The CHD proteins have distinct structural motifs that implicate specific functional roles in a variety of DNA transactions that include replication, transcription, and DNA repair. To further study the physiological role of Chd2 in a mammalian model, we generated Chd2 mutant mouse model using the Baygenomics gene trap ES cell resource. Characterization of the gene-trap used in the generation of the Chd2 mutant mouse model indicated that it was not completely effective in disrupting the expression of the Chd2 gene suggesting the possibility of a hypomorphic or a dominant negative Chd2 mutant mouse model. We have shown via protein interaction studies that the truncated Chd2-β-gal-neomycin has a potential to interact with the native Chd2 protein. However our studies do not rule out the possibility that the mutant Chd2 fusion protein can either act as a dominant negative mutant or a gain of function mutant. Future studies aimed at identifying functional partners of Chd2 will allow us to determine the effect of this mutation. The expression of wild type CHD2 mRNA in the homozygous mutant cells is in contrast to the results reported earlier (Marfella et al., 2006
) and we believe that the discrepancy is due to the low number of cycles in the RT-PCR analysis used by the other group in comparison to ours (26 cycles versus 30 cycles in this study). Our results indicate that the Chd2 mutation leads to pleiotropic effects that impinge on hematopoietic and lymphoid development pathways in mammals. We have shown that the CHD2 protein is involved in the regulation of hematopoietic stem cell differentiation and its loss may lead to an imbalance in the various downstream compartments that include the erythroid, myeloid and lymphoid compartments. Interestingly, earlier studies have shown the importance of differentiation and cell type specific transcriptional programming during the terminal differentiation of hematopoietic cells and our studies point to the role of chromatin remodeling and its effects on transcription on hematopoietic stem cell differentiation (Heyworth et al., 2002
; Iwasaki et al., 2003
; Ney, 2006
More importantly, the Chd2 mutant mice develop primarily lymphomas and lymphoid hyperplasias. A few of the phenotypes we have described in this report are similar to the ones reported in a recent study involving the phenotypic characterization of the Chd2 mutant mouse model (Marfella et al., 2006
). However, the earlier study has not reported and susceptibility of lymphomas in the Chd2 mutant mice and the differences between the two studies may relate to the fact that our study is more extensive that involved the analysis of a larger set of mutant animals. While the other study does report the presence of lymphoid hyperplasia (a precursor for lymphomas) in the mutants, the reasons for the differences in lymphoma diagnoses between the two studies are yet to be ascertained. Interestingly, the human CHD2 chromosomal locus (15q26.2) is also implicated in a rare genetic disorder that leads to growth retardation, cardiac defects, and early post natal lethality (Whiteford et al., 2000
; Wilson et al., 1985
). The data we have compiled on human chromosomal aberrations provide preliminary evidence that the Chd2 protein may play a role in the etiology of human lymphoid tumors. Furthermore and consistent with our observations, the recent characterization of a T-cell Hodgkin lymphoma cell line using array comparative genomic hybridization (aCGH) analysis has also shown the homozygous loss of the Chd2 chromosomal locus (Feys et al., 2007
). The above mentioned data and the enhanced tumor susceptibility of the Chd2 heterozygous mice raise the possibility that CHD2 is a potential tumor suppressor gene involved in the suppression of lymphomas.
Our data also show that the Chd2 protein affects DNA damage signaling and processing at the chromatin level by modulating the levels of γH2AX induced by DNA damage. While several studies have shown that a decrease in the γH2AX foci often mirrors a decrease in the number of DNA strand breaks, we cannot rule out the possibility that the persistence of γH2AX foci may relate to the inability of the Chd2 mutant cells to displace γH2AX subsequent to DNA repair (Banath & Olive, 2003
; Jin et al., 2005
; Lukas et al., 2004
; Rogakou et al., 1998
; Rothkamm et al., 2003
). Consistent with this notion, a recent study has shown that the removal of γH2AX after DNA damage is mediated by the Tip60 chromatin remodeling complex (Kusch et al., 2004
). In addition, DNA damage processing in lower eukaryotes is mediated by the INO80 complex and this complex requires the HMG1 domain containing Nhp10 subunit protein for its interaction with the γH2AX (Morrison et al., 2004
; Tsukuda et al., 2005
; van Attikum et al., 2004
). Interestingly, the Chd2 protein contains a similar domain and the ability of Chd1 to transfer histones to DNA also suggests a parallel and mutually exclusive role for CHD2 in the removal of γH2AX (Lusser et al., 2005
). Whether Chd2 plays a functional role in γH2AX removal during the attenuation of DNA damage response or directly affects DNA repair processes remains to be seen.
The functional roles of CHD family members and other chromatin remodeling proteins in transcriptional regulation have been well established. However, our data suggest that the CHD2 protein may play an additional role in DNA damage signaling besides affecting transcription. Determining the tissue specific transcriptional targets and the role of Chd2 in downstream DNA damage response pathways will provide further insights on its functions in development, hematopoietic stem cell differentiation, and tissue specific tumor suppression.