cells with extensive DNA damage [20]. Menin has often been implicated in the DNA damage response and studies using Men1-/-MEFs have shown that these cells are hypersensitive to intrastrand crosslinking agents and fail to activate cellular checkpoints after c-irradiation (c-IR) [21,22]. Menin has been found to interact with p53-containing complexes and to function as a transcriptional activator of CDKN1A (p21) after c-IR treat- ment [22,23]. Several pieces of evidence also support a role for menin in DNA repair including interactions with FANCD2 and RPA [21,24]. Men1 deficient Drosophila mutants were found to be hypermutable in response to DNA damage [25]. Menin is phosphorylated at two Ser residues, Ser543 and Ser583, and it is possible that post-translational modifications may mediate distinct cellular functions [26]. In this study we describe menin association with 59 regions of DNA repair and cell cycle genes in the absence of DNA damage. Following DNA damage, we observed a significant increase in menin association with these genes at both the 59 and 39 regions. We also confirm that menin is phosphorylated in response to DNA damage at Ser394 and describe a novel phosphorylation site at Ser487 that is dynamically regulated following different forms of DNA damage. A subset of MEN1-associated missense point mutants failed to become phosphorylated after DNA damage and may link altered tumor suppressor function to the DNA damage response. The post-translational modification of menin may play an as yet undetermined functional role in the tumor suppressor function of menin. Results Menin is associated with the 59 regions of genes involved in DNA repair The tumor suppressor protein menin has been best character- ized as a transcription regulatory factor. We utilized chromatin immunoprecipitation (ChIP) assays as a means to investigate a role for menin in the DNA damage response. The DNA damage induced transcription response is mediated in large part through the tumor suppressor p53. Upon activation and stabilization, p53 can increase the expression of genes involved in cell-cycle arrest, DNA repair and apoptosis among others [27]. ChIP assays in the p53 wildtype expressing cell line U2OS revealed that even in the absence of DNA damage, menin is found to associate with the 59 regions of genes involved in cell cycle control (CDKN1A and GADD45A), p53-regulation (MDM2) and apoptosis (BBC3, TP53I3, and FAS) (Figure 1A). A very low amount of detectable menin was associated with the previously identified target gene CDKN1B suggesting a cell-type specific role for menin association with this locus [14]. The non-expressed hemoglobin HBG1 gene is shown for comparison. While varying amounts of RNA polymerase II (RNAPII) were observed at the 59 regions of these genes, a strong correlation exists between menin and RNAPII association at these genes (Figure 1B). We next sought to examine the interaction between menin and RNAPII before and after DNA damage. Previous co-immuno- precipitation studies had revealed that menin was able to interact with RNAPII that was phosphorylated at Ser5 within the carboxyl-terminal domain (CTD) of the large subunit of RNAPII and more weakly with RNAPII phosphorylated at Ser2 of the CTD [12], however by including a sonication step in the lysate preparation we were now able to see a more robust interaction between menin and RNAPII phosphorylated at Ser2 (Figure 1C). The interaction between menin and RNAPII was present before treatment (untreated) and remained intact for at least 3 hours following c-irradiation (c-IR). Interestingly, the interaction between Menin Is Phosphorylated in Response to DNA Damage menin and pSer2-RNAPII is reduced within 15 minutes and remains diminished until about 2 hours post-exposure. During the same timecourse after c-IR, the interaction between menin and pSer5-RNAPII is initially increased followed by a gradual decline. However exposure to 25 J/m2 of ultraviolet (UV) irradiation resulted in a transient increase in the association of menin and pSer2- and pSer5-RNAPII until approximately 2 hours, after which time the interaction was diminished presumably due to UV-induced RNAPII degradation (Figure 1C). DNA damage triggers an increase in menin association at DNA damage responsive genes After finding a correlation between menin and RNAPII association with DNA damage response genes, we next wanted to look at the effects DNA damage would have on menin localization. RNA was harvested from U2OS cells treated with 1000 Rads of c-IR or 10 J/m2 UV after 0.5 h and 6 h to look at time dependent changes in transcription. Quantitative RT-PCR showed that the mRNA levels of the cell cycle regulator Cyclin- Dependent Kinase Inhibitor 1A gene (CDKN1A) were elevated 7- fold at 6 hours after c-IR treatment while we were unable to detect a change in CDKN1A mRNA after UV (Figure 2A). Immunoblots of U2OS cells under the same conditions show phosphorylation of p53 at Ser15 indicating that the DNA damage response was initiated following c-IR and UV treatment (Figure 2B). We next performed ChIP after c-IR and UV treatment to understand differences in early and late menin association with the CDKN1A locus. Following c-IR, a time dependent increase in menin association was observed at the proximal promoter that correlated with increased p53 association at the upstream p53 binding sites (Figure 2C). UV treatment revealed a strikingly different pattern in association whereby an increase in menin was found after a short incubation time and was dramatically reduced after 6 hours mirroring what was observed for RNAPII (Figure 2D). Interestingly, we were able to consistently detect an association of menin along the length of the locus under conditions of high expression. Histone H3 tri-methyl-lysine 4 (histone H3- K4me3) levels 6 hours after c-IR were not significantly altered (Figure S1). To further examine the role of menin in DNA damage- dependent transcription, we looked at menin association along two additional DNA damage responsive genes including, the Growth Arrest and DNA-Damage-inducible Alpha gene (GADD45A) and the Mouse Double Minute 2 (MDM2) gene. GADD45A mRNA levels were found to be quickly elevated following both c-IR and UV treatment (Figure 3A). ChIP studies revealed that menin was significantly increased at the GADD45A locus 6 hours after c-IR and 30 min after UV, consistent with a gene undergoing induction of expression (Figure 3B and C). Analysis of MDM2 expression revealed an increase in mRNA levels after c-IR and only a modest short-term increase after UV treatment (Figure 3D). ChIP studies found that menin was significantly enriched at the 59 regions of the MDM2 locus 6 hours after treatment with c-IR and 30 min after UV treatment (Figure 3E and F). Menin association with the 39 region of the MDM2 locus was observed after UV treatment but not following c-IR (Figure 3E and F). We were able to use shRNA to knockdown menin in U2OS cells, however we were unable to consistently see an effect on CDKN1A, MDM2 or GADD45A transcription following DNA damage. One possible interpretation of the lack of transcriptional consequences of MEN1 knockdown may be attributed to the off- target effects of MEN1 RNAi described previously [28]. PLoS ONE | www.plosone.org 2 January 2011 | Volume 6 | Issue 1 | e16119