ATM-Dependent Phosphorylation of Nemo SQ Motifs is Dispensable for Nemo-Mediated Gene Expression Changes in Response to DNA Double Strand Breaks

In response to DNA double strand breaks (DSBs), the ATM kinase activates NF-?B factors to stimulate gene expression changes that promote survival and allow time for cells to repair damage. In cell lines, ATM can activate NF-?B transcription factors via two independent, convergent mechanisms. One is ATM-mediated phosphorylation of nuclear NF-?B essential modulator (Nemo) protein, which leads to monoubiquitylation and export of Nemo to the cytoplasm where it engages the I?B kinase (IKK) complex to activate NF-?B. Another is DSB-triggered migration of ATM into the cytoplasm where it promotes monoubiquitylation of Nemo and resulting IKK-mediated activation of NF-?B. ATM has many other functions in the DSB response beyond activation of NF-?B, and Nemo activates NF-?B downstream of diverse stimuli, including developmental or proinflammatory stimuli such as lipopolysaccharides (LPS). To elucidate the in vivo role of DSB-induced, ATM-dependent changes in expression of NF-?B-responsive genes, we generated mice expressing phosphomutant Nemo protein lacking consensus SQ sites for phosphorylation by ATM or related kinases. We demonstrate that these mice are viable/healthy, fertile, and exhibit overall normal B and T lymphocyte development. Moreover, treatment of their B lineage cells with LPS induces normal NF-?B-regulated gene expression changes. Furthermore, in marked contrast to results from a pre-B cell line, primary B lineage cells expressing phosphomutant Nemo treated with the genotoxic drug etoposide induce normal ATM- and Nemo-dependent changes in expression of NF-?B-regulated genes. Our data demonstrate that ATM-dependent phosphorylation of Nemo SQ motifs in vivo is dispensable for DSB-signaled changes in expression of NF-?B-regulated genes. Overall design: RNA-Seq was performed on isolated CD19+ splenocytes from WT, Nemo3SA?T, or Mb1cre+Nemoflox (MN) mice