O-GlcNAcylation of MLL5β is essential for MLL5β-AP-1 transcription complex assembly at the HPV16/18-long control region.

Dear Editor, Cervical cancer is one of the leading causes of cancer death in women (Munoz et al., 2004), and current treatment strategies though effective still possess limitations. Thus, identification of more molecular-targeted therapeutics for cervical cancer is urgently needed. We recently identified an isoform of mixed lineage leukemia 5 (MLL5b, 503 amino acids) that is specifically expressed in HPV16/ 18+ cervical cancer cell lines and patient samples (Yew et al., 2011). MLL5b was found to regulate E6/E7 oncogene transcription activation through its interaction with AP-1 at the 5′ segment of the HPV16/ 18-long control region (LCR). With no known DNA-binding motifs on MLL5b, interaction of MLL5b with AP-1 (c-Fos/ c-Jun, AP-1 dimer) was thought to be essential for its recruitment to the HPV16/ 18-LCR (Yew et al., 2011). As E6 and E7 are the main pathological factors of HPV16/18+ cervical cancer, the development of new therapeutics for this type of cancer has been focused on the inhibition of E6/E7 expression (Butz et al., 2003; Putral et al., 2005). The identification of the essential role of MLL5b in E6/E7 transcriptional regulation (Yew et al., 2011) poses MLL5b as an attractive molecular target. A further understanding of the molecular mechanisms underlying recruitment and assembly of the MLL5b–AP-1 transcriptional activation complex to the HPV16/18-LCR could provide new insights into MLL5b-targeted therapeutics. Here, we report the essential role of MLL5b O-GlcNAcylation in MLL5b–AP-1 complex assembly at the HPV16/18-LCR and the selective cytotoxic effects of O-GlcNAcylation inhibition on HPV16/18+ cervical cancer cells. Our findings highlight the potential of targeting MLL5b O-GlcNAcylation as a novel therapeutic approach for HPV16/ 18+ cervical cancer. Several studies have proposed that O-GlcNAcylation is important in mediating histone-modifying activity of SET domaincontaining transcription complexes and their subsequent effects on transcription activation (Deplus et al., 2013; Zhou et al., 2013). To establish the role of O-GlcNAcylation in MLL5b-mediated E6/E7 transcriptional activation, we determined the effects of O-GlcNAcylation inhibition or activation, by utilizing known O-GlcNAcylation inhibitor Azaserine and activator PUGNAc, respectively, on E6/E7 expression in SiHa (HPV16+) and HeLa (HPV18+) cells. We found that inhibition of O-GlcNAcylation decreased E6/E7 levels and activation of O-GlcNAcylation increased E6/E7 expression (Figure 1A). These were linked to MLL5b O-GlcNAcylation levels and MLL5b assembly into the MLL5b–AP-1 complex at the HPV16/18-LCR. While Azaserine inhibited MLL5b O-GlcNAcylation and its ability to associate with AP-1 and the LCR, enhancement of MLL5b O-GlcNAcylation by PUGNAc was accompanied by increased association with AP-1 and the LCR in coimmunoprecipitation (co-IP) and ChromatinIP (ChIP) assays (Figure 1B and C). To further determine whether O-GlcNAcylated MLL5b was indeed recruited to the HPV16/ 18-LCR, a re-ChIP assay was performed where O-GlcNAcylated proteins were first immunoprecipitated with anti-O-GlcNAc antibody and eluted. Elutes were then subjected to a second IP with anti-Flag antibody, and the amount of bound DNA was analyzed by quantitative PCR. The results confirmed that O-GlcNAcylated MLL5b was indeed recruited to the LCR, and the modulation of O-GlcNAcylation affected the amount of O-GlcNAcylated MLL5b associated with the LCR (Figure 1D). We also examined effects of direct OGT inhibition with OGT-siRNA (siOGT) on MLL5b O-GlcNAcylation status and its recruitment to the MLL5b–AP-1 complex at the LCR. OGT knockdown inhibited MLL5b O-GlcNAcylation resulting in loss of MLL5b–AP-1 interaction and MLL5b recruitment to the LCR (Figure 1E and F), cumulating to the downregulation of E6/E7 (Figure 1G). Interestingly, OGT was noted to be a part of the complex (Figure 1E), suggesting that it was required for E6/E7 transcription activation. To understand the role of MLL5bO-GlcNAcylation in MLL5b–AP-1 complex assembly at the HPV16/18-LCR, we went on to identify the key O-GlcNAcylation site on MLL5b that was essential for the complex assembly. Initial analysis of MLL5b protein sequence using a O-GlcNAcylation site prediction software (http://cbsb.lombardi. georgetown.edu/OGAP.html) identified four potential O-GlcNAcylation sites in MLL5b (Supplementary Figure S1A). Sitedirected mutagenesis of these sites to alanine residues followed by immunoblotting with anti-O-GlcNAc antibody of the immunoprecipitated Flag-tagged mutants indicated T440 as the key O-GlcNAcylation site in MLL5b (Figure 1H). Computational modeling further suggested the location of T440 in the solvent-exposed flexible loop of the MLL5b-SET domain, which was predicted to have a similar fold to the MLL1-SET domain (Supplementary Figure S1B). This concurred with a recent report that O-GlcNAcylation preferentially occurred at the solvent flexible loop of 22 proteins with known crystal structures (Liu et al., 2014). To further delineate the role of O-GlcNAcylation at T440 in MLL5b–AP-1 complex assembly at the HPV16/18-LCR, we studied the ability of MLL5b-T440A and 180 | Journal of Molecular Cell Biology (2015), 7(2), 180–183 doi:10.1093/jmcb/mjv009 Published online February 10, 2015