E2A acts in cis in G1 phase of cell cycle to promote Ig gene diversification

Abstract

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 lambda(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 lambda(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 lambda(R)/E2A colocalizations are most prominent in G(1). We further show that expression of the E2A antagonist Id1 prevents lambda(R)/E2A colocalizations in G(1) and impairs diversification but not transcription of lambda(R). Thus, E2A acts in cis to promote Ig gene diversification, and G(1) phase is the critical window for E2A action.

FIGURE 1. E2A associates with the rearranged Igλ locus

A. Schematic of the rearranged and unrearrranged Igλ loci in the chicken B cell lymphoma line, DT40. Shown are promoter (P), leader (L), variable (V), joining (J) and constant (Cλ) regions; the putative matrix attachment region (M) in the J-C intron; the 3’-enhancer (E-3’); and the most proximal (ψV1) and distal (ψV25) of the upstream nonfunctional pseudo-variable regions which are templates for gene conversion. The rearranged and unrearranged alleles can readily be distinguished by PCR, using primers indicated by arrows. B. ChIP analysis of E2A enrichment at the rearranged λ_R and unrearranged λ_U loci in DT40 cells, relative to ovalbumin gene control amplicon (Ova). Fold enrichment is shown below. NTC, no template control.

FIGURE 2. E2A acts in cis to regulate Igλ gene diversification

A. Schematic of the PolyLacO-tagged rearranged Igλ locus. PolyLacO is integrated between ψV17-20; other notions as in Figure 1A. B. Cell cycle profile of DT40 and DT40 PolyLacO-λ_R cells. C. Accumulation of sIgM-loss variants by DT40 and DT40 PolyLacO-λ_R cells. Frequencies of sIgM-loss variants in 24 subclones from each line were quantitated by flow cytometry following 6 wk clonal expansion. Frequencies shown were normalized to DT40; mean sIgM-loss frequencies were 0.8% and 0.9%, respectively. D. Cell cycle profile of DT40 PolyLacO-λ_R GFP-LacI and DT40 PolyLacO-λ_R E47-LacI cells. E. RT-PCR analysis of expression of Igλ, AID or β-actin mRNAs in DT40 PolyLacO-λ_R GFP-LacI and DT40 PolyLacO-λ_R E47-LacI transfectants. Triangles indicate 30, 10, 3 and 1x relative concentrations of cDNA templates. F. Mean sIgM-loss of independent clonal DT40 PolyLacO-λ_R GFP-LacI (n = 13) and DT40 PolyLacO-λ_R E47-LacI (n = 19) transfectants, cultured for 3 wk in the absence or presence of 100 µM IPTG. Values were normalized to DT40 PolyLacO-λ_R GFP-LacI cells cultured without IPTG.

FIGURE 3. Accelerated diversification depends upon the E2A activation domains

A. Schematic of wild-type E47 and its mutants. AD1, activation domain 1; AD2, activation domain 2; bHLH, basic-helix-loop-helix. B. Mean sIgM-loss of independent clonal DT40 PolyLacO-λ_R cells expressing RFP-LacI (n = 17), E47-LacI (n = 10), E47ΔAD1-LacI (n = 22), E47ΔAD2-LacI (n = 9), or E47ΔAD1/2-LacI (n = 7), analyzed 3 wk posttransfection. The result shown is representative of two independent experiments. Values were normalized to DT40 PolyLacO-λ_R RFP-LacI cells.

FIGURE 4. Nuclear radius correlates with cell cycle

A. Representative images of G1- and G2/M-enriched cells. G1 (left) and G2/M (right) cells were stained with Hoechst 33342 (10 µM; Molecular Probes) then sorted based on DNA content. Bar, 10 µm. B. Representative cell cycle profile of DT40 PolyLacO-λ_R cells. Mean radii ± s.d. are shown as horizontal bars within the representative profile. Dotted vertical lines indicate cut-offs for G1 and G2 used in experimental analyses: G1, r < 4 µm; G2, r > 5.2 µm.

FIGURE 5. E2A localizes to λ_R in G1 phase of cell cycle

A. Representative image of colocalization of λ_R and E2A in DT40 PolyLacO-λ_R GFP-LacI cells. Nuclear perimeter as determined by DAPI staining is outlined by the dashed white line. Bar, 5 µm. B. Fraction of λ_R/E2A colocalizations occurring in each phase of cell cycle in DT40 PolyLacO-λ_R GFP-LacI cells. C. Representative image of colocalization of λ_R and active Pol II (P*-Pol II) in DT40 PolyLacO-λ_R RFP-LacI cells. Notations as in panel A. D. Fraction of λ_R/P*-Pol II colocalizations occurring in each phase of cell cycle in DT40 PolyLacO-λ_R RFP-LacI cells.

FIGURE 6. Id1 expression inhibits Ig gene diversification and λ_R/E2A colocalizations

A. Western blot assaying Id1 expression in DT40 PolyLacO-λ_R RFP-LacI (left) and DT40 PolyLacO-λ_R RFP-LacI Id1 cells (right). Marker polypeptide sizes (kDa) at left. B. Cell cycle profile of DT40 PolyLacO-λ_R RFP-LacI and DT40 PolyLacO-λ_R RFP-LacI Id1 cells. C. Mean sIgM-loss of independent clonal DT40 PolyLacO-λ_R RFP-LacI Id1 transfectants (n = 6), cultured for 6 wk. Values were normalized to DT40 PolyLacO-λ_R RFP-LacI cells. D. RT-PCR analysis of mRNA expression of E proteins in DT40 cells. Arrows indicate predicted sizes of products; smudge below (HEB, E2-2) is due to unincorporated primers. E. Id1 expression does not alter Igλ or AID transcript levels. RT-PCR analysis of expression of Igλ, AID or β-actin control mRNA in DT40 PolyLacO-λ_R RFP-LacI and DT40 PolyLacO-λ_R RFP-LacI Id1 cells. F. Id1 expression diminishes λ_R/E2A colocalizations. Colocalizations of λ_R/E2A and λ_R/P*-Pol II in asynchronous populations of DT40 PolyLacO-λ_R RFP-LacI cells and a derivative stably expressing Id1 (Id1 − and +, respectively).

FIGURE 7. Id1 expression diminishes λ_R/E2A colocalizations in G1 phase

A. Effect of Id1 expression on cell cycle-dependence of λ_R/E2A colocalizations. The Colocalizations in DT40 PolyLacO-λ_R RFP-LacI cells and a derivative stably expressing Id1 (Id1 − and +, respectively) in each stage of cell cycle are graphed, showing the percent of total cells in which colocalizations were evident. Note that Id1 expression diminished total colocalizations by about half. B. Effect of Id1 expression on cell cycle-dependence of λ_R/P*-Pol II colocalizations. Details as in Figure 7A. C. Fluorescence analysis of Id1-GFP levels in the course of cell cycle. DT40 cells were transiently transfected with Id1-GFP or its derivative carrying mutations in the D-box, which determines cell cycle-dependent instability; and GFP intensity and DNA content analyzed at 24 hr posttransfection. Fold increases of mean GFP fluorescence intensity in Id1^DBM-GFP relative to Id1-GFP is shown in the rightmost panel.