pires hrgfp ii tet1 cd  (Addgene inc)

Journal: Nature genetics

Article Title: TETs compete with DNMT3 activity in pluripotent cells at thousands of methylated somatic enhancers

doi: 10.1038/s41588-020-0639-9

Figure Lengend Snippet: a) Bar chart showing the proportion of CpGs with varying methylation loss in DKO cells compared to WT. CpGs that lost >60% methylation were generally located in closer proximity compared to CpGs that lost 10–20% methylation. b) The proportion of cDKO-DMRs with respective mean methylation levels in single DNMT3A −/− (3AKO) or DNMT3B −/− (3BKO) knockout ESCs. c) Composite plot showing methylation difference between passage (P) 20 and 6 in PKO cells as distance from cDKO-DMR. There is a small background loss (−0.045) with a slightly greater focal decrease (−0.07), but methylation levels stay generally high. d) The methylation level in HUES64 DKO-A, HUES64 WT and HUES8 TKO cells including the 2 kb either side is displayed. For regions where the neighboring 2 kb is hypomethylated in WT cells (class 1), almost every region shows an increase in methylation following TET loss. e) Violin plots showing methylation at TKO-DMRs across different HUES8 lines. We used stringent parameters to define TKO-DMRs that aberrantly gain methylation upon loss of TET expression (also described later in ). Methylation levels were rescued by re-expression of TET. Violin plots extend from the data minima to the maxima, white dot indicates median, thick bar shows the interquartile range and thin bar shows 1.5x interquartile range. f) Methylation levels across 1 kb tiles for PKO ESCs rescued with either TET1s or TET2. Intensity of blue shading indicates density of data points. Pearson correlation coefficient (cor) is displayed. g) ChIP-seq enrichment (from Ref ) for TET1 and DNMT3B over CpG islands (CGI), H1-specific enhancers and cDKO-DMRs. h) Representative browser tracks displaying methylation levels in WT and DKO cells as well as 5hmC levels in WT ESCs. Increased 5hmC is observed over the cDKO-DMR and at the border of the hypomethylated CGI, where TETs are known to localize.

Article Snippet: The TET1 coding sequence was obtained by PCR amplification of pIRES-hrGFP II-TET1 (Addgene #83568) using Gibson primers to create overhangs for pPiggyBac MCS digested with XbaI ( ).

Techniques: Methylation, Knock-Out, Expressing, ChIP-sequencing

Journal: Nature genetics

Article Title: TETs compete with DNMT3 activity in pluripotent cells at thousands of methylated somatic enhancers

doi: 10.1038/s41588-020-0639-9

Figure Lengend Snippet: a) TET1 enrichment (from Ref ) for LINE 5’UTRs and active H1 typical enhancers showing a greater enrichment at enhancers. b) A region within the gene NCOR2 that has many cDKO-DMRs. These lose methylation in different somatic tissues as shown below. H3K27ac and H3K4me1 tracks display ENCODE data derived from H1 ESCs. Methylation tracks are from Ref . c) The percentage of overlap with putative somatic enhancers (defined in Ref ) for class 1 and class 2 cDKO-DMRs, an equal number of same-sized randomly selected regions, H3K4me1 peaks in ESCs (H1) and 1 kb tiles with matched CpG density to cDKO-DMRs (3.1–3.3%). d) B cell enhancers (defined in Ref ) separated by methylation levels in WT ESCs, DKO ESCs and B cells. e) Composites showing methylation levels in passage (P) 3 and 28 DKO ESCs for P3 and P28 DKO-DMRs. f) For 86 different previously defined putative enhancer sets , the stacked bar plots display the proportion that are already hypomethylated in WT ESCs, lose methylation in DKO cells (WT – DKO difference > 0.2) or remain methylated in DKO cells. For this analysis, P28 DKO was used. g) Schematic of in vitro pancreatic islet cell differentiation (from Ref ). h) The proportion of cDKO-DMRs or P28 DKO-DMRs that overlap with each set of cell type specific enhancers. Enhancers were previously defined . i) For regions defined as showing dynamic methylation changes during differentiation to beta islet cells , methylation levels are shown for these cell types as well as HUES64 WT and DKO-A ESCs.

Article Snippet: The TET1 coding sequence was obtained by PCR amplification of pIRES-hrGFP II-TET1 (Addgene #83568) using Gibson primers to create overhangs for pPiggyBac MCS digested with XbaI ( ).

Techniques: Methylation, Derivative Assay, In Vitro, Cell Differentiation

Journal: Nature genetics

Article Title: TETs compete with DNMT3 activity in pluripotent cells at thousands of methylated somatic enhancers

doi: 10.1038/s41588-020-0639-9

Figure Lengend Snippet: a) Sequence mutations introduced into the DKO ESCs. Green box = PAM sequence, grey box = sgRNA. Codons are colored black or blue, grey text denotes non-coding DNA. Amino acid (aa) and protein sequence number are displayed above the DNA. b) Western blots for WT, DNMT3A −/− (3AKO), DNMT3B −/− (3BKO) and double knockout (DKO) mouse ESCs. GAPDH or H3 were used as loading controls. Full blots in . Western blots were performed three times with consistent results. c) Overlap between DKO-DMRs called in each Dnmt3a −/− tissue. d) The difference in methylation between WT and Dnmt3a −/− tissues, ESCs or EpiSCs. e) Methylation levels for EpiSC DKO-DMRs in EpiSCs and ESCs. Violin plots extend from the data minima to the maxima, white dot indicates median, thick bar shows the interquartile range and thin bar shows 1.5x interquartile range. f) Methylation levels for tissue-specific DKO-DMRs in WT and Dnmt3a −/− E6.5 epiblast and 8 day old tissues. Somatic DKO-DMRs were fully methylated in Dnmt3a −/− embryos following implantation due to DNMT3B expression hence must have lost methylation at a later stage. Violin plots extend from the data minima to the maxima, white dot indicates median, thick bar shows the interquartile range and thin bar shows 1.5x interquartile range. g) The number of DKO-DMRs identified when using a reduced stringency of 0.25 differential methylation instead of 0.6 to compensate for mixed cell types. We identified more DKO-DMRs (1,186–2,182) but still many less than in ESCs, and this may include false positives. h) The percentage of DKO-DMRs that fall into class 1 or 2 (described in ). i) The overlap of DKO-DMRs with genomic features. Categories are not exclusive. CGI = CpG island, TSS = transcription start site. j) Schematic showing the differentiation of human ESCs to motor neurons (MNs) via neuronal progenitor cells (NPCs). For WT and 3AKO ESCs expression of DNMT3A and DNMT3B is displayed across the time course. After day two, 3AKO cells are “DKO-like” as they do not express either DNMT3. k) The percentage of cDKO-DMRs with mean methylation <0.2 in each sample during differentiation. Only 1.3% and 2.4% of cDKO-DMRs lost methylation for WT and 3AKO MNs respectively. WGBS is from Ref . l) Expression of TET1–3 in WT and 3AKO ESCs during differentiation. RNA-seq is from Ref .

Article Snippet: The TET1 coding sequence was obtained by PCR amplification of pIRES-hrGFP II-TET1 (Addgene #83568) using Gibson primers to create overhangs for pPiggyBac MCS digested with XbaI ( ).

Techniques: Sequencing, Western Blot, Double Knockout, Methylation, Expressing, RNA Sequencing

Journal: International Journal of Biological Sciences

Article Title: MSTN Mutant Promotes Myogenic Differentiation by Increasing Demethylase TET1 Expression via the SMAD2/SMAD3 Pathway

doi: 10.7150/ijbs.40551

Figure Lengend Snippet: MSTN mutant demethylates myogenesis-specific genes by up-regulating demethylase TET1 . (A, B) Methylation levels of myogenesis-specific genes of MyoD , MyoG , PAX3 , and PAX7 in promoters (A) and gene bodies (B). (C) Expression of demethylase TET1 , TET2 , and TET3 at the mRNA levels. (D, E) Expression of demethylase TET1 at the protein level. *P<0.05, **P<0.01, ***P<0.001; t-tests were used to calculate the p-values.

Article Snippet: The TET1 overexpression vector pIRES-hrGFPII-TET1 (#83568) was purchased from Addgene.

Techniques: Mutagenesis, Methylation, Expressing

Journal: International Journal of Biological Sciences

Article Title: MSTN Mutant Promotes Myogenic Differentiation by Increasing Demethylase TET1 Expression via the SMAD2/SMAD3 Pathway

doi: 10.7150/ijbs.40551

Figure Lengend Snippet: MSTN regulates TET1 expression via SMAD2/SMAD3. (A) The binding region of SMAD2/SMAD3 with the promoter of TET1 . (B) The combination of TET1 promoter with SMAD2/SMAD3 detected by ChIP-qPCR. The detected binding region was from ‒1613 to ‒1437 on the TET1 promoter. (C) Luciferase reporter assays. HEK293T cells were transfected with luciferase reporter plasmids containing the promoter of TET1 (1800 bp). *P<0.05, **P<0.01, ***P<0.001; t-tests were used to calculate the p-values.

Article Snippet: The TET1 overexpression vector pIRES-hrGFPII-TET1 (#83568) was purchased from Addgene.

Techniques: Expressing, Binding Assay, ChIP-qPCR, Luciferase, Transfection

Journal: International Journal of Biological Sciences

Article Title: MSTN Mutant Promotes Myogenic Differentiation by Increasing Demethylase TET1 Expression via the SMAD2/SMAD3 Pathway

doi: 10.7150/ijbs.40551

Figure Lengend Snippet: Overexpression of TET1 promotes myogenic differentiation. (A-C) The expression of TET1 at the mRNA and protein level after the overexpression of TET1 in the wild type muscle satellite cells. (D, E) Methylation levels of myogenesis-specific genes in the promoter and gene body. (F) Expression of myogenic genes at the mRNA level. (G, H) Expression of myogenic genes at the protein level. (I) The differentiated myotubes. (J) Myotube fusion index after the overexpression of TET1 . (K) Length of myotubes. *P<0.05, **P<0.01, ***P<0.001; t-tests were used to calculate the p-values.

Article Snippet: The TET1 overexpression vector pIRES-hrGFPII-TET1 (#83568) was purchased from Addgene.

Techniques: Over Expression, Expressing, Methylation

Journal: International Journal of Biological Sciences

Article Title: MSTN Mutant Promotes Myogenic Differentiation by Increasing Demethylase TET1 Expression via the SMAD2/SMAD3 Pathway

doi: 10.7150/ijbs.40551

Figure Lengend Snippet: Knockdown of TET1 inhibits myogenic differentiation. (A-C) The expression of TET1 at the mRNA and protein level after knockdown of TET1 in MSTN mutant muscle satellite cells. (D, E) Methylation level of myogenesis-specific genes in promoter and gene body. (F) Expression of myogenic genes at the mRNA level. (G, H) Expression of myogenic genes at the protein level. (I) The differentiated myotubes. (J) Myotube fusion index after TET1 knockdown. (K) Length of myotubes. *P<0.05, **P<0.01, ***P<0.001; t-tests were used to calculate the p-values.

Article Snippet: The TET1 overexpression vector pIRES-hrGFPII-TET1 (#83568) was purchased from Addgene.

Techniques: Knockdown, Expressing, Mutagenesis, Methylation

Journal: International Journal of Biological Sciences

Article Title: MSTN Mutant Promotes Myogenic Differentiation by Increasing Demethylase TET1 Expression via the SMAD2/SMAD3 Pathway

doi: 10.7150/ijbs.40551

Figure Lengend Snippet: Schematic of mechanisms for the inhibition of SMAD2/SMAD3 and the activation of TET1 signaling by MSTN mutation. In the wild type model (left), MSTN combined with ActRIIB, followed by the combination of the complex with type I receptor, the activation of the GS region (Ser/Thr) of the receptor, and the transmission of the MSTN signal. The activated MSTN signal then combined and phosphorylated SMAD2/SMAD3, whereby pSMAD2/SMAD3 was activated and combined with SMAD4. This complex entered the nucleus to combine with the promoter of demethylase TET1 , and inhibited the activity of TET1 promoter. In the MSTN mutant model (the right), the MSTN mutant decreased the binding between pSMAD2/SMAD3 and the promoter of TET1 and enhanced TET1 expression, which increased the demethylation of myogenesis-specific genes, including PAX3 , PAX7 , MyoD , and MyoG .

Article Snippet: The TET1 overexpression vector pIRES-hrGFPII-TET1 (#83568) was purchased from Addgene.

Techniques: Inhibition, Activation Assay, Mutagenesis, Transmission Assay, Activity Assay, Binding Assay, Expressing