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. 2015 Apr;7(2):180-3.
doi: 10.1093/jmcb/mjv009. Epub 2015 Feb 10.

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

Dawn Sijin Nin  1 , Weitai Huang  1 , Muzaffar Ali  2 , Chow Wenn Yew  1 , Tatiana G Kutateladze  2 , Lih-Wen Deng  3
Affiliations

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

Dawn Sijin Nin et al. J Mol Cell Biol. 2015 Apr.
No abstract available

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Figures

Figure 1
Figure 1
O-GlcNAcylation is critical for MLL5β–AP-1 recruitment to the HPV16/18-LCR and inhibition of O-GlcNAcylation exhibits selective cytotoxicity to HPV16/18+ cells. (A) O-GlcNAcylation inhibitor Azaserine (45 μM, 20 h) downregulated E6/E7 levels, while O-GlcNAcylation activator PUGNAc (150 μM, 20 h) upregulated E6/E7 levels in HeLa and SiHa cells. (B) Azaserine decreased MLL5β O-GlcNAcylation levels resulting in the loss of MLL5β–AP-1 interaction, while PUGNAc increased MLL5β O-GlcNAcylation and MLL5β–AP-1 interaction in co-immunoprecipitation (IP) assays. Competition blots using 0.5 M free GlcNAc were included to control for the specificity of the anti-O-GlcNAc antibody. (C) Azaserine decreased MLL5β recruitment to the LCR, while PUGNAc increased MLL5β–LCR interaction in quantitative chromatin IP (qChIP) assays. (D) Re-ChIP assay of vehicle-, Azaserine-, or PUGNAc-treated cells with O-GlcNAc IP followed by Flag-MLL5β IP. (E and F) Knockdown of OGT decreased MLL5β O-GlcNAcylation levels and inhibited MLL5β–AP-1 interaction (co-IP) and MLL5β recruitment to the LCR (qChIP). (G) Relative E6/E7 transcription levels in OGT-siRNA (siOGT)-treated HeLa and SiHa cells. (H) Site-directed mutagenesis of predicted O-GlcNAcylation sites to alanine in MLL5β followed by immunoblotting with anti-O-GlcNAc antibody identified T440 as the key O-GlcNAcylation site. (I) MLL5β-T440A and MLL5β-T440E were not able to co-IP c-Jun. (J) Inability of MLL5β-T440A and MLL5β-T440E to immunoprecipitate the HPV18-LCR as indicated by qChIP. (K) HeLa cells expressing exogenously introduced MLL5β-WT but not MLL5β-T440A or MLL5β-T440E were able to maintain E6/E7 levels in MLL5β knockdown cells. (L) A schematic model summarizing the role of O-GlcNAcylation at T440 of MLL5β in the assembly of the MLL5β–AP-1 transcription complex at the HPV16/18-LCR (upper panel) and the proposed action of O-GlcNAcylation inhibitors on complex recruitment (lower panel). (M and N) MLL5β-expressing SiHa (HPV16+) and HeLa (HPV18+) cells showed more prominent loss of cell viability, compared with MLL5β-negative C33A, primary human keratinocytes (HK), and normal diploid fibroblast WI38, at 72 h after OGT knockdown (M) and Azaserine treatment (N) as determined by MTT assays. All results are reported as mean ± SD. All qChIP data are reported as % of input chromatin. A P-value <0.05 indicates statistically significant (*P < 0.05).
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