription (8–12). Btk is involved in multiple signaling pathways following the activation Lysine acetylation of histones in chromatin is regulated by histone http://www.jimmunol.org/ of diverse cell membrane receptors. The role of Btk in BCR activation, deacetylases (HDACs) and histone acetyltransferases (HATs) and is however, has been the most studied, and results indicate that Btk is essential for the activation of gene transcription (13–16). Mammalian critical for B cell survival, proliferation, and differentiation (2, 3). Btk HDACs are divided into four classes according totheir homology with was originally identified as a disease gene for human X-linked agam- yeast counterparts. Among these, class I HDACs include HDAC-1, -2, maglobulinemia (also referred to as Bruton’s agammaglobulinemia) in -3, and -8, whereas class II HDACs include HDAC-4, -5, -6, -7, -9, and which B cell development is blocked at the pre-B cell stage, resulting in -10. The third class of HDACs is the sirtuin family with seven a near total absence of peripheral B cells and a failure to mount Ab members (SIRT1-7). HDAC-11 is classified in the fourth class due to responses (4, 5). A spontaneous point mutation at arginine 28 to cys- its similarity to both class I and II enzymes. HDACs decrease histone teine in the pleckstrin homology domain of murine Btk leads to the acetylation and generally mediate transcriptional repression. Many by guest on September 27, 2021 condition xid, which involves impaired B cell development and re- HATs have been identified and are divided into three major families: sponse and is similar to phenotypes observed in Btk knockout mice (6). E1A-associated 300-kDa protein (p300)/CREB-binding protein, Upon BCR signaling, Btk is translocated to the plasma membrane Gcn5-related N-acetyltransferases, and MYST proteins. These pro- bybindingofitspleckstrinhomologydomaintoPtdIns-3,4,5-P3.Btk teins acetylate histones and activate transcription. It has been reported isthenphosphorylatedbytheSrckinaseLynattyrosine551(Y551)in that some HDACs and HATs modulate the lysine acetylation of its kinase domain, followed by autophosphorylation at Y223 in its nonhistone proteins such as p53, tubulin, GATA-1, STAT3, MyoD, SH3 domain. Phosphorylated Btk activates PLC-r2 by binding to and E2F1 (17–20). Therefore, the histone lysine-modified enzymes BLNK and modulating Ca2+ influx, playing an important role in B are also termed lysine deacetylases and lysine acetyltransferases, cell function (1, 5, 7). In addition to phosphorylation, Btk expression respectively (21, 22). Lysine acetylation has emerged as an important posttranslational modification for nonhistone proteins and could in- teract with other posttranslational modifications to constitute multi- *Laboratory of B-Cell and Autoantibody, Institute of Health Sciences, Shanghai Institutes for Biological Sciences, and {Shanghai Institute of Immunology, Institutes site modification programs and regulate cell function (17, 22). of Medical Sciences, Shanghai JiaoTong University School of Medicine, Shanghai; In this study, we asked whether histone acetylation regulates Btk †Institute of Genetics and Developmental Biology, and ‡Graduate School, Chinese x transcription, contributing to BCR-induced B cell activation. Our Academy of Sciences, Beijing, China; and Dipartimento di Studi Farmaceutici, Universita degli Studi di Roma La Sapienza, Rome, Italy results demonstrate that the regulation of histone acetylation mod- Received for publication July 21, 2009. Accepted for publication October 26, 2009. ulates Btk transcription and expression. In short, p300 promoted, whereas HDAC-1, -2, and -3 inhibited, the transcription and ex- This work was supported by research grants from the National Natural Science Foundation of China (30872314), the Knowledge Innovation Program of the Chinese pression of Btk. Moreover, BCR signaling markedly recruited p300 Academy of Sciences (KSCX1-YW-R-45), and the Shanghai Science and Technol- to the Btk promoter and induced local histone acetylation. BCR- ogy Committee (074319112 and 08JC1421200). induced local acetylation, however, was not associated with reduced Address correspondence to Dr. Jian Sun, Institute of Health Sciences, Shanghai recruitment of HDAC-1 to the Btk locus. Consistent with these Institutes for Biological Sciences, 225 South Chongqing Road, Shanghai 200025, China. E-mail address: [email protected] results, B cell activation in vitro with anti-m or LPS and in vivo by The online version of this article contains supplemental material. OVA led to global histone acetylation, which was coordinated with Abbreviations used in this paper: Btk, Bruton’s tyrosine kinase; ChIP, chromatin an increase in p300 expression and total HAT activity, but was not immunoprecipitation; HATs, histone acetyltransferases; HDACs, histone deacety- a result of reduced HDAC-1 expression or reduced activity of total lases; p300, E1A-associated 300-kDa protein; pol II, polymerase II; siRNA, small HDACs. These results indicate that histone acetylation regulates interfering RNA; TSA, trichostatin A. Btk transcription. Furthermore, we found that BCR signaling in- Copyright Ó 2009 by The American Association of Immunologists, Inc. 0022-1767/10/$16.00 duces Btk lysine acetylation mediated by p300 through interaction www.jimmunol.org/cgi/doi/10.4049/jimmunol.0902324 The Journal of Immunology 245 with Btk and that Btk acetylation promotes its phosphorylation via (purity .95%) were isolated from C57BL/6 mice by negative selection using Lyn signaling. These results identify a novel posttranslational mod- a B cell separation kit (Miltenyi Biotec, Bergisch Gladbach, Germany). ification of the Btk protein and an association between Btk acetylation Analysis of mRNA expression and phosphorylation. Together, our study links lysine acetylation with 3 6 Btk regulation and sheds new light on B cell activation. Splenic B cells (2 10 /ml) were treated with TSA, MS-275, or MC1568. In some experiments, B cells were activated by F(ab9)2 anti-m Ab plus IL-4, or LPS alone, in the presence or absence of TSA. Total RNA was extracted with Trizol (Invitrogen, Carlsbad, CA), and cDNA was prepared Materials and Methods with the Promega cDNA Synthesis kit (Promega, Madison, WI). The Abs, chemicals, plasmids, primers, and small interfering RNAs mRNA levels were measured by RT-PCR using gene-specific primers. Abs