In this study we evaluated the transcriptional response induced by physiologic and genotoxic DSBs in developing B cells. By comparing these different types of damage we found that there are important similarities as well as striking differences in the cellular responses to these different forms of DNA lesions.
In previous work [6
], we observed a lymphocyte-specific response to physiologically generated RAG-induced dsDNA breaks. Highlights of this response include changes in the expression of genes important in immune function and maturation. Changes in these genes suggest an increase in the signalling of the CD40 and NFκB pathways, suggesting a role for DNA breaks in the progression of B cell maturation. After the observation that RAG-induced DSBs trigger a response to move the B cells toward maturation, the next obvious question is whether or not other types of DSBs induce the same lymphocyte-specific maturation profile. Here we observed 288 probes that were differentially regulated in the same direction after induction of breaks regardless of the source of the damage. These changes include increased expression of Cd40
, as well as other immune related genes. Increased expression of these genes and others associated with Cd40-, Cd69-, and NFκB-related pathways suggest that the B cells are preparing to undergo maturation irrespective of the source of the DNA damage. While a core set of genes is regulated in the same direction after both types of damage, the response to genotoxic damage is generally more robust than the response to RAG-induced breaks. We hypothesize this is due to the greater amount of DNA damage induced by IR exposures, perhaps initiating a stronger signal towards maturation.
While we see similarities in the response to physiologic and genotoxic breaks, we recognize there are potential biological and technical differences in comparing RAG-induced and ionization radiation-induced DSBs. RAG-induced DSBs are tightly regulated and only a small number of breaks in very specific locations in the genome are induced. In contrast, IR exposure can induce a broad range of DNA damage, including DSBs, that is not restricted to specific locations but occurs throughout the genome and can involve DNA lesions with damaged nucleotide ends as opposed to the “clean” ends generated by the RAG endonucleases. We have attempted to mitigate these differences by using exposures to a low dosage of IR and by ensuring the cells are in the same phase of the cell cycle to ensure that similar DNA damage repair processes would be available under both conditions. Despite the differences in the nature of the DSBs it appears that the cells undergo a similar response to that damage by initiating a central lymphocyte-specific transcriptional response that is common to both.
In addition to these similarities, the genotoxic damage also induces changes in 1694 unique probes representing almost 900 genes. Broad expression changes in transcription factors and protein kinases suggest genotoxic DSBs induce a myriad of changes in both gene expression and physiological pathways. Gene expression changes and alterations in pathways associated with B cell activation, increased proliferation, and oxidative stress responses are seen in the unique response to IR. Many of the pathways altered on a transcriptional level are known to be involved in the generation of cancers. Also, this study highlights the importance of additional layers of regulation, which has become obvious with the discovery of genotoxic regulation of small regulatory RNAs such as microRNAs (miRNAs). Their role in a wide variety of physiological processes has revealed their vital importance in proper cellular function, and disregulation has been linked to human diseases, including cancer and immune disorders (reviewed in [22
In addition to the lymphocyte specific transcriptional pattern induced by both types of DNA damage, genotoxic damage induces a potentially oncogenic combination of alterations of genes and biological response pathways. We found that genotoxic, but not physiologic, damage induces increased expression of several proto-oncogenes such as Kras
and the oncomiR miR-155. This suggested to us that the cellular response to double strand DNA damage could be specific to the method of generation of that damage, recognizing that IR-induced genotoxic damage causes many types of DNA damage. miR-155 is a known oncogenic miRNA and its increase has been correlated with formation of B cell malignancies. The up-regulation of miR-155, at both the primary transcript and mature microRNA level, seen after IR-induced breaks suggests a potential for the development of cancer after genotoxic damage. Additionally, miR-155 has been identified to suppress a number of tumor suppressors, including Socs1
, which we found to be suppressed after genotoxic damage. MiR-155 up-regulation has been associated with B cell cancers and B cell transformation [11
], as well as with normal immune response [26
]. Altered expression of this miRNA and its target Socs1
suggests that an increase in proliferation may be triggered after IR exposure. Interestingly, this increase in proliferation and up-regulation of miR-155 has been seen in mature B cells as a result of their response to antigen [27
Another noteworthy difference between genotoxic and physiologic damage is the significant change in regulation of genes in B cell activation pathways and Nrf2-mediated signalling. Nrf2 signalling is a known response to IR but has also been seen in the activation of mature B cells [19
]. The Nrf2 pathway is a critical regulator of the defense against oxidative stress. Activation of Nrf2 pathways is an important component in the clearing of oxidative stress and in a cytoprotective outcome. There has been some suggestion that Nrf2 also has a role in rescuing cells from cell cycle arrest that can be generated in response to oxidative damage [30
]. These responses to genotoxic damage in both the B cell activation and Nrf2 signalling pathways could combine to result in serious deleterious consequences to the immune system.