Journal: bioRxiv

Article Title: TET1 dioxygenase is required for FOXA2-associated chromatin remodeling in pancreatic beta-cell differentiation

doi: 10.1101/2020.05.20.107532

Figure Lengend Snippet: a TET knock-out mutants were generated using CRISPR gRNAs (green arrowheads) targeting exon 7 of TET1, exon 3 of TET2 , and exon 3 of TET3 . Translation start sites are indicated by red arrows. b Analysis of global 5-hydroxymethylcytosine (5hmC) levels (top) in WT, TET1 knock-out (TET1KO), TET2 knock-out (TET2KO), TET3 knock-out (TET3KO), TET1/TET2 double knock-out (TET1/2DKO), TET1/TET3 double knock-out (TET1/3DKO), TET2/TET3 double knock-out (TET2/3DKO), and TET1/TET2/TET3 triple knock-out (TKO) hESCs by 5hmC dot blot analysis. The bottom panel shows methylene blue staining using the total amount of input DNA as the loading control. c Representative plots of flow cytometry for the expression of pluripotency marker (OCT4) and endoderm marker (SOX17) at the DE stage are shown in the top panel. Quantification of the percentage of SOX17 + cells is shown in the bottom panel. n = 3 independent differentiation. d Immunostaining of OCT4 and FOXA2 in WT and TKO cells at the DE stage. Scale bar = 50 μm. e Expression analysis by RT-qPCR for specific genes in WT and TKO cells at the ES, DE, PP, and PE stages. RT-qPCR validation was performed with three independent batches of samples. f Heatmap showing stage-specific DEGs in TKO compared with WT cells at the ES, DE, and PP stages (|fold change| ≥ 2; FDR < 0.05). Two mutant lines were used for TKO (clones 2 and 6). Each column represents one biological replicate for each cell line. Genes located in green rectangle are DEGs in TKO_PP compared with WT_PP cells. g Volcano plot of ATAC-seq data illustrating differentially accessible regions in TKO_PP cells (FDR < 0.05). h Transcription factor (TF) motif enrichment analysis of genomic regions showed significantly decreased ATAC-seq signals in TKO_PP cells. The top 10 significant motifs are shown after removing redundant motifs.

Article Snippet: To generate overexpression constructs for TET1, the C-terminal FLAG-tagged human full-length TET1 (TET1FL), TET1 catalytic domain (TET1CD), and TET1 catalytic inactive mutant (TET1CD mut ) were amplified from FH-TET1-pEF (Addgene #49792) or pIRES-hrGFP II-mTET1 (Addgene #83569), and cloned into the NotI digested PCDH-CAG-MCS-P2A-Puro vector (a kind gift from Dr. R. Xu, University of Macau) .

Techniques: Knock-Out, Generated, CRISPR, Dot Blot, Staining, Control, Flow Cytometry, Expressing, Marker, Immunostaining, Quantitative RT-PCR, Biomarker Discovery, Mutagenesis, Clone Assay

Journal: bioRxiv

Article Title: TET1 dioxygenase is required for FOXA2-associated chromatin remodeling in pancreatic beta-cell differentiation

doi: 10.1101/2020.05.20.107532

Figure Lengend Snippet: a Stepwise differentiation of hESCs to pancreatic endocrine cells. Wild-type (WT) and TET1 / TET2 / TET3 triple knock-out (TKO) hESC lines were differentiated and analyzed by RNA-seq, WGBS, CMS-IP-seq, ATAC-seq, and ChIP-seq at the indicated stages (ES: embryonic stem cell; DE: definitive endoderm; GT: primitive gut tube; PP: pancreatic progenitor; PE: pancreatic endocrine). b Representative flow cytometry plots for PDX1 and NKX6.1 in WT or TKO cells at the PP and PE stages. Quantification of the percentage of PDX1 + NKX6.1 + cells is shown in the right panel. n = 3 independent differentiation. c Immunostaining of PDX1/NKX6.1 at the PP stage and insulin (INS)/glucagon (GCG) at the PE stage are shown in WT and TKO cells. Scale bar = 50 μm. d Principal component analysis showing variance in normalized transcriptome between WT and TKO cells at the ES, DE, GT, and PP stage. Each plotted point represents one biological replicate. e Genes with significant changes in expression in TKO cells relative to WT cells at the PP stage. Differentially expressed genes (DEGs) are classified into down- and upregulated groups. Each row corresponds to one individual gene and each column to a different biological replicate. The color scale from white to blue represents Z-score normalized gene expression levels from low to high (|fold change| ≥ 2; FDR < 0.05). f Functional analysis of DEGs in TKO_PP cells showing the top 10 KEGG pathways. Benjamini-Hochberg corrected p -values were used. g Number of regions with significant changes in chromatin accessibility upon TET depletion at proximal (≤ 1 kb from TSS) and distal (> 1 kb from TSS) regions (FDR < 0.05). h Dot plots depicting the ratios (WT_PP over TKO_PP) of ATAC-seq signals (y-axis) at proximal (left) or distal (right) regions of DEGs that were downregulated (blue) or upregulated (red) in TKO_PP cells compared with WT_PP cells. Values indicate the fraction of increased or decreased chromatin accessibility regions in down- and upregulated genes, respectively. i Average density plots (top) and heatmaps (bottom) of FOXA2 binding at decreased accessible regions (DARs) and total identified ATAC-seq peaks (± 5 kb). Heatmaps are ranked by decreased FOXA2 binding. The color scale from white to red represents the normalized signal from low to high.

Article Snippet: To generate overexpression constructs for TET1, the C-terminal FLAG-tagged human full-length TET1 (TET1FL), TET1 catalytic domain (TET1CD), and TET1 catalytic inactive mutant (TET1CD mut ) were amplified from FH-TET1-pEF (Addgene #49792) or pIRES-hrGFP II-mTET1 (Addgene #83569), and cloned into the NotI digested PCDH-CAG-MCS-P2A-Puro vector (a kind gift from Dr. R. Xu, University of Macau) .

Techniques: Knock-Out, RNA Sequencing, ChIP-sequencing, Flow Cytometry, Immunostaining, Expressing, Gene Expression, Functional Assay, Binding Assay

Journal: bioRxiv

Article Title: TET1 dioxygenase is required for FOXA2-associated chromatin remodeling in pancreatic beta-cell differentiation

doi: 10.1101/2020.05.20.107532

Figure Lengend Snippet: a Immunostaining and representative plots of flow cytometry of PDX1 at the PP stage for WT, TET1KO, TET2KO, TET3KO, TET1/2DKO, TET1/3DKO, and TET2/3DKO cells. Quantifications of the percentage of PDX1 + cells are shown in the right panel. n = 3 independent differentiation. Scale bar = 50 μm. b Immunostaining and representative plots of flow cytometry of NKX6.1 at the PE stage for WT, TET1KO, TET2KO, TET3KO, TET1/2DKO, TET1/3DKO, and TET2/3DKO cells. Quantifications of the percentage of NKX6.1 + cells are shown in the right panel. n = 3 independent differentiation. Scale bar = 50 μm. c Expression of PAX4 , PTF1A , SOX9 , ARX , and FOXA2 in WT, TET1KO, TET2KO, TET3KO, TET1/2DKO, TET1/3DKO, and TET2/3DKO cells at the PP stage by RT-qPCR. RT-qPCR validation was performed with three independent batches of samples.

Article Snippet: To generate overexpression constructs for TET1, the C-terminal FLAG-tagged human full-length TET1 (TET1FL), TET1 catalytic domain (TET1CD), and TET1 catalytic inactive mutant (TET1CD mut ) were amplified from FH-TET1-pEF (Addgene #49792) or pIRES-hrGFP II-mTET1 (Addgene #83569), and cloned into the NotI digested PCDH-CAG-MCS-P2A-Puro vector (a kind gift from Dr. R. Xu, University of Macau) .

Techniques: Immunostaining, Flow Cytometry, Expressing, Quantitative RT-PCR, Biomarker Discovery

Journal: bioRxiv

Article Title: TET1 dioxygenase is required for FOXA2-associated chromatin remodeling in pancreatic beta-cell differentiation

doi: 10.1101/2020.05.20.107532

Figure Lengend Snippet: a Analysis of global 5-hydroxymethylcytosine (5hmC) levels (top) in WT, TKO-TET1CD mut , TKO-TET1CD, and TKO-TET1FL cells by 5hmC dot blot analysis. The bottom panel shows methylene blue staining using the total amount of input DNA as the loading control. b Immunostaining of insulin (INS) and glucagon (GCG) at the PE stage for WT, TKO-TET1CD mut , TKO-TET1CD, and TKO-TET1FL cells. Scale bar=50 μm. c Representative plots of flow cytometry of NKX6.1, human C-peptide (CPEP), and glucagon (GCG) in WT, TKO-TET1CD mut , TKO-TET1CD, and TKO-TET1FL cells at the PE stage. Quantifications of the percentage of NKX6.1 + , CPEP + , or GCG + cells are shown in the right panel. n = 3 independent differentiation. d Western blots showing immunoprecipitation with antibodies against FLAG on cell extract of TKO-TET1FL cells followed by immunoblotting with antibodies against TET1, SOX17, GATA6, or FOXA1. TKO is included as control. e Locus-specific decreases in 5-methylcytosine (5mC) at the PAX4 enhancer in TKO or TET1KO samples compared with TET2/3DKO samples. Percentages of unmethylated cytosine and 5mC at CCGG sites are shown. n = 3 independent differentiation.

Article Snippet: To generate overexpression constructs for TET1, the C-terminal FLAG-tagged human full-length TET1 (TET1FL), TET1 catalytic domain (TET1CD), and TET1 catalytic inactive mutant (TET1CD mut ) were amplified from FH-TET1-pEF (Addgene #49792) or pIRES-hrGFP II-mTET1 (Addgene #83569), and cloned into the NotI digested PCDH-CAG-MCS-P2A-Puro vector (a kind gift from Dr. R. Xu, University of Macau) .

Techniques: Dot Blot, Staining, Control, Immunostaining, Flow Cytometry, Western Blot, Immunoprecipitation

Journal: bioRxiv

Article Title: TET1 dioxygenase is required for FOXA2-associated chromatin remodeling in pancreatic beta-cell differentiation

doi: 10.1101/2020.05.20.107532

Figure Lengend Snippet: a Immunostaining of PDX1 and NKX6.1 at the PP stage for WT, TKO-TET1CD mut , TKO-TET1CD, and TKO-TET1FL cells. Scale bar=50 μm. b Expression of PDX1 , NKX6.1 , PAX4 , and INS in WT, WT, TKO-TET1CD mut , TKO-TET1CD, and TKO-TET1FL cells at the PE stage. c Western blots showing immunoprecipitation with antibodies against FLAG on cell extract of TKO-TET1FL cells followed by immunoblotting with antibodies against TET1 or FOXA2. TKO is included as control. d Genome-browser view of the PAX4 locus with increased methylation and decreased chromatin associability upon TET depletion at a FOXA2-bound site featured enhancer signatures H3K4me1 and H3K27ac. e Locus-specific increase in 5mC at the PAX4 enhancer in TKO or TKO-TET1CD samples compared with WT and TKO-TET1FL samples. Percentages of unmethylated cytosine and 5mC at CCGG sites are shown. n = 3 independent differentiation. f Schematic model depicting cooperative interaction among TET1 and TFs in lineage-specific enhancer activation. Pioneer TF FOXA2 transiently binds to enhancers. Lineage-specific TFs, such as PTF1A and NEUROD1, stabilize the binding of FOXA2 which recruits TET1 to induce DNA demethylation and subsequent chromatin opening.

Article Snippet: To generate overexpression constructs for TET1, the C-terminal FLAG-tagged human full-length TET1 (TET1FL), TET1 catalytic domain (TET1CD), and TET1 catalytic inactive mutant (TET1CD mut ) were amplified from FH-TET1-pEF (Addgene #49792) or pIRES-hrGFP II-mTET1 (Addgene #83569), and cloned into the NotI digested PCDH-CAG-MCS-P2A-Puro vector (a kind gift from Dr. R. Xu, University of Macau) .

Techniques: Immunostaining, Expressing, Western Blot, Immunoprecipitation, Control, Methylation, Activation Assay, Binding Assay

Journal: Scientific Reports

Article Title: Epigenetic inactivation of the CpG demethylase TET1 as a DNA methylation feedback loop in human cancers

doi: 10.1038/srep26591

Figure Lengend Snippet: ( A ) Representative methylome data. TET1 gene structure, promoter and exon 1 (NCBI database GRCh37.p13) are shown on the top panel. E1: exon 1. Positive methylation signal peaks identified by MeDIP-chip are shown in pink shadow for: NPC xenografts (C15, C18) and primary tumor (OCT83), ESCC cell lines (KYSE140, KYSE510), HCC cell lines (HuH7, HepG2) and primary tumor (HCC418T), NKTCL cell lines (SNK6, NK-YS) and primary tumor (NK1). ( B ) Expression of TET family genes ( TET1, −2, −3 ) in human normal adult and fetal tissues by semi-quantitative RT-PCR, with GAPDH as a control. Sk. M., skeleton muscle.

Article Snippet: Human TET1 catalytic domain (TET1-CD) cDNA and its catalytic domain mutant (TET1- CD-mut) clones (Addgene, Cambridge, MA) were used as templates to generate TET1 constructs with an N-terminal Flag tag, and subcloned into pcDNA3.1 vector (Invitrogen, Carlsbad, Ca).

Techniques: Methylation, Methylated DNA Immunoprecipitation, Expressing, Quantitative RT-PCR

Journal: Scientific Reports

Article Title: Epigenetic inactivation of the CpG demethylase TET1 as a DNA methylation feedback loop in human cancers

doi: 10.1038/srep26591

Figure Lengend Snippet: ( A ) Structure of the TET1 promoter CpG island (CGI). CpG sites are shown as short vertical lines. MSP primer sites and BGS region analyzed are also indicated. ( B ) TET1 methylation was not detected in not-bisulfited DNA samples, indicating that the MSP system is specific. m4/m8 represents specific MSP primer set of TET1 methylation detection. ( C , D ) TET1 was frequently silenced and methylated in multiple carcinoma and lymphoma cell lines, detected by semi-quantitative RT-PCR and MSP, but expressed and unmethylated in immortalized but non-transformed normal epithelial cell lines (with names green underlined). M, methylated; U, unmethylated. ( E ) Abundant expression of TET2 and TET3 in TET1 -downregulated tumor cell lines. Ca, carcinoma; NPC, nasopharyngeal carcinoma; ESCC, esophageal squamous cell carcinoma; CRC, colorectal cancer; RCC, renal cancer; NKTCL, nasal NK/T-cell lymphoma.

Article Snippet: Human TET1 catalytic domain (TET1-CD) cDNA and its catalytic domain mutant (TET1- CD-mut) clones (Addgene, Cambridge, MA) were used as templates to generate TET1 constructs with an N-terminal Flag tag, and subcloned into pcDNA3.1 vector (Invitrogen, Carlsbad, Ca).

Techniques: Methylation, Quantitative RT-PCR, Transformation Assay, Expressing

Journal: Scientific Reports

Article Title: Epigenetic inactivation of the CpG demethylase TET1 as a DNA methylation feedback loop in human cancers

doi: 10.1038/srep26591

Figure Lengend Snippet: Summary of TET1 methylation in cell lines, tumor and normal tissues.

Article Snippet: Human TET1 catalytic domain (TET1-CD) cDNA and its catalytic domain mutant (TET1- CD-mut) clones (Addgene, Cambridge, MA) were used as templates to generate TET1 constructs with an N-terminal Flag tag, and subcloned into pcDNA3.1 vector (Invitrogen, Carlsbad, Ca).

Techniques: Methylation

Journal: Scientific Reports

Article Title: Epigenetic inactivation of the CpG demethylase TET1 as a DNA methylation feedback loop in human cancers

doi: 10.1038/srep26591

Figure Lengend Snippet: ( A ) Detection of TET1 methylation in multiple tumor cell lines and normal cell lines by BGS. ( B ) Treatment with Aza or combined with TSA (A + T) demethylated TET1 promoter in silenced cell lines of multiple tissue types. Expression and methylation changes were detected by semi-quantitative RT-PCR and MSP. ( C ) BGS analysis of TET1 promoter in cell lines with or without treatment. NPC, nasopharyngeal carcinoma; ESCC, esophageal squamous cell carcinoma; CRC, colorectal cancer; BrCa, breast cancer; RCC, renal cancer; Ca, carcinoma.

Article Snippet: Human TET1 catalytic domain (TET1-CD) cDNA and its catalytic domain mutant (TET1- CD-mut) clones (Addgene, Cambridge, MA) were used as templates to generate TET1 constructs with an N-terminal Flag tag, and subcloned into pcDNA3.1 vector (Invitrogen, Carlsbad, Ca).

Techniques: Methylation, Expressing, Quantitative RT-PCR

Journal: Scientific Reports

Article Title: Epigenetic inactivation of the CpG demethylase TET1 as a DNA methylation feedback loop in human cancers

doi: 10.1038/srep26591

Figure Lengend Snippet: TET1 promoter methylation in ( A ) multiple primary tumors and ( B ) nose swab samples from NPC patients, detected by MSP. ( C ) TET1 methylation is barely seen in normal tissues by MSP analysis. ( D ) Representative BGS analysis of TET1 promoter methylation in primary tumors and normal tissues. Circles, CpG sites analyzed; row of circles, an individual promoter allele that was cloned, randomly selected and sequenced; filled circle, methylated CpG site; open circle, unmethylated site. ( E ) Levels of TET1 mRNA expression in representative paired tumor (T)/normal (N) tissues, and primary tumor tissues (NPC), measured by semi-quantitative RT-PCR. Ca, carcinoma; NPC, nasopharyngeal carcinoma; CRC, colorectal cancer; RCC, renal cancer; GsCa, gastric cancer; Sk. muscle, skeleton muscle; S. intestine, small intestine.

Article Snippet: Human TET1 catalytic domain (TET1-CD) cDNA and its catalytic domain mutant (TET1- CD-mut) clones (Addgene, Cambridge, MA) were used as templates to generate TET1 constructs with an N-terminal Flag tag, and subcloned into pcDNA3.1 vector (Invitrogen, Carlsbad, Ca).

Techniques: Methylation, Clone Assay, Expressing, Quantitative RT-PCR

Journal: Scientific Reports

Article Title: Epigenetic inactivation of the CpG demethylase TET1 as a DNA methylation feedback loop in human cancers

doi: 10.1038/srep26591

Figure Lengend Snippet: Somatic mutations of TET1 gene in human cancers were analyzed using the COSMIC database. ( A ) Frequencies and ( B ) distributions of TET1 mutations. ( C ) Diagram displaying complete TET1 mutation spectrum identified and their distribution in the coding region of TET1.

Article Snippet: Human TET1 catalytic domain (TET1-CD) cDNA and its catalytic domain mutant (TET1- CD-mut) clones (Addgene, Cambridge, MA) were used as templates to generate TET1 constructs with an N-terminal Flag tag, and subcloned into pcDNA3.1 vector (Invitrogen, Carlsbad, Ca).

Techniques: Mutagenesis

Journal: Scientific Reports

Article Title: Epigenetic inactivation of the CpG demethylase TET1 as a DNA methylation feedback loop in human cancers

doi: 10.1038/srep26591

Figure Lengend Snippet: ( A ) Structure and functional domains of the human TET1 protein, containing a C-terminal CD domain including the Cys-rich and DSBH regions, and a CXXC domain. The positions of three nuclear localization sequences (NLS) are shown. TET1 catalytic domain (TET1-CD) containing the Cys-rich and DSBH regions and TET1 mutant (TET1-CD-mut) with two amino acid substitutions (H1672A; D1674A) in the catalytic domain are also shown. ( B ) Ectopic expression of TET1-CD inhibited tumor cell growth of multiple tissue types. Representative colony formation assays of TET1-CD- and TET1-CD-mut-expressing tumor cells of nasopharyngeal, esophageal, gastric, colon, and breast cancers are shown. Quantitative analyses of colony numbers are shown as values of mean ± S.D. (lower panel), ***p < 0.001. NPC, nasopharyngeal carcinoma; ESCC, esophageal squamous cell carcinoma; GsCa, gastric cancer; CRC, colorectal cancer; BrCa, breast cancer. ( C ) Ectopic expression of TET1-CD induced tumor cell apoptosis. TET1-CD, TET1-CD-mut, and vector-expressing NPC tumor cells (HONE1) were analyzed by TUNEL assays. ( D ) TET1-CD upregulated multiple TSGs expression in tumor cells, as examined by semi-quantitative RT-PCR. ( E ) TET1-CD upregulated multiple TSGs expression as measured by qRT-PCR in NPC (HNE1) cells. Fold changes of TSGs expression in TET1-CD and TET1-CD-mut-transcfected cells were calculated by normalizing towards vector-expressing cells (set 1.0). GAPDH was used as an internal control. Data are shown as mean ± SD of three independent experiments. *p < 0.05; **p < 0.01; ***p < 0.001. ( F ) Detection of promoters methylation of HOXA9, SLIT2 and ZNF382 genes by MSP in TET1-CD and TET1-CD-mut-expressing tumor cells.

Article Snippet: Human TET1 catalytic domain (TET1-CD) cDNA and its catalytic domain mutant (TET1- CD-mut) clones (Addgene, Cambridge, MA) were used as templates to generate TET1 constructs with an N-terminal Flag tag, and subcloned into pcDNA3.1 vector (Invitrogen, Carlsbad, Ca).

Techniques: Functional Assay, Mutagenesis, Expressing, Plasmid Preparation, TUNEL Assay, Quantitative RT-PCR, Methylation

Journal: Scientific Reports

Article Title: Epigenetic inactivation of the CpG demethylase TET1 as a DNA methylation feedback loop in human cancers

doi: 10.1038/srep26591

Figure Lengend Snippet: When normal cells are exposed to carcinogens (chemical carcinogens, tumor viruses, etc), DNA methyltransferases (DNMTs) are induced, upregulated or overactivated, which further generates higher levels of DNA CpG methylation (5 mC). Elevated level of 5 mC on tumor suppressor gene (TSG) promoters lead to TSGs silencing and functional inactivation, ultimately to tumorigenesis. Ten-eleven-translocation (TET) proteins catalyze DNA CpG demethylation through converting 5 mC to 5-hydroxymethylcytosine (5 hmC), maintaining a delicate balance between CpG methylation and demethylation in normal cells. While in premalignant or tumor cells, CpG demethylation by TET would induce TSG promoter demethylation and functional restoration for further tumor suppression. Thus unlike normal cells where TET proteins are abundant, loss of TET1 expression through promoter CpG methylation frequently occurs in tumor cells, which in turn, increases 5 mC levels and promotes TSG inactivation in tumor pathogenesis.

Article Snippet: Human TET1 catalytic domain (TET1-CD) cDNA and its catalytic domain mutant (TET1- CD-mut) clones (Addgene, Cambridge, MA) were used as templates to generate TET1 constructs with an N-terminal Flag tag, and subcloned into pcDNA3.1 vector (Invitrogen, Carlsbad, Ca).

Techniques: CpG Methylation Assay, Functional Assay, Translocation Assay, Expressing