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. 2019 Apr 1;79(7):1612-1623.
doi: 10.1158/0008-5472.CAN-18-2705. Epub 2019 Jan 4.

Smarcal1 and Zranb3 Protect Replication Forks From Myc-Induced DNA Replication Stress

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Free PMC article

Smarcal1 and Zranb3 Protect Replication Forks From Myc-Induced DNA Replication Stress

Matthew V Puccetti et al. Cancer Res. .
Free PMC article

Abstract

The cellular DNA replication stress response functions to stabilize DNA replication forks and inhibits genome instability and tumorigenesis induced by oncogenes. However, the specific proteins required for resolving oncogenic stress remain poorly understood. Here we report that Smarcal1 and Zranb3, closely related replication fork-remodeling proteins, have nonredundant functions in resolving Myc-induced DNA replication stress. In Myc-overexpressing primary cells, significant differences in replication fork stalling, collapse, and DNA damage were detected between cells deficient in Smarcal1 or Zranb3, leading to changes in proliferation and apoptosis. These differences were also reflected in Myc-induced lymphoma development; haploinsufficiency of Smarcal1 resulted in accelerated lymphomagenesis, whereas haploinsufficiency of Zranb3 inhibited lymphoma development. Complete loss of either protein resulted in disparate survival outcomes. Our results reveal that endogenous replication stress from Myc in primary cells requires both alleles of Smarcal1 and Zranb3 and demonstrate the requirement of both proteins to stabilize replication forks upon Myc dysregulation in a nonredundant manner. SIGNIFICANCE: Smarcal1 and Zranb3 are essential, but nonredundant, for responding to DNA replication stress and stabilizing replication forks following Myc overexpression.See related commentary by Sotiriou and Halazonetis, p. 1297.

Conflict of interest statement

Conflict of Interest Statement: The authors have no conflicts of interest.

Figures

Figure 1.
Figure 1.. Myc overexpression leads to replication stress in primary B cells.
(A-E) B cells purified from bone marrow of Eμ-myc and littermate wild-type (WT) mice were subjected to single-molecule DNA fiber analysis. A) Design of DNA fiber analysis and representative fiber images. B) Quantification of total fiber length from two independent experiments; red line is median. C) Binned fiber length frequencies from (B). D) Quantification of fibers that only incorporated IdU from (B); SEM. E) Ratios of measured CldU tracks from left and right moving forks from the same replication origin; median indicated with red bar. F) Quantification of immunofluorescence of γH2AX foci in purified bone marrow B cells; SEM. G) Box-and-whisker plots of tail moments of purified bone marrow B cells. Box is the 25th and 75th percentiles, whiskers are 5th and 95th percentiles, line is the median. H) Whole cell lysates from spleens of littermate-matched Eμ-myc and WT mice were Western blotted for the indicated proteins. B cell lymphoma treated with etoposide or vehicle served as positive (+) and negative (–) controls, respectively, for the replication stress response. Total number (n) of fibers (B), replication structures (D, E), or cells (F, G) noted. Student’s t-tests (B, E, G) or chi-square tests (D, F); *p<0.0001, **p=0.0037, ***p=0.0052.
Figure 2.
Figure 2.. Deficiency of Smarcal1 or Zranb3 significantly alters Myc-induced lymphomagenesis.
A, B) Kaplan-Meier survival curves of mice of the indicated genotypes. Overall P values in figures and p=0.0142 (+/+ vs. +/Δ), p=0.0002 (+/Δ vs. Δ/Δ), p=0.3224 (+/+ vs. Δ/Δ) (B), and p=0.0066 (+/+ vs. +/−), p=0.0199 (+/+ vs. −/−), log-rank tests. Number (n) of mice indicated. C) Representative flow cytometry contour plots of the early precursor B cell lymphomas arising in a fraction of the Smarcal1+/Δ Eμ-myc, Zranb3+/− Eμ-myc, and Zranb3−/− Eμ-myc mice (also see Table 1) using fluorochrome-linked antibodies specific for B cell surface markers (indicated).
Figure 3.
Figure 3.. Smarcal1-deficient B cells have increased rates of replication fork collapse with Myc dysregulation.
B cells from Smarcal1+/+ Eμ-myc, Smarcal1+/Δ Eμ-myc, and Smarcal1Δ/Δ Eμ-myc mice were purified from bone marrow (A-C, G, I) or spleen (K, M). Pro-B cell cultures from Smarcal1+/+, Smarcal1+/Δ, and Smarcal1Δ/Δ mice expressing MycER (D-F, H, J, L, N) with MycER activated by the addition of 4-hydroxytamoxifen (4-OHT) for 8 hours. (A, D) Design of DNA fiber labeling experiments and images of representative fiber tracks. B, E) Quantification of total fiber length in purified Eμ-myc B cells (B) and MycER pro-B cells (E); median indicated with red bar. C, F) Fiber length frequencies from (B) and (E), respectively. G, H) Quantification of DNA fibers that only incorporated IdU from Eμ-myc B cells (G) and MycER pro-B cells (H); SEM. I, J) Ratios of measured CldU track lengths from left and right moving forks from the same replication origin from Eμ-myc B cells (I) and MycER pro-B cells (J); median indicated with red bar. K, L) Quantification of immunofluorescence of γH2AX foci in Eμ-myc B cells (K) and MycER pro-B cells (L); SEM. M, N) Box-and-whisker plots of tail moments of Eμ-myc B cells (M) and MycER pro-B cells (N). Line is the median, boxes are the 25th and 75th percentiles, whiskers are the 5th and 95th percentiles. The total number (n) of fibers (B, E), replication structures (G-J), or cells (K-N) analyzed is indicated. All data are from 2–3 independent experiments. One-way ANOVA with Bonferroni correction (B, E, M, N, I, J) or chi-square test (G, H, K, L); *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.
Figure 4.
Figure 4.. Zranb3 protects replication forks during Myc-induced replication stress.
B cells from Zranb3+/+ Eμ-myc, Zranb3+/− Eμ-myc, and Zranb3−/− Eμ-myc mice were purified from bone marrow (A-C, G, I, K, M). Pro-B cells from Zranb3+/+, Zranb3+/− and Zranb3−/− mice expressing MycER (D-F, H, J, L, N) with MycER activated by the addition of 4-hydroxytamoxifen (4-OHT) for 8 hours. (A, D) Design of DNA fiber labeling experiments and images of representative fiber tracks. B, E) Quantification of total fiber length in purified Eμ-myc B cells (B) and MycER pro-B cells (E); median indicated with red bar. C, F) Fiber length frequencies from (B) and (E), respectively. G, H) Quantification of DNA fibers that only incorporated IdU from Eμ-myc B cells (G) or pro-B cells (H); SEM. I, J) Ratios of measured CldU track lengths from left and right moving forks from the same replication origin from Eμ-myc B cells (I) and MycER pro-B cells (J); median indicated with red bar. K, L) Quantification of immunofluorescence of γH2AX foci in Eμ-myc B cells (K) and MycER pro-B cells (L); SEM. M, N) Box-and-whisker plots of tail moments of Eμ-myc B cells (M) and MycER pro-B cells (N). Line is the median, boxes are the 25th and 75th percentiles, whiskers are the 5th and 95th percentiles. The total number (n) of fibers (B, E), replication structures (G-J), or cells (K-N) analyzed is indicated. All data are from 2–3 independent experiments. One-way ANOVA with Bonferroni correction (B, E, M, N, I, J) or chi-square test (G, H, K, L); *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.
Figure 5.
Figure 5.. Loss of Smarcal1 reduces cell cycle progression of B cells, whereas loss of Zranb3 does not when Myc is overexpressed.
A, C) Quantification of total splenic leukocytes from litters of the indicated genotypes. B, D) Representative contour plots and quantification of mature (B220+/IgM+) B splenocytes from litters of the indicated genotypes. E) Quantification of BrdU+ splenic B cells (CD19+; left) and the percentage of purified B cells in each phase of the cell cycle (right) from litters of the Eμ-myc mice of the indicated Smarcal1 genotype. Cell cycle was determined using Dean-Jett-Fox analysis. F) Quantification of BrdU+ splenic B cells (CD19+) from litters of the Eμ-myc mice of the indicated Zranb3 genotype. Total number (n) of mice indicated; data are from 3–7 independent litters. Error bars are SEM; *p<0.05, one-way ANOVA with Bonferroni post-test.
Figure 6.
Figure 6.. Loss of Smarcal1 or Zranb3 sensitizes B cells to Myc-induced apoptosis.
Bone marrow derived pro-B cells expressing MycER of the indicated genotypes. MycER was activated with 4-hydroxytamoxifen (4-OHT) and at intervals MTS assays (A, F), cell numbers and viability (B, G), Annexin-V positivity (C, H), and sub-G1 DNA content (D, I) were evaluated. Student’s t-tests determine significance, *p<0.0001. E, J) Western blots of whole cell lysates for the indicated proteins at intervals after MycER activation with 4-OHT.

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