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E2A Acts in cis in G₁ Phase of Cell Cycle to Promote Ig Gene Diversification¹

Munehisa Yabuki^2,*, Ellen C. Ordinario^2,, W. Jason Cummings^*, Monica M. Fujii^* and Nancy Maizels^3,*,

^* Department of Immunology and Department of Biochemistry, University of Washington School of Medicine, Seattle, WA 98195

Rearranged Ig genes undergo diversification in sequence and structure initiated by the DNA deaminase, activation-induced deaminase. Ig genes must be transcribed for diversification to occur, but whether there are additional requirements for cis activation has not been established. Here we show, by chromatin immunoprecipitation, that the regulatory factor E2A associates with the rearranged Ig_R gene in the chicken DT40 B cell line, which performs constitutive Ig gene diversification. By analysis of a DT40 derivative in which polymerized lactose operator tags the rearranged _R gene, we show that E2A must function in cis to promote diversification and that stimulation of diversification in cis depends on the E2A activation domains. By direct imaging, we show that _R/E2A colocalizations are most prominent in G₁. We further show that expression of the E2A antagonist Id1 prevents _R/E2A colocalizations in G₁ and impairs diversification but not transcription of _R. Thus, E2A acts in cis to promote Ig gene diversification, and G₁ phase is the critical window for E2A action.

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.

¹ This work was supported by National Institutes of Health Grants R01 GM39799 and R01 GM41712 (to N.M.) and National Institutes of Health Predoctoral Training Programs T32 GM07223 and T32 AG00057 (to E.C.O.).

² M.Y. and E.C.O. contributed equally to this work.

³ Address correspondence and reprint requests to Dr. Nancy Maizels, Department of Immunology, 474A HSB, University of Washington School of Medicine, Seattle, WA 98195-7650. E-mail address: maizels{at}u.washington.edu

⁴ Abbreviations used in this paper: CSR, class switch recombination; AD, activation domain; AID, activation-induced cytidine deaminase; ChIP, chromatin immunoprecipitation; IPTG, isopropyl-β-D-thiogalactopyranoside; LacI, lactose repressor; PI, propidium iodide; PolyLacO, polymerized lactose operator; RFP, red-fluorescent protein; sIgM, surface IgM; V, pseudo-V.