Figure 4

EZH2β represses transcription through H3-K27 trimethylation of gene promoters and increases cellular proliferation. (A) Luciferase values are shown relative to control (pcDNA) upon nucleofection of EZH2β or with co-factors SUZ12 and EED (EZH2β/S/E) in cells with an integrated FOXP3 luciferase reporter. EZH2α alone or with co-factors SUZ12 and EED (EZH2α/S/E) is included. *P <0.05. (B) Quantification of flow cytometry analysis of primary mouse naïve T cells transduced with empty vector, EZH2β or EZH2α for FOXP3 expression. *P <0.05. (C) qPCR of FOXP3 expression in primary mouse naïve T cells indicates that transcription is reduced by transduction of EZH2β and EZH2α. Glyceraldehyde-3-phosphate dehydrogenase and hypoxanthine phosphoribosyltransferase were used as expression controls. (D) ChIP assay of H3-K27me3 on the FOXP3 promoter in primary mouse naïve T cells. Transduction with EZH2α or EZH2β increases H3-K27me3 on the FOXP3 promoter relative to empty vector control. ChIP performed using an antibody against the HIS-tag demonstrates that only the EZH2α- and EZH2β-infected cells amplify a band to indicate their presence on the FOXP3 promoter, whereas empty vector-infected cells serve as a negative control. A representative gel is shown from triplicate experiments with associated qPCR quantification. These results reveal that novel EZH2 isoforms can regulate gene expression through H3-K27 trimethylation of gene promoters. (E) Incorporation of ³H-thymidine in primary mouse naïve T cells transduced with empty vector, EZH2α or EZH2β after 5 days of stimulation. Representative data shown from experiments performed in duplicate, representing the mean and SD of technical triplicates. These results indicate that EZH2β increases cellular proliferation in a similar fashion as EZH2α. 3HT: ³H-thymidine; ChIP: chromatin immunoprecipitation; EV: empty vector; EZH2: enhancer of zeste homologue 2; FACS: fluorescence-activated cell sorting; H3-K27me3: trimethylation of histone 3 at lysine 27; HIS: histidine; qPCR: quantitative polymerase chain reaction.