Levels of Polyadenylation Factor CstF-64 Control IgM Heavy Chain mRNA Accumulation and Other Events Associated with B Cell Differentiation  Yoshio Takagaki, James L Manley  Molecular Cell  Volume 2, Issue 6, Pages 761-771 (December 1998) DOI: 10.1016/S1097-2765(00)80291-9

Figure 1 CstF-64 Is Essential for Growth and Viability of DT40 Cells (A) Gene-targeting vectors. The structures of gene-targeting vectors are shown together with the wild-type CstF-64 allele. Gene-targeting vectors contain neomycin (neor) or hygromycin resistance gene (hygr) under the control of chicken β-actin promoter (dotted boxes), which replaces exons 2 through 8 of the CstF-64 gene. A probe used to screen for homologous recombinants (1.1 kbp) and the lengths of EcoRI fragments are shown. Positions of relevant restriction sites (E, EcoRI; H, HindIII; S, SacII) and the direction of transcription (arrows) are also indicated. (B) Southern blot analysis. Genomic DNA obtained from wild-type DT40 cells (lane 1), after the first round of gene targeting and cotransfection with ptTA and p tet O-flu64K (lane 2) or after the second round of gene targeting (lane 3) was digested with EcoRI, transferred to a nitrocellulose membrane and hybridized with the probe shown in (A). Positions of DNA size markers and wild-type (wt) and mutant (mut) gene fragments are indicated. (C) Cell growth. Wild-type (DT40, top) and gene-targeted (DT40–64, bottom) cells were cultured initially in the absence of tet (from day −2 to day 0) and then in the presence of 10 μg/ml tet (from day 0 to day 6). Cells were diluted when densities reached ∼1 × 106 cells/ml as indicated by dotted lines. At the times of dilution (Tet-0, +Tet-1, -2) and at the end of culture (+Tet-3), cells were harvested for protein analysis. (D) Western blot analysis. Wild-type (DT40, lanes 1–4) and gene-targeted (DT40–64, lanes 5–8) cells were harvested as indicated in (C). Total cellular proteins (18 μg) were fractionated on a 10% SDS-polyacrylamide gel, transferred to a nitrocellulose membrane, and probed with an anti-chicken CstF-64 polyclonal antibody. Positions of protein size markers and CstF-64 (arrowhead) are indicated. Molecular Cell 1998 2, 761-771DOI: (10.1016/S1097-2765(00)80291-9)

Figure 2 Depletion of CstF-64 Causes Cell Cycle Arrest and Apoptotic Cell Death (A) DNA content. DT40–64 cells cultured for 9 days in the absence of tet (left) or for 6 (center) or 9 days (right) in the presence of 10 μg/ml tet were stained with propidium iodide and analyzed by flow cytometry. DNA content and numbers of nuclei are plotted on x and y axes, respectively. Cells at G0/G1, S, and G2/M phases and cells containing less than diploid amount of DNA (subdiploid) are indicated. (B) TUNEL assay. Wild-type (DT40, top) and gene-targeted (DT40–64, bottom) cells were cultured as in (A), labeled with FITC-dUTP, and stained with propidium iodide. DNA content and fluorescence derived from incorporated FITC-dUTP are plotted on x and y axes, respectively. Molecular Cell 1998 2, 761-771DOI: (10.1016/S1097-2765(00)80291-9)

Figure 3 Reduced CstF-64 Levels Reversibly Arrest Cell Growth (A) Cell growth. DT40–64 cells were cultured in the absence of tet for 9 days (circles, lines) or in the presence of 1 μg/ml tet for 6 days (triangles, dotted lines) followed by in the absence of tet for 3 days (triangles, lines). Cells were diluted when densities reached ∼1.0–1.5 × 106 cells/ml as in Figure 1C. (B) Western blot analysis. DT40–64 cells were cultured in the absence of tet for 9 days (lane 3), in the presence of 1 μg/ml tet for 6 days (lane 4), or in the presence of 1 μg/ml tet for 6 days followed by in the absence of tet for 3 days (lane 5) as shown in (A). Total cellular proteins were analyzed as in Figure 1D. Proteins derived from wild-type cells (DT40), cultured for 9 days in the absence (lane 1) or in the presence of 1 μg/ml tet (lane 2), were used as controls. (C) DNA content. DNA content of DT40–64 cells, cultured in the absence of tet for 9 days (left), in the presence of 1 μg/ml tet for 6 days (center), or in the presence of 1 μg/ml tet for 6 days then in the absence of tet for 3 days (right), were analyzed as in Figure 2A. Note that there is no significant increase in the subdiploid fraction during culture in the presence of 1 μg/ml tet. Molecular Cell 1998 2, 761-771DOI: (10.1016/S1097-2765(00)80291-9)

Figure 4 A Low Concentration of CstF-64 Is Sufficient for Cell Growth (A) Cell growth. DT40–64 cells were cultured for 8–9 days in the absence of tet (circles) or in the presence of 1 (triangles), 10 (inverted triangles), or 100 ng/ml tet (squares). Cells were diluted when densities reached ∼1 × 106 cells/ml as in Figure 1C. Since growth rate in the presence of 1 or 10 ng/ml tet was essentially the same as in the absence of tet for up to 4 days of culture, it is not shown in this figure. (B) Western blot analysis. DT40–64 cells were cultured in the absence of tet (lane 2) or in the presence of 0.1 (lane 3), 1 (lane 4), or 10 ng/ml tet (lane 5) and harvested as indicated in (A). Total cellular proteins were analyzed as in Figure 1D. Proteins derived from wild-type cells (DT40), cultured in the absence of tet (lane 1), were used as controls. Molecular Cell 1998 2, 761-771DOI: (10.1016/S1097-2765(00)80291-9)

Figure 5 Decreased CstF-64 Levels Specifically Influence Accumulation of IgM H-chain mRNA (A) S1 nuclease analysis of IgM H-chain mRNA. DT40–64 cells were cultured in the absence of tet (lane 4) or in the presence of 0.1 (lane 5), 1 (lane 6), or 10 ng/ml of tet (lane 7) and harvested as indicated in Figure 4A. Total cellular RNA (10 μg) was hybridized with an end-labeled DNA probe (lane 1) and digested with S1 nuclease, and protected DNA fragments were fractionated on an 8.3 M urea–5% polyacrylamide gel. Positions of DNA size markers and probe DNA fragments corresponding to the spliced μm and polyadenylated μs mRNAs are indicated. tRNA (10 μg, lane 2) or total cellular RNA isolated from wild-type cells (DT40) grown in the absence of tet (lane 3) was used as controls. Structures of probe and protected DNA fragments are shown at the bottom. Asterisks indicate the position of 3′-end label. (B–D) RNase protection assays of IgM H-chain (Cμ1 and Cμ2 exon probes [B]), Ig L-chain (constant region exon probe [C]), and β-actin (exon 3 and 6 probes [D]) mRNAs. tRNA (lanes 2 and 9) or total cellular RNA derived from DT40 cells and DT40–64 cells (lanes 3–7 and 10–14), cultured and harvested as in (A), was hybridized with uniformly labeled RNA probes (lanes 1 and 8) and digested with RNases, and protected RNA fragments were fractionated on denaturing polyacrylamide gels as in (A). The RNA fragments corresponding to exon sequences are indicated by arrowheads. The amounts of total cellular RNA used and the lengths of exposure of X-ray films are as follows: IgM H-chain (10 μg, 3 hr); Ig L-chain (1 μg, 1 hr); β-actin (3 μg, 6 hr). Molecular Cell 1998 2, 761-771DOI: (10.1016/S1097-2765(00)80291-9)

Figure 6 Roles of CstF-64 in B Cell Differentiation (A) Differentiation of B cells. Resting B cells, arrested at G0/G1 phase, start to proliferate and differentiate into plasma cells when stimulated with antigens or inducers such as LPS. During this process, the CstF-64 concentration increases dramatically, switching IgM H-chain gene expression from μm to μs mRNA and increasing the amounts of IgM H-chain mRNA synthesized. In contrast, without antigen stimulation, resting B cells die by apoptosis. (B) Changes in CstF-64 concentration in DT40 cell reproduce key events in B cell differentiation. An increase in the CstF-64 level switches IgM H-chain gene expression from μm to μs mRNA. In contrast, a decrease in CstF-64 concentration first specifically influences μ gene expression by reducing the total amount of IgM H-chain mRNA. Further decrease reversibly arrests the cell cycle at G0/G1 phase, and depletion of CstF-64 causes cell death by apoptosis. Molecular Cell 1998 2, 761-771DOI: (10.1016/S1097-2765(00)80291-9)