Skip to main content
Fig. 5 | Epigenetics & Chromatin

Fig. 5

From: Post transcriptional control of the epigenetic stem cell regulator PLZF by sirtuin and HDAC deacetylases

Fig. 5

PLZF epigenetic effects on its endogenous targets and their related gene expression. Histone H3 enrichment at PLZF promoter targets (a). Transfected 293T cells were used for chromatin immunoprecipitation with an antibody against histone H3 or an antibody against acetylated forms of histone H3. For each condition, the amount of LINE-1, CRABPI and c-myc promoters bound by each antibody was amplified and quantified by real-time PCR. This was expressed relative to the signal obtained from the 5 % input chromatin sample and corrected by the signal obtained with the total histone H3 immunoprecipitation. DNA methylation enrichment of CpG promoters (b). MeDIP assay of the LINE-1, c-myc, CRABPI and H19 promoter regions in the PLZF with or without HDAC3 or SIRT1 expression cells. Cells were harvested 24 h after transfection and genomic DNA for MeDIP analysis with specific 5 mC antibody was isolated. Immunoprecipitated DNA was amplified by gene specific quantitative PCR. To quantify the amount of DNA methylation in these regions, the ratio of ΔCT of the MeDIP and input samples are calculated by comparing MeDIP samples against input (sonicated library DNA was set aside before MeDIP was performed for use as input DNA). The data are normalized to the DNA methylation at the UBE locus and fold enrichment ratio calculated in comparison to the untransfected cells. A representative dataset from these experiments, which were repeated 3 times, is shown. H19 locus was used as control of DNA methylation (known to be methylated in human) and as a non-PLZF targeted promoter. White bars represent transfection with the empty vector and the black bar with PLZF expression vector in presence (+) or absence (−) of HDAC3 or SIRT1 co-transfection. Relative gene expression of endogenous PLZF targets (c). To measure expression of PLZF targets, mRNA expression was measured at 12 h post-transfection in 293T cells using SYBR green quantitative real-time PCR. After normalization to GAPDH, expression levels of LINE-1, c-myc and CRABPI genes are presented as mean ± standard deviation. The experiments were conducted in triplicate

Back to article page