analyzed. Menin immunoprecipitations were tested for HMT activity through the transfer of a radiolabelled methyl group to histone H3 (Figure 6B). We found that the Ser394Ala, Ser487Ala, Ser394/Ser487Ala and Ser394/Thr397/Ser399Ala menin mu- tants were still able to immunoprecipitate HMT activity. The MEN1-associated missense mutant A309P maintained the inter- action with HMT activity while the A242V mutant did not, consistent with previous reports [12]. These results strongly suggest that Ser394 and Ser487 phosphorylation do not mediate or interfere with the interaction between menin and KMT2A/ KMT2D. We also detected a number of peptides corresponding to KMT2A/KMT2D in menin immunoprecipitations following treatment with c-IR and UV (Figure S5). The Ser543Ala mutant has been previously reported to not impact the interaction of menin with KMT2A and KMT2B as well [26]. A number of different approaches were undertaken to gain insight into the functional significance of these menin phosphor- ylation sites including BrdU incorporation and proliferation in Men12/2 MEF cells expressing the Ser-to-Ala menin mutants; however, the mutants did not display significant alterations compared to wild type menin in these assays. We also examined changes in DNA damage-induced Cdkn1A expression and cell cycle effects in these MEF cells and were unable to detect any differences between menin MEFs and those expressing wildtype or Ser-to-Ala mutations (data not shown). We next looked to see if Ser394, Ser487 and Ser543 played a role in the interaction with RNAPII. Flag-tagged-wild type, - Ser394Ala, -Ser487Ala, or -Ser543Ala forms of menin were immunoprecipitated from 293T cells following treatment with either Adr or UV and the resulting complexes were immuno- blotted for phosphorylated RNAPII. Under the conditions tested, phosphorylation of Ser-394 or Ser-487 did not correlate with either increased or decreased ability of menin to interact with RNAPII. The Flag-Ser543Ala menin mutant had a higher affinity for pSer2-RNAPII while the Flag-Ser394Ala and Ser487Ala mutants exhibited a modest increase in affinity for pSer2-RNAPII when compared to wild type menin in the absence of DNA damage (Figure 6C). Menin does not colocalize with cH2Ax foci Several reports have suggested that menin may be involved in DNA repair [21,24,25]. To investigate a potential role for menin in the DNA repair process we performed immunofluorescence on cells treated with c-IR, UV and HU and looked for menin relocalization to sites of DNA damage as visualized using phospho- Ser139 histone H2Ax (cH2Ax). We were unable to observe menin colocalization at cH2Ax foci in HeLa cells (Figure 7A) or in a number of other cell lines tested with additional menin antibodies (data not shown). An increase in menin staining at the nuclear matrix is observed in the c-IR and UV treated cells consistent with previous reports [21,32]. We attempted to visualize menin at DNA-damage foci using phospho-menin antibodies but the antibody specificity was not sufficient to distinguish between menin and other phosphorylated SQ motifs. Upon further investigation it was found that menin colocalized under all treatment conditions with SC35, a protein found within nuclear speckle domains in the nucleus (Figure 7B) [33]. Discussion Menin has long been implicated in the DNA damage response, either through direct interactions with DNA repair factors like FANCD2 and RPA or in the activation of genes involved in cell- cycle arrest or apoptosis [21,22,23,24,25]. Our results show that Menin Is Phosphorylated in Response to DNA Damage menin functions in the transcriptional response to DNA damage. We cannot conclusively determine whether or not menin plays an essential role in DNA repair given that functional experiments such as proliferation experiments in response to DNA damage showed no effect, and menin did not colocalize with c-H2Ax foci, however it is possible that the conditions and cell lines tested in the study were not sensitive to menin perturbation. ChIP experiments have found that menin is associated with the 59 regions of many DNA damage responsive genes in the absence of damage and this is suggestive of a mechanism where these important transcription components are ‘‘pre-loaded’’ in order to allow for immediate activation upon DNA damage [34]. Interestingly, we were able to see an interaction between menin and both the initiating (pSer5) and elongating (pSer2) forms of RNAPII and these data along with our ability to detect menin within the 39 regions of these genes suggests that menin may associate with an elongation complex. Additional support for a role in elongation is provided by the colocalization of menin with nuclear speckle bodies as denoted by SC35. Nuclear speckles have been shown to contain both elongation and splicing factors, and menin could be interacting with any of the proteins involved in these nuclear bodies [35]. Furthermore, under conditions of elevated transcription, and increased menin association with promoter regions, we were unable to detect significant changes in histone methylation. This is suggestive of a function outside of histone methylation and consistent with the model proposed by Bres et al. whereby menin can activate transcription independently of KMT2A (MLL) [19]. It is also important to note that menin association with 59 regions did not correlate with p53 binding suggesting that the two transcription regulatory factors function independently. During the course of these studies we were able to confirm that menin undergoes a DNA damage dependent phosphorylation at Ser394. When Ser394 was mutated to an alanine, phosphorylation was also detected at either Thr397 or Thr399 suggesting that any SQ/TQ cluster can be modified. We were also able to identify an additional phosphorylation site at Ser487 and to confirm the phosphorylation of Ser543 [26]. Phosphorylation of Ser487 was dynamically regulated after DNA damage suggesting a tightly regulated function for this modification. Ser543 did not appear to undergo any noticeable change in phosphorylation level in the presence or absence of DNA damage and may be a constitutive phosphorylation site. The presence of Ser487 and Ser543 phosphorylation on menin purified from a mouse b cell line suggest that these modifications are likely to be important in menin function in the neuroendocrine lineages. MEN1-associated missense menin point mutants can be classified based upon their ability to interact with KMT2A/D and it is believed that those mutants that retain the ability to interact with histone methyltransferase complexes have defects in other functions [14]. Surprisingly, we observed that most of the point mutants that had HMT activity failed to be phosphorylated at Ser394 following Adr and UV treatment. The presence or absence of phosphorylation at these sites does not appear to aid or interfere with the ability of menin to interact with KMT2A/D or RNAPII and is supportive of the concept that these tumor mutants are defective in other functions. It is believed that phosphorylation of these sites may be important for stabilizing or destabilizing protein complexes between menin and other currently unknown factors, however what those complexes are remains elusive. It is also possible that menin Ser394 phosphorylation occurs solely because the DNA damage transducing kinases ATM, ATR and DNA-PK are promiscuous and will phosphorylate any protein containing an SQ/TQ cluster whether they are functionally important or not. The overall significance of these menin PLoS ONE | www.plosone.org 9 January 2011 | Volume 6 | Issue 1 | e16119