Temporal modulation of the NF-κB RelA network in response to different types of DNA damage.

Different types of DNA damage can initiate phosphorylation-mediated signalling cascades that result in stimulus specific pro- or anti-apoptotic cellular responses. Amongst its many roles, the NF-κB transcription factor RelA is central to these DNA damage response pathways. However, we still lack understanding of the co-ordinated signalling mechanisms that permit different DNA damaging agents to induce distinct cellular outcomes through RelA. Here, we use label-free quantitative phosphoproteomics to examine the temporal effects of exposure of U2OS cells to either etoposide (ETO) or hydroxyurea (HU) by monitoring the phosphorylation status of RelA and its protein binding partners. Although few stimulus-specific differences were identified in the constituents of phosphorylated RelA interactome after exposure to these DNA damaging agents, we observed subtle, but significant, changes in their phosphorylation states, as a function of both type and duration of treatment. The DNA double strand break (DSB)-inducing ETO invoked more rapid, sustained responses than HU, with regulated targets primarily involved in transcription, cell division and canonical DSB repair. Kinase substrate prediction of ETO-regulated phosphosites suggest abrogation of CDK and ERK1 signalling, in addition to the known induction of ATM/ATR. In contrast, HU-induced replicative stress mediated temporally dynamic regulation, with phosphorylated RelA binding partners having roles in rRNA/mRNA processing and translational initiation, many of which contained a 14-3-3ε binding motif, and were putative substrates of the dual specificity kinase CLK1. Our data thus point to differential regulation of key cellular processes and the involvement of distinct signalling pathways in modulating DNA damage-specific functions of RelA.

Missense mutations that cause Van der Woude syndrome and popliteal pterygium syndrome affect the DNA-binding and transcriptional activation functions of IRF6.

Cleft lip and cleft palate (CLP) are common disorders that occur either as part of a syndrome, where structures other than the lip and palate are affected, or in the absence of other anomalies. Van der Woude syndrome (VWS) and popliteal pterygium syndrome (PPS) are autosomal dominant disorders characterized by combinations of cleft lip, CLP, lip pits, skin-folds, syndactyly and oral adhesions which arise as the result of mutations in interferon regulatory factor 6 (IRF6). IRF6 belongs to a family of transcription factors that share a highly conserved N-terminal, DNA-binding domain and a less well-conserved protein-binding domain. To date, mutation analyses have suggested a broad genotype-phenotype correlation in which missense and nonsense mutations occurring throughout IRF6 may cause VWS; in contrast, PPS-causing mutations are highly associated with the DNA-binding domain, and appear to preferentially affect residues that are predicted to interact directly with the DNA. Nevertheless, this genotype-phenotype correlation is based on the analysis of structural models rather than on the investigation of the DNA-binding properties of IRF6. Moreover, the effects of mutations in the protein interaction domain have not been analysed. In the current investigation, we have determined the sequence to which IRF6 binds and used this sequence to analyse the effect of VWS- and PPS-associated mutations in the DNA-binding domain of IRF6. In addition, we have demonstrated that IRF6 functions as a co-operative transcriptional activator and that mutations in the protein interaction domain of IRF6 disrupt this activity.