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Results: 7

1.
FIGURE 5

FIGURE 5. E2A localizes to λR in G1 phase of cell cycle. From: E2A Acts in cis in G1 Phase of Cell Cycle to Promote Ig Gene Diversification.

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

Munehisa Yabuki, et al. J Immunol. ;182(1):408-415.
2.
FIGURE 3

FIGURE 3. Accelerated diversification depends upon the E2A activation domains. From: E2A Acts in cis in G1 Phase of Cell Cycle to Promote Ig Gene Diversification.

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-λR 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-λR RFP-LacI cells.

Munehisa Yabuki, et al. J Immunol. ;182(1):408-415.
3.
FIGURE 4

FIGURE 4. Nuclear radius correlates with cell cycle. From: E2A Acts in cis in G1 Phase of Cell Cycle to Promote Ig Gene Diversification.

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-λR 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.

Munehisa Yabuki, et al. J Immunol. ;182(1):408-415.
4.
FIGURE 1

FIGURE 1. E2A associates with the rearranged Igλ locus. From: E2A Acts in cis in G1 Phase of Cell Cycle to Promote Ig Gene Diversification.

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 λR and unrearranged λU loci in DT40 cells, relative to ovalbumin gene control amplicon (Ova). Fold enrichment is shown below. NTC, no template control.

Munehisa Yabuki, et al. J Immunol. ;182(1):408-415.
5.
FIGURE 7

FIGURE 7. Id1 expression diminishes λR/E2A colocalizations in G1 phase. From: E2A Acts in cis in G1 Phase of Cell Cycle to Promote Ig Gene Diversification.

A. Effect of Id1 expression on cell cycle-dependence of λR/E2A colocalizations. The Colocalizations in DT40 PolyLacO-λR 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 λR/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 Id1DBM-GFP relative to Id1-GFP is shown in the rightmost panel.

Munehisa Yabuki, et al. J Immunol. ;182(1):408-415.
6.
FIGURE 2

FIGURE 2. E2A acts in cis to regulate Igλ gene diversification. From: E2A Acts in cis in G1 Phase of Cell Cycle to Promote Ig 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-λR cells.
C. Accumulation of sIgM-loss variants by DT40 and DT40 PolyLacO-λR 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-λR GFP-LacI and DT40 PolyLacO-λR E47-LacI cells.
E. RT-PCR analysis of expression of Igλ, AID or β-actin mRNAs in DT40 PolyLacO-λR GFP-LacI and DT40 PolyLacO-λR E47-LacI transfectants. Triangles indicate 30, 10, 3 and 1x relative concentrations of cDNA templates.
F. Mean sIgM-loss of independent clonal DT40 PolyLacO-λR GFP-LacI (n = 13) and DT40 PolyLacO-λR E47-LacI (n = 19) transfectants, cultured for 3 wk in the absence or presence of 100 µM IPTG. Values were normalized to DT40 PolyLacO-λR GFP-LacI cells cultured without IPTG.

Munehisa Yabuki, et al. J Immunol. ;182(1):408-415.
7.
FIGURE 6

FIGURE 6. Id1 expression inhibits Ig gene diversification and λR/E2A colocalizations. From: E2A Acts in cis in G1 Phase of Cell Cycle to Promote Ig Gene Diversification.

A. Western blot assaying Id1 expression in DT40 PolyLacO-λR RFP-LacI (left) and DT40 PolyLacO-λR RFP-LacI Id1 cells (right). Marker polypeptide sizes (kDa) at left.
B. Cell cycle profile of DT40 PolyLacO-λR RFP-LacI and DT40 PolyLacO-λR RFP-LacI Id1 cells.
C. Mean sIgM-loss of independent clonal DT40 PolyLacO-λR RFP-LacI Id1 transfectants (n = 6), cultured for 6 wk. Values were normalized to DT40 PolyLacO-λR 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-λR RFP-LacI and DT40 PolyLacO-λR RFP-LacI Id1 cells.
F. Id1 expression diminishes λR/E2A colocalizations. Colocalizations of λR/E2A and λR/P*-Pol II in asynchronous populations of DT40 PolyLacO-λR RFP-LacI cells and a derivative stably expressing Id1 (Id1 − and +, respectively).

Munehisa Yabuki, et al. J Immunol. ;182(1):408-415.

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