Journal: Clinical Epigenetics

Article Title: Epigenetic therapy of novel tumour suppressor ZAR1 and its cancer biomarker function

doi: 10.1186/s13148-019-0774-2

Figure Lengend Snippet: Epigenetic therapy of ZAR1 induces ZAR1 targets p21 and WEE1 . a ZAR1 promoter activity is inhibited in comparison to pRLnull empty reporter construct by luciferase assay. b ZAR1 promoter activity can be epigenetically modified by the overexpression of guide RNAs targeting ZAR1 together with epigenetic activators p300, VP160, and epigenetic inhibitors EZH2 and DNMT3A by luciferase assay. c ZAR1 endogenous expression in HeLa can be reactivated by epigenetic activators p300, VP160, and TET1 targeted to ZAR1 by RNA guides by RT-PCR. d Expression of ZAR1 targets WEE1/p21 by RT-PCR is stimulated by overexpression of ZAR1 guided VP160. e, f, g Epigenetic reexpression of ZAR1 by overexpression of ZAR1 guide directed p300, VP16,0 and TET1 or empty control for 24, 48, and 72 h as well as expression dynamics of reexpressed ZAR1 targets WEE1 and p21 by ZAR1-guided epigenetic activators

Article Snippet: Epigenetic modifier plasmids were ordered from Addgene and modified if indicated: pcDNA-dCas9-p300 Core (61357), pdCas9-DNMT3A-EGFP (71666) with deletion of U6 promoter (site-directed mutagenesis), pdCas9-Tet1-CD (83340) as wildtype, with deletion of U6 promoter (site-directed mutagenesis) or as wildtype with cloned ZAR1 guides in Bbs I restriction site (ZAR1-guided-TET1), pcDNA3.1-MS2-Tet1-CD (83341), Ezh2[SET]-dCas9 (100087), DNMT3A-dCas9 (100090).

Techniques: Activity Assay, Construct, Luciferase, Modification, Over Expression, Expressing, Reverse Transcription Polymerase Chain Reaction

Journal: Clinical Epigenetics

Article Title: Epigenetic therapy of novel tumour suppressor ZAR1 and its cancer biomarker function

doi: 10.1186/s13148-019-0774-2

Figure Lengend Snippet: Modulation of ZAR1 promoter methylation offers therapeutic approach to ZAR1 reactivation. a Hypermethylation of ZAR1 by DNMT3A using ZAR1 promoter (pRLnull) in HEK and HeLa for indicated time points followed by CoBRA methylation analysis and b according quantification by pyrosequencing. c Pharmacological DNMT inhibition by 0.5 μM/1 μM 5-aza-2′-deoxycytidine (Aza) together with ZAR1 targeted VP160 overexpression activates ZAR1 expression. d ZAR1 endogenous reexpression by epigenetic editing through overexpression of ZAR1 guided p300, VP160, and TET1 in HeLa, HEK, and HCT116. e Promoter methylation of HeLa, HEK, and HCT116 by CoBRA and f quantified by pyrosequencing. g ZAR1 endogenous reexpression by epigenetic editing through ZAR1-guided TET1 upon overexpression h is guide-combination dependent. i, j ZAR1 reexpression by overexpressed TET1 (guided by ZAR1 oligos) is accompanied by ZAR1 demethylation by pyrosequencing

Article Snippet: Epigenetic modifier plasmids were ordered from Addgene and modified if indicated: pcDNA-dCas9-p300 Core (61357), pdCas9-DNMT3A-EGFP (71666) with deletion of U6 promoter (site-directed mutagenesis), pdCas9-Tet1-CD (83340) as wildtype, with deletion of U6 promoter (site-directed mutagenesis) or as wildtype with cloned ZAR1 guides in Bbs I restriction site (ZAR1-guided-TET1), pcDNA3.1-MS2-Tet1-CD (83341), Ezh2[SET]-dCas9 (100087), DNMT3A-dCas9 (100090).

Techniques: Methylation, Combined Bisulfite Restriction Analysis Assay, Inhibition, Over Expression, Expressing