Journal: ACS Synthetic Biology

Article Title: A Transcription Factor-Based Biosensor for Detection of Itaconic Acid

doi: 10.1021/acssynbio.8b00057

Figure Lengend Snippet: Influence of ItcR on inducible gene expression. Absolute normalized fluorescence (in arbitrary units) of (A) E. coli MG1655 and (B) C. necator H16 harboring the Y. pseudotuberculosis ( Yp ) and P. aeruginosa ( Pa ) itaconate-inducible systems composed of promoter and transcriptional regulator (ItcR/P), and promoter-only (P) implementation in the absence and presence of 5 mM itaconate. Single time-point fluorescence measurements were taken 6 h after inducer addition. The promoterless reporter plasmid pEH006E was employed as negative control. Error bars represent standard deviations of three biological replicates. Asterisks indicate statistically significant induction values for p < 0.01 (unpaired t test).

Article Snippet: RFP fluorescence assays for biosensor characterization were performed in wild type E. coli MG1655 (DSMZ 18039) and C. necator H16 (ATCC 17699).

Techniques: Expressing, Fluorescence, Plasmid Preparation, Negative Control

Journal: ACS Synthetic Biology

Article Title: A Transcription Factor-Based Biosensor for Detection of Itaconic Acid

doi: 10.1021/acssynbio.8b00057

Figure Lengend Snippet: Kinetics and dynamics of the Yp ItcR/P ccl inducible system. (A) Absolute normalized fluorescence of E. coli MG1655 harboring the Yp ItcR/P ccl inducible system (pEH086) in response to different concentrations of itaconate added at time zero. The standard deviation of three biological replicates is shown as a lighter color ribbon displayed lengthwise of the induction kinetics curve. For the lower concentrations, the standard deviation is too small to be visible. (B) Dose response curve of the Yp ItcR/P ccl inducible system in E. coli MG1655, illustrating the correlation between inducer concentration and fluorescence output 4 and 8 h post-induction (hpi) with itaconate. Error bars represent standard deviations of three biological replicates.

Article Snippet: RFP fluorescence assays for biosensor characterization were performed in wild type E. coli MG1655 (DSMZ 18039) and C. necator H16 (ATCC 17699).

Techniques: Fluorescence, Standard Deviation, Concentration Assay

Journal: ACS Synthetic Biology

Article Title: A Transcription Factor-Based Biosensor for Detection of Itaconic Acid

doi: 10.1021/acssynbio.8b00057

Figure Lengend Snippet: Inducer-dependent orthogonality of the Yp ItcR/P ccl inducible system. (A) Compounds that were investigated for cross-induction with the Yp ItcR/P ccl inducible system: itaconic acid (1), succinic acid (2), d -malic acid (3), l -malic acid (4), fumaric acid (5), oxaloacetic acid (6), l -aspartic acid (7), methylsuccinic acid (8), mesaconic acid (9), citraconic acid (10), α-ketoglutaric acid (11), l -glutamic acid (12), acetic acid (13), propionic acid (14), butyric acid (15), 3-butenoic acid (16), valeric acid (17), acrylic acid (18), methacrylic acid (19), tiglic acid (20), citric acid (21), cis -aconitic acid (22), trans -aconitic acid (23), tricarballylic acid (24), isocitric acid (25). (B) Normalized fluorescence (in %) of E. coli MG1655 harboring the Yp ItcR/P ccl inducible system 12 hours after addition of different compounds at a final concentration of 5 mM, relative to the fluorescence output obtained by adding 5 mM itaconate. (−) uninduced sample. Error bars represent standard deviations of three biological replicates. Asterisks indicate statistically significant induction values for p < 0.01 (unpaired t test).

Article Snippet: RFP fluorescence assays for biosensor characterization were performed in wild type E. coli MG1655 (DSMZ 18039) and C. necator H16 (ATCC 17699).

Techniques: Fluorescence, Concentration Assay

Journal: Frontiers in Genetics

Article Title: Argonaute 4 as an Effector Protein in RNA-Directed DNA Methylation in Human Cells

doi: 10.3389/fgene.2019.00645

Figure Lengend Snippet: Association of the AGO4-binding genes and methylation levels. (A) Scheme showing that tetracycline-treated cells switch off AGO4 protein expression (Tet+) from day 1 to 12 and that 5’-azacytidine treatment from day 7 to 9 inhibits DNA methyltransferase (5-AC). (B) Detection of AGO4 mRNA in Tet- (AGO4 expression) and Tet+ (No AGO4 expression) cells; AGO4 was significantly expressed in the absence of Tet. (C) Confirmation of AGO4-binding genes showed that AGO4 only bounds to the selected gene from bioinformatics data as exemplified by C16ORF89 . (D) However, AGO4 did not bind to a gene lacking the AGO4-binding site, MSN . (E) The AGO4 methylation level of the gene, which contains AGO4-binding sites, was increased due to the presence of the AGO4 protein (Tet-). This increased methylation level was not associated with the presence of DNA methyltransferase (5-AC+). (F) These results were not observed for a gene lacking AGO4-binding sites, MSN . (G to H) Recovery of DNA methylation levels is found in cells expressing the AGO4 protein (Tet-) with 5’-azacytidine withdrawal in both of AGO4-binding gene, C16ORF89 and non-AGO4-binding gene, MSN ; AGO4 is inferred to methylate previously demethylated loci. The above data are the representative dataset from three independent experiments presented as the mean ± SEM. Statistical analyses were performed using a paired-sample t -test, where p < 0.05, p < 0.01, and p < 0.005 are represented as *, **, and ***, respectively, where Tet+ means tetracycline treatment, 5-AC+ means azacytidine treatment, and Tet- and 5-AC- mean untreated groups (I). Schematic overview of the role of AGO4 in de novo methylation. After DNA replication, DNA methylation is maintained by DNMT1 by the recognition of hemimethylated CpG on the parental strand as a template and the addition of newly methylated CpG to the daughter strand. DNMT1 is inhibited by the action of 5-AC, and the methylation level should be decreased by half under 5-AC treatment. In our study, when tetracycline was added, AGO4 shRNA was manipulated, resulting in AGO4 expression repression. Thus, without tetracycline, AGO4 is upregulated and binds to DNA loci, and DNA methylation is maintained, although 5-AC is added, suggesting that AGO4 involved in de novo methylation

Article Snippet: Overexpression of AGO4 was performed by the using pIRESneo-FLAG/HA AGO4 plasmid (Addgene, MA, USA).

Techniques: Binding Assay, Methylation, Expressing, DNA Methylation Assay, shRNA

Journal: Frontiers in Genetics

Article Title: Argonaute 4 as an Effector Protein in RNA-Directed DNA Methylation in Human Cells

doi: 10.3389/fgene.2019.00645

Figure Lengend Snippet: Alu methylation upon Alu siRNA transfection in Tet-controlled AGO4-expressing cells. The Alu methylation level was significantly increased when AGO4 was upregulated under Alu siRNA transfection. The above data are the representative dataset from six independent experiments presented as the mean ± SEM. Statistical analysis was performed using an unpaired t -test where p < 0.01 is represented as **.

Article Snippet: Overexpression of AGO4 was performed by the using pIRESneo-FLAG/HA AGO4 plasmid (Addgene, MA, USA).

Techniques: Methylation, Transfection, Expressing

Journal: Frontiers in Genetics

Article Title: Argonaute 4 as an Effector Protein in RNA-Directed DNA Methylation in Human Cells

doi: 10.3389/fgene.2019.00645

Figure Lengend Snippet: Overexpression of HA-AGO4 increases global genomic methylation. (A) Confirmation of AGO4 mRNA expression in AGO4- or PC-overexpressing cells after transfection for 72 h by using real-time PCR showed that AGO4 mRNA was significantly upregulated in AGO4-overexpressing cells, and (B) Western blot analysis showed the expression of the HA-AGO4 protein in a time-course experiment. β-Actin was used to confirm equal protein loading of each lane. AGO4 protein expression was at the highest level at 48 h after transfection. (C) Overexpression of HA-AGO4 resulted in a significant increase in interspersed repetitive sequence methylation in the input (cell lysate) HA-AGO4 compared with untaransfected HeLa cells, detected at 72 h post-transfection. The above data in A and C are the representative dataset from five independent experiments presented as the mean ± SEM. Statistical analyses were performed using a paired t -test where p < 0.05, p < 0.01, and p < 0.005 are represented as *, **, and ***, respectively.

Article Snippet: Overexpression of AGO4 was performed by the using pIRESneo-FLAG/HA AGO4 plasmid (Addgene, MA, USA).

Techniques: Over Expression, Methylation, Expressing, Transfection, Real-time Polymerase Chain Reaction, Western Blot, Sequencing

Journal: Frontiers in Genetics

Article Title: Argonaute 4 as an Effector Protein in RNA-Directed DNA Methylation in Human Cells

doi: 10.3389/fgene.2019.00645

Figure Lengend Snippet: AGO4 localization is involved in RdDM by introducing genomic DNA methylation in human cells. (A) ChIP experiments using a hemagglutinin (HA) antibody showed elevation of the levels of AGO4-binding LINE-1 or ALU found in AGO4-overexpressing cells compared with their control counterparts. (B) Moreover, the binding of the AGO4 protein to interspersed repetitive sequence increased DNA methylation.

Article Snippet: Overexpression of AGO4 was performed by the using pIRESneo-FLAG/HA AGO4 plasmid (Addgene, MA, USA).

Techniques: DNA Methylation Assay, Binding Assay, Sequencing

Journal: Frontiers in Genetics

Article Title: Argonaute 4 as an Effector Protein in RNA-Directed DNA Methylation in Human Cells

doi: 10.3389/fgene.2019.00645

Figure Lengend Snippet: AGO4 protein specifically induced DNA methylation of target loci. By using CPP-AGO4 conjugated with Alu sgRNA (CPP-AGO4-Alu) transfected to HeLa cells, we found that (A) Alu methylation level was significantly increased in CPP-AGO4-Alu transfected cells, while Alu methylation was not promoted in control counterpart groups. (B) While LINE-1 methylation level was not changed, this means the complex has no off targets to LINE-1. The representative dataset was obtained from five independent experiments. Statistical analyses were performed using a paired-sample T -test where p < 0.05 and p < 0.01 are represented as * and **, respectively, where buffer means non-inducible CPP-AGO4 transfection.

Article Snippet: Overexpression of AGO4 was performed by the using pIRESneo-FLAG/HA AGO4 plasmid (Addgene, MA, USA).

Techniques: DNA Methylation Assay, Transfection, Methylation