Menin Is Phosphorylated in Response to DNA Damage Figure 1. Menin is associated with the 59 regions of DNA damage responsive genes in the absence of damage. (A) ChIPs were performed in untreated U2OS cells with antibodies for menin, or RNA polymerase II (RNAPII) or no antibody (-Ab) as a background control. Precipitated chromatin was used for quantitative real-time PCR using primers that amplify 59 regions of the indicated genes. Results are the average of at least 3 independent experiments and represent the amount of DNA immunoprecipitated with each antibody relative to the amount of input DNA. Error bars indicate standard error of the mean. (B) Scatter plot showing the correlation between menin and RNAPII at the 59 regions of the indicated genes. (C) Menin immunoprecipitations (IPs) were performed with anti-menin antibody over the course of 3 hours in 293T cells treated with either 1000 Rads of c-IR or 25J/m2 UV. The resulting immunocomplexes were resolved and immunoblotted with antibodies targeting phosphorylated residues within the carboxyl-terminal domain (CTD) of RNAPII. doi:10.1371/journal.pone.0016119.g001 Menin is phosphorylated in response to DNA damage We next wanted to explore whether post-translational modifi- cations could be mediating the increases in menin association with specific gene loci after DNA damage. Large-scale mass spectrom- etry studies found menin to be phosphorylated at Ser394 (Ser399 in isoform1) in response to c-IR and UV treatment [29,30]. To expand upon this finding, we immunoprecipitated endogenous menin from 293T cells following treatment with different means of inducing DNA damage and then used mass spectrometry to identify phosphorylation sites. DNA damage-specific phosphory- lation was detected at Ser394, a residue in the region containing the SQ/TQ cluster. A new phosphorylation site at Ser487 was identified in all samples regardless of DNA damage as well as the previously reported site at Ser543 (Figure S2) [26]. Ser394 resides within an SQ/TQ cluster, the motif recognized by DNA damage transducing kinases. To ascertain whether or not nearby Thr397 or Ser399 were also phosphorylated, we generated the Ser394Ala substitution and performed an immunoprecipita- tion on Flag-tagged-Ser394Ala menin purified after treatment with the topoisomerase II inhibitor adriamycin (Adr). Mass spectro- metric analysis revealed the presence of a phospho-peptide within the cluster suggesting that either Thr397 or Ser399 or both may also be phosphorylated after DNA damage. This finding is important because Ser394 is conserved in larger mammals but not conserved in rodents (Figure S2A and S2C). Mass spectrometry also detected phosphorylation at the conserved Ser487 and Ser543 sites on menin immunopurified from the mouse islet b-cell line bTC3 under normal cell culture conditions suggesting that these sites may be functionally important (Figure S2B). In order to investigate the significance of these phosphorylation sites, phospho-specific antibodies were generated against phospho- peptides corresponding to each of these sites. To examine the function of each phosphorylation site, and to confirm the specificity of the phospho-specific antibodies, we generated Flag- tagged menin mutants with non-phosphorylatable Serine to Alanine substitutions at each site. We were unable to detect a signal with the phospho-Ser394 and phospho-Ser487 antibodies in Flag immunoprecipitations from cells expressing the Ser394Ala and Ser487Ala mutants, respectively, demonstrating antibody specificity (Figure 4B and S3A). Cells transiently expressing these phospho-deficient mutants were treated with Adr, UV or c-IR and differences in phosphorylation at the remaining unsubstituted PLoS ONE | www.plosone.org 3 January 2011 | Volume 6 | Issue 1 | e16119