Role of chromodomain helicase DNA-binding protein 2 in DNA damage response signaling and tumorigenesis

Abstract

The chromodomain helicase DNA-binding proteins (CHDs) are known to affect transcription through their ability to remodel chromatin and modulate histone deacetylation. In an effort to understand the functional role of the CHD2 in mammals, we have generated a Chd2 mutant mouse model. Remarkably, the Chd2 protein appears to play a critical role in the development, hematopoiesis and tumor suppression. The Chd2 heterozygous mutant mice exhibit increased extramedullary hematopoiesis and susceptibility to lymphomas. At the cellular level, Chd2 mutants are defective in hematopoietic stem cell differentiation, accumulate higher levels of the chromatin-associated DNA damage response mediator, gamma H2AX, and exhibit an aberrant DNA damage response after X-ray irradiation. Our data suggest a direct role for the chromatin remodeling protein in DNA damage signaling and genome stability maintenance.

Figure 1. Analysis of developmental phenotypes in Chd2 mutant mice

a, Morphological phenotypes of Chd2 mutant embryos. E12.5 embryos were harvested and photographed. b, Hematopoietic cell distribution and organization defects in mutant neonates. Representative images of hemotoxylin and eosin (H&E) stained sections of WT and homozygous mutant neonatal liver sections are shown. The megakaryocytes are circled. c, Increased megakaryocytes in Chd2 mutant mice. A total of 10 different fields were counted from hematoxylin and eosin stained sections of neonatal fetal livers from each group (n=7). The differences between the wild-type and the mutants were statistically significant as determined by single tailed t-test (WT v. +/m P<0.002 and WT v. m/m P<0.005). Error bars represent s.e. d, Hematopoietic stem cell differentiation defects in Chd2 mutants. Burst forming unit —erythroid (BFU-E) and Colony-forming unit-granulocyte macrophage (CFU-GM) formation in wild-type (n=5), Chd2^+/m (n=5) and Chd2^m/m (n=8) were assayed using E13.5 fetal liver progenitor cells as described. The differences between the wild-type and the homozygous mutants were statistically significant as determined by single tailed t-test (WT v. m/m P<0.00005). Error bars represent s.e.

Figure 2. Lymphoid tumor susceptibility and hematopoietic defects in Chd2 mutant mice

a, Kaplan-Meier survival curves of Chd2 mutant and wild-type littermates (n=50). The percentages of survival are plotted as a function of age in weeks. Animals were monitored for tumors, morbidity, or spontaneous death over a period of 105 weeks. Of the 74 animals analyzed for each group, 50 of the heterozygous mutants have died in comparison to 6 for the wild-type controls during a period of two years. All mice were of mixed inbred C57BL/6X129/Sv background. b-f, Representative images of hematoxylin and eosin stained sections of a normal spleen from an age matched wild-type mouse (b), lymphoid hyperplasia (c), lymphoma (d and e) and extra medullary hematopoiesis from heterozygous mice (f) is shown.

Figure 3. Characterization of lymphoid hyperplasias and lymphomas in Chd2 mutant mice

a, Total number of activated T cells from the spleens of age-matched wild type, Chd2 mutants with hyperplasia, and Chd2 mutants with lymphoma. b, Total number of B cells from spleens of indicated mice. c, Representative FACS profile showing CD4 and CD44 expression of cells from the lymph nodes of wild type, Chd2 heterozygous mutant with lymphoid hyperplasia, and Chd2 heterozygous mutant with lymphoma. In each group 3-4 mice were used; ** indicates p<0.01 and *** indicates p<0.001.

Figure 4. Defective DNA damage response in Chd2 mutant cells

Wild-type and Chd2 homozygous mutant littermate mouse embryonic fibroblasts (MEFs) were treated with 4Gy X-ray irradiation and analyzed for γH2AX foci formation at 30 minutes, 3 (data not shown) and 6 hours. For immuno-fluorescence analysis, wild-type and mutant cells were grown in different chambers on the same slide. Results were confirmed with four experiments using two sets of independently derived cell lines. At least 30 cells were analyzed per experiment. All cells were photographed using the same exposure time and microscope settings. Bar equals 10 microns.