Selective Inhibition of Class Switching to IgG and IgE by Recruitment of the HoxC4 and Oct-1 Homeodomain Proteins and Ku70/Ku86 to Newly Identified ATTT cis-Elements

doi:10.1074/jbc.M212952200

Selective Inhibition of Class Switching to IgG and IgE by Recruitment of the HoxC4 and Oct-1 Homeodomain Proteins and Ku70/Ku86 to Newly Identified ATTT cis-Elements^*

^‡Division of Molecular Immunology, Department of Pathology and Laboratory Medicine, Joan and Sanford I. Weill Medical College, Cornell University, New York, New York 10021 and the ^∥Center for Immunology, School of Biological Sciences and College of Medicine, University of California, Irvine, California 92697

‡‡To whom correspondence should be addressed. Tel.: 949-824-9648; E-mail: pcasali{at}uci.edu.

Abstract

Immunoglobulin (Ig) class switching is central to the maturation of the antibody response as IgG, IgA, and IgE are endowed with more diverse biological effector functions than IgM. It is induced upon engagement of CD40 on B lymphocytes by CD40L expressed by activated CD4⁺ T cells and exposure of B cells to T cell-secreted cytokines including interleukin-4 and transforming growth factor-β. It begins with germ line I_H-C_H transcription and unfolds through class switch DNA recombination (CSR). We show here that the HoxC4 and Oct-1 homeodomain proteins together with the Ku70/Ku86 heterodimer bind as a complex to newly identified switch (S) regulatory ATTT elements (SREs) in the Iγ and Iϵ promoters and downstream regions to dampen basal germ line Iγ-Cγ and Iϵ-Cϵ transcriptions and repress CSR to Cγ and Cϵ. This mechanism is inactive in the Cα1/Cα2 loci because of the lack of SREs in the Iα1/Iα2 promoters. Accordingly, in resting human IgM⁺IgD⁺ B cells, HoxC4, Oct-1, and Ku70/Ku86 can be readily identified as bound to the Iγ and Iϵ promoters but not the Iα1/Iα2 promoters. CD40 signaling dissociates the HoxC4·Oct-1·Ku complex from the Iγ and Iϵ promoter SREs, thereby relieving the I_H-C_H transcriptional repression and allowing CSR to unfold. Dissociation of HoxC4·Oct-1·Ku from DNA is hampered by CD153 engagement, a CD40-signaling inhibitor. Thus, these findings outline a HoxC4·Oct-1·Ku-dependent mechanism of selective regulation of class switching to IgG and IgE and further suggest distinct co-evolution and shared CSR activation pathways in the Cγ and Cϵ as opposed to the Cα1/Cα2 loci.

Footnotes

↵1 The abbreviations used are: GC, germinal center; AID, activation-induced cytidine deaminase; C_H, heavy chain constant region; ChIP, chromatin immunoprecipitation assay; CHX, cycloheximide; CSR, class switch DNA recombination; HD, homeodomain; HIM, HD interaction motif; I_H, intervening region of C_H gene; Ig, immunoglobulin; NHEJ, non-homologous end-joining; S, switch (region); SC, switch circle; SRE, S-regulatory ATTT element; V_HDJ_H, variable/diversity/joining regions of heavy chain gene; IL, interleukin; GFP, green fluorescent protein; mAb, monoclonal antibody; GST, glutathione S-transferase; Ab, antibody; RT, reverse transcriptase; EMSA, electrophoretic mobility shift assay; DBB, DNA binding buffer; DAC, DNA affinity chromatography; mut, mutated; WT, wild type; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight; RE, responsive element; ht, human trimeric; Stat, signal transducer and activator of transcription.
↵2 E. C. Kim, X. Wu, A. Schaffer, L. Testoni, H. Zan, and P. Casali, manuscript in preparation.
↵* This work was supported in part by National Institutes of Health Grants AI 45011, AR 40908, AI 07621, and AG 13910 (to P. C.), a Cancer Research Institute predoctoral fellowship in tumor immunology (to A. S.), National Institutes of Health Grant AR 47872, and a New Investigator Award from the Leukemia Research Foundation (to A. C.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵ The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. 2, A and B, and 7, A and B.
↵§ Present address: Dept. of Pathology, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02114.
↵¶ Both authors contributed equally to this work.
↵** Present address: Dept. of Internal Medicine, University of Milan Medical School, Milano 20122, Italy.
- Received December 19, 2002.
- Revision received March 20, 2003.
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