Journal: Nature

Article Title: RNA m 5 C oxidation by TET2 regulates chromatin state and leukaemogenesis

doi: 10.1038/s41586-024-07969-x

Figure Lengend Snippet: a , b , Average histone modification levels, as well as input signals, in WT and Tet2 -KO mESCs at DNA hypermethylated regions ( a ) or hypomethylated regions ( b ), detected in Tet2 -KO mESCs. c , Average ATAC signals in WT, Tet2 -KO, and Pspc1 -KO mESCs at DNA 5mC hypermethylated regions or hypomethylated regions detected in Tet2 -KO mESCs. d , Correlation between DNA methylation differences in Tet2 -KO versus WT mESCs and changes in their downstream gene transcription. carRNAs were categorized into different groups, including eRNA, paRNA, and repeat RNA. Within each group of carRNAs, they were further divided into 50 bins based on the ranked DNA methylation differences upon Tet2 KO in mESCs. Error band represents standard errors. e , Schematics showing the dual mode of chromatin regulation by TET2 when bound to different protein partners. f , Overlapping ratios of regions marked by different histone modifications with DNA hypermethylated regions (detected in the Tet2 -KO mESCs). g , H2AK119ub and H3K27me3 chromatin bindings at IAP, MERVL, and LINE loci measured through CUT&Tag protocol. Comparisons against “0 h” group. h - i , Total number of ATAC-seq peaks in mESCs. h , Number of ATAC-seq peaks marked with H2AK119ub modification in mESCs; i , ATAC-seq peaks were categorized into four groups: differentially upregulated (Up), downregulated (Down), or unchanged (Invar) after Tet2 KO in mESCs. Additionally, they were classified based on their association with H2AK119ub as either marked (H2AK119ub) or not marked (notH2AK119ub). j , Volcano plot displaying changes in H2AK119ub peak intensity for Tet2 -KO versus WT mESCs. k , Overlap of ATAC-seq peaks with increased chromatin accessibility (up ATAC) or decreased H2AK119ub (Down H2AK119ub) in Tet2 -KO mESCs. l , Genomic distribution of H2AK119ub peaks in WT and Tet2 -KO mESCs. Line centre ( g ) represents mean and whiskers s.d. P values, Wald test ( j ), two-tailed unpaired t test with Welch’s correction ( g ), two-tailed t-distribution with n-2 degrees of freedom ( d ). NS, P > 0.05. n = 3 biological replicates ( g ). The depicted genome-wide data represent an integration of three biological replicates ( c , d , f , h - l ) or two biological replicates ( a , b ).

Article Snippet: The antibodies used in this study are summarized below: rabbit monoclonal anti-H2AK119ub antibody (Cell Signaling Technology, 8240S, 1:1,000 for western blot, 1:50 for CUT&Tag); rabbit monoclonal anti-H3 antibody (Cell Signaling Technology, 4499S, 1:1,000); mouse monoclonal anti-TET2 antibody (MilliporeSigma, MABE462, 1:500); rabbit monoclonal anti-GAPDH antibody, HRP conjugate (Cell Signaling Technology, 8884S, 1:1,000); rabbit monoclonal anti-DDDDK tag antibody (Abcam, ab205606, 1:1,000 for western blot, 1:50 for immunoprecipitation); rabbit polyclonal anti-SNRP70/U1-70K antibody (Abcam, ab83306, 1:1,000); mouse monoclonal anti-5-methylcytosine antibody (Diagenode, C15200081-100, 1:1,000 for dot blot, 1:50 for meRIP); mouse monoclonal anti-hm 5 C antibody (Diagenode, C15200200-100, clone Mab-31HMC, 1:1,000 for dot blot); rabbit monoclonal anti-H3K27me3 antibody (Cell Signaling Technology, 9733S, only for CUT&Tag experiments, 1:50); mouse monoclonal anti-BAP1 antibody (Santa Cruz, sc-28383, 1:50 for CUT&Tag).

Techniques: Modification, DNA Methylation Assay, Two Tailed Test, Genome Wide

Journal: Nature

Article Title: RNA m 5 C oxidation by TET2 regulates chromatin state and leukaemogenesis

doi: 10.1038/s41586-024-07969-x

Figure Lengend Snippet: a , b , Feature enrichment in hypermethylated differentially methylated regions (DMRs) compared with hypomethylated DMRs (hyperspecific; a ), and in hypomethylated DMRs compared with hypermethylated DMRs (hypospecific; b ) using the odds ratio. c , Overlapping ratios of histone modifications with DNA hypomethylated regions for Tet2 -KO versus WT mES cells. d , The H2AK119ub levels at IAP loci after TET2-CD (catalytic domain; WT or catalytically dead HxD) tethering by dCas13. e , Representative image showing nucleic acids cross-linked to MBD6. f , Spike-in-calibrated ATAC–seq signals or H2AK119ub signals in WT (siNC WT) and Tet2- KO (siNC Tet2 KO) mES cells, as well as in Mbd6- KD Tet2 -KO (siMbd6 Tet2 KO) mES cells. g , Changes in H2AK119ub on m 5 C hypomethylated (hypo.), unchanged (invar.) and hypermethylated (hyper.) repeat RNA after Tet2 KO versus WT. h , H2AK119ub changes in m 5 C hypermethylated repeat RNA in Tet2 -KO versus WT compared with Mbd6- KD Tet2 -KO versus Tet2 -KO mES cells. i , The correlation between ATAC signal fold changes in Tet2 -KO versus WT mES cells, and comparing Mbd6 KD with control Tet2 -KO mES cells. PCC, Pearson correlation coefficient. j , The correlation between changes in ATAC and H2AK119ub signals, comparing Mbd6 KD with control Tet2- KO mES cells. k , The H2AK119ub levels and ATAC signal at IAP RNA after dCas13–MBD6-MBD tethering. l , Schematics of the proposed pathway of MBD6 regulating chromatin state through caRNA m 5 C binding. Data are mean ± s.d. ( d and k ) and mean ± s.e.m. ( f ). For g and h , the box plots show the median (centre line), the upper and lower quartiles (box limits) and 1.5× the interquartile range (whiskers). P values were calculated using two-tailed unpaired t -tests with Welch’s correction ( d and k ), two-tailed Wilcoxon–Mann–Whitney tests ( g and h ), two-tailed t -distribution with n − 2 d.f. ( i and j ). n = 3 biological replicates ( d – f and k ). The depicted genome-wide data represent an integration of three biological replicates.

Article Snippet: The antibodies used in this study are summarized below: rabbit monoclonal anti-H2AK119ub antibody (Cell Signaling Technology, 8240S, 1:1,000 for western blot, 1:50 for CUT&Tag); rabbit monoclonal anti-H3 antibody (Cell Signaling Technology, 4499S, 1:1,000); mouse monoclonal anti-TET2 antibody (MilliporeSigma, MABE462, 1:500); rabbit monoclonal anti-GAPDH antibody, HRP conjugate (Cell Signaling Technology, 8884S, 1:1,000); rabbit monoclonal anti-DDDDK tag antibody (Abcam, ab205606, 1:1,000 for western blot, 1:50 for immunoprecipitation); rabbit polyclonal anti-SNRP70/U1-70K antibody (Abcam, ab83306, 1:1,000); mouse monoclonal anti-5-methylcytosine antibody (Diagenode, C15200081-100, 1:1,000 for dot blot, 1:50 for meRIP); mouse monoclonal anti-hm 5 C antibody (Diagenode, C15200200-100, clone Mab-31HMC, 1:1,000 for dot blot); rabbit monoclonal anti-H3K27me3 antibody (Cell Signaling Technology, 9733S, only for CUT&Tag experiments, 1:50); mouse monoclonal anti-BAP1 antibody (Santa Cruz, sc-28383, 1:50 for CUT&Tag).

Techniques: Methylation, Control, Binding Assay, Two Tailed Test, MANN-WHITNEY, Genome Wide

Journal: Nature

Article Title: RNA m 5 C oxidation by TET2 regulates chromatin state and leukaemogenesis

doi: 10.1038/s41586-024-07969-x

Figure Lengend Snippet: a , Sequence alignment of human MBD family proteins (MBD1-6 and MeCP2) showing differences in the MBD region. b , Validation of Flag-MBD5 or Flag-MBD6 overexpressing mESCs using RT-qPCR. Averaged transcript expressions were indicated. Comparisons against “WT” group. c , Representative gel image quantifying nucleic acids crosslinked to MBD5 or MBD6. Comparisons against “RNase-, DNase-” group. d , EMSA showing the binding preferences of the purified MBD domain of MBD6 fused to maltose binding protein (MBP) with C, m 5 C, or hm 5 C-containing oligonucleotide probes. The purity of the single-stranded probe (ss probe) and double-stranded probe (ds probe) was validated by electrophoresis. Gradient for MBP-MBD protein was 0, 0.1, 0.5, 2.5, 12, 50, and 100 μM. e , EMSA showing that ASXL1 binding to MBD6 does not affect MBD6-m 5 C binding. MBD6 MBD domain (amino acid 1–100 fused by MBP-His) and ASXL1 ASXH domain (amino acid 1509–1541 fused by His-SUMO) were used. f , m 5 C abundance in MBD5 and MBD6 enriched RNA. g , Overlap between the RNA binding sites of MBD5 and MBD6. h - j , Repeat RNA expression ( h ), H2AK119ub ( i ) or caRNA m 5 C ( j ) level upon Mbd5 or Mbd6 KD. Comparisons against “siNC” group. k , IAP RNA lifetime upon Tet2 KO or Mbd6 KD in mESCs. l , qPCR results assessing Mbd6 or Nsun2 RNA abundance in mESCs. Comparisons against “siNC” groups. m , Chromatin openness measured by DNase-TUNEL assay upon Nsun2 or Mbd6 KD. n - r , Spike-in calibrated ATAC-seq signals in WT (WT [siNC]) and Tet2 KO ( Tet2 KO [siNC]) mESCs, as well as Mbd6 KD ( Tet2 KO siMbd6) and Nsun2 KD ( Tet2 KO siNsun2) in Tet2 -KO mESC cells. Metagene profile at genes regulated by different RNA polymerases ( n ), protein-coding genes ( o ), overall signal ( p , q ), or clustered ATAC peaks ( r ) were shown. s , Correlation fold changes in spike-in calibrated ATAC-seq signals in Tet2 KO versus WT mESCs, and comparing Nsun2 KD or Mbd6 KD with control Tet2 KO mESCs. t , Venn diagram displaying the significantly up-regulated ATAC-seq peaks in Tet2 KO compared with WT mESC cells, and down-regulated ATAC-seq peaks in Nsun2 KD or Mbd6 KD compared with control mESC cells following Tet2 KO. Bar ( b , c , f , h - j , l , p ) or line centre ( k ) represents mean and whiskers s.d. P values, two-tailed unpaired t test with Welch’s correction ( b , c , f , h - l ), two-tailed t-distribution with n-2 degrees of freedom ( s ). NS, P > 0.05. n = 3 biological replicates ( b - f , h - r ). The depicted genome-wide data represent an integration of three biological replicates.

Article Snippet: The antibodies used in this study are summarized below: rabbit monoclonal anti-H2AK119ub antibody (Cell Signaling Technology, 8240S, 1:1,000 for western blot, 1:50 for CUT&Tag); rabbit monoclonal anti-H3 antibody (Cell Signaling Technology, 4499S, 1:1,000); mouse monoclonal anti-TET2 antibody (MilliporeSigma, MABE462, 1:500); rabbit monoclonal anti-GAPDH antibody, HRP conjugate (Cell Signaling Technology, 8884S, 1:1,000); rabbit monoclonal anti-DDDDK tag antibody (Abcam, ab205606, 1:1,000 for western blot, 1:50 for immunoprecipitation); rabbit polyclonal anti-SNRP70/U1-70K antibody (Abcam, ab83306, 1:1,000); mouse monoclonal anti-5-methylcytosine antibody (Diagenode, C15200081-100, 1:1,000 for dot blot, 1:50 for meRIP); mouse monoclonal anti-hm 5 C antibody (Diagenode, C15200200-100, clone Mab-31HMC, 1:1,000 for dot blot); rabbit monoclonal anti-H3K27me3 antibody (Cell Signaling Technology, 9733S, only for CUT&Tag experiments, 1:50); mouse monoclonal anti-BAP1 antibody (Santa Cruz, sc-28383, 1:50 for CUT&Tag).

Techniques: Sequencing, Biomarker Discovery, Quantitative RT-PCR, Binding Assay, Purification, Electrophoresis, RNA Binding Assay, RNA Expression, TUNEL Assay, Control, Two Tailed Test, Genome Wide

Journal: Nature

Article Title: RNA m 5 C oxidation by TET2 regulates chromatin state and leukaemogenesis

doi: 10.1038/s41586-024-07969-x

Figure Lengend Snippet: a , Metagene profile of the spike-in calibrated ATAC-seq signals of WT and Tet2 −/− HSPCs. b , qPCR results validating efficiency of shRNA-enabled Mbd6 KD in HSPCs. c , Colony formation of serial replating assays of HSPCs upon Mbd6 KD. d , Representative flow cytometry results characterizing HSPC frequency in suspension cultures (top, KIT and Lineage as markers, day 7) and differentiation (bottom, CD11b as marker, day14) upon Mbd6 KD. e , f , Spike-in calibrated ATAC-seq signals in WT (shNC WT) and Tet2 -KO (shNC Tet2 KO) HSPCs, as well as in Mbd6 -KD (shMbd6 Tet2 KO) Tet2 -KO HSPCs. Overall levels ( e ) and metagene profiles ( f ) were shown. g , Volcano plot comparing spike-in calibrated ATAC-seq peak signals in Mbd6 -KD versus control Tet2 -KO HSPCs. h , i Spike-in calibrated H2AK119ub levels in WT (shNC WT) and Tet2 -KO (shNC Tet2 KO) HSPCs, as well as in Mbd6 -KD (shMbd6 Tet2 KO) Tet2 -KO HSPCs. Overall levels ( h ) and metagene profiles ( i ) were shown. j , Volcano plot comparing spike-in calibrated H2AK119ub peak signals in Mbd6 KD compared with control Tet2 -KO HSPCs. k , Quantifications of in vitro differentiation and maintenance of HSPCs with Nsun2 , Nsun5 or Trdmt1 KD. l , qPCR results assessing efficacies of shRNA-enabled Nsun2 , Nsun5 or Trdmt1 KD in HSPCs. m , Half life measurements of IAP RNA in HSPCs of different genotypes. WT denotes wild-type, Tet2 KO denotes Tet2 KO, EE mutant lost hm 5 C oxidation activities and HxD mutant is catalytically inactive. Bar ( b , e , h , l , m ) or line centre ( k , m ) represents mean and whiskers s.d. ( b , k - m ) or s.e.m. ( e , h ). P values, Wald test ( g , j ), two-tailed unpaired t test with Welch’s correction ( b , e , h , k - m ). NS, P > 0.05. n = 3 biological replicates ( a - c , e - m ). The depicted genome-wide data represent an integration of three biological replicates.

Article Snippet: The antibodies used in this study are summarized below: rabbit monoclonal anti-H2AK119ub antibody (Cell Signaling Technology, 8240S, 1:1,000 for western blot, 1:50 for CUT&Tag); rabbit monoclonal anti-H3 antibody (Cell Signaling Technology, 4499S, 1:1,000); mouse monoclonal anti-TET2 antibody (MilliporeSigma, MABE462, 1:500); rabbit monoclonal anti-GAPDH antibody, HRP conjugate (Cell Signaling Technology, 8884S, 1:1,000); rabbit monoclonal anti-DDDDK tag antibody (Abcam, ab205606, 1:1,000 for western blot, 1:50 for immunoprecipitation); rabbit polyclonal anti-SNRP70/U1-70K antibody (Abcam, ab83306, 1:1,000); mouse monoclonal anti-5-methylcytosine antibody (Diagenode, C15200081-100, 1:1,000 for dot blot, 1:50 for meRIP); mouse monoclonal anti-hm 5 C antibody (Diagenode, C15200200-100, clone Mab-31HMC, 1:1,000 for dot blot); rabbit monoclonal anti-H3K27me3 antibody (Cell Signaling Technology, 9733S, only for CUT&Tag experiments, 1:50); mouse monoclonal anti-BAP1 antibody (Santa Cruz, sc-28383, 1:50 for CUT&Tag).

Techniques: shRNA, Flow Cytometry, Suspension, Marker, Control, In Vitro, Mutagenesis, Two Tailed Test, Genome Wide

Journal: Nature

Article Title: RNA m 5 C oxidation by TET2 regulates chromatin state and leukaemogenesis

doi: 10.1038/s41586-024-07969-x

Figure Lengend Snippet: a , Schematics of the mixed chimera transplantation assay. b , Quantification of CD45.2 + cells in the PB of recipient mice at different timepoints after transplantation. c , Quantification of CD45.2 + cells in the BM (left) or spleen (right) of recipient mice. d , e , Representative image ( d ) and quantification ( e ) of spleen size isolated from recipient mice at 24 weeks after transplantation. f , g , Colony formation analysis of the serial replating assay ( f ) and flow cytometry analyses of suspension cultures ( g ) of WT and Tet2 −/− HSPCs with (shMbd6) or without (shNC) Mbd6 KD. h , The correlation between changes in ATAC–seq signal and H2AK119ub signal in response to Mbd6 KD in Tet2 -KO HSPCs. i , Integrative Genome Viewer visualization of the H2AK119ub and ATAC signal around the Socs3 or Nfkbia genes in WT, Tet2 KO and shMbd6 after Tet2 KO in HSPCs. The values in parentheses represent the scale of signal in each track. Data are mean ± s.d. ( b , c and e – g ). P values were calculated using two-tailed unpaired t -tests with Welch’s correction ( b , c and e – g ) and two-tailed t -distribution with n − 2 d.f. ( h ). n = 3 biological replicates ( b – g ). The depicted genome-wide data represent an integration of three biological replicates.

Article Snippet: The antibodies used in this study are summarized below: rabbit monoclonal anti-H2AK119ub antibody (Cell Signaling Technology, 8240S, 1:1,000 for western blot, 1:50 for CUT&Tag); rabbit monoclonal anti-H3 antibody (Cell Signaling Technology, 4499S, 1:1,000); mouse monoclonal anti-TET2 antibody (MilliporeSigma, MABE462, 1:500); rabbit monoclonal anti-GAPDH antibody, HRP conjugate (Cell Signaling Technology, 8884S, 1:1,000); rabbit monoclonal anti-DDDDK tag antibody (Abcam, ab205606, 1:1,000 for western blot, 1:50 for immunoprecipitation); rabbit polyclonal anti-SNRP70/U1-70K antibody (Abcam, ab83306, 1:1,000); mouse monoclonal anti-5-methylcytosine antibody (Diagenode, C15200081-100, 1:1,000 for dot blot, 1:50 for meRIP); mouse monoclonal anti-hm 5 C antibody (Diagenode, C15200200-100, clone Mab-31HMC, 1:1,000 for dot blot); rabbit monoclonal anti-H3K27me3 antibody (Cell Signaling Technology, 9733S, only for CUT&Tag experiments, 1:50); mouse monoclonal anti-BAP1 antibody (Santa Cruz, sc-28383, 1:50 for CUT&Tag).

Techniques: Transplantation Assay, Isolation, Flow Cytometry, Suspension, Two Tailed Test, Genome Wide

Journal: Nature

Article Title: RNA m 5 C oxidation by TET2 regulates chromatin state and leukaemogenesis

doi: 10.1038/s41586-024-07969-x

Figure Lengend Snippet: a , Proliferation of WT or TET2 -KO K-562 or THP-1 cells with (shMBD6) or without (shNC) MBD6 KD. n = 4. b , The K-562 H2AK119ub level changes after MBD6 KD. n = 3. c , NSG mice were transplanted with K-562 (left) or THP-1 (right) cells and their overall survival is shown as the Kaplan–Meier estimator. n = 5 mice. d , Heat map illustrating the spike-in-calibrated ATAC–seq signals on ATAC–seq peak regions in WT (siNC WT) and TET2 -KO (siNC TET2 KO) K-562 cells, as well as in NSUN2 KD (siNSUN2 TET2 KO) and MBD6 -KD (siMBD6 TET2 KO) TET2 -KO K-562 cells. n = 3. Data are row-normalized using z scores. e , The correlation of changes in ATAC signals between TET2- KO versus WT K-562 cells, and comparing NSUN2 KD or MBD6 KD with control in TET2- KO K-562 cells. f , m 5 C methylation level changes of K-562 cells in different repeat RNA families after TET2 KO. The size of the circle represents the number of loci methylated by m 5 C. g , The H2AK119ub signals around HERVH-int genomic loci. The colour bar shows H2AK119ub signal. Data are mean ± s.d. P values were calculated using two-tailed unpaired t -tests with Welch’s correction ( a and b ), log-rank Mentel–Cox tests ( c ) and two-tailed t -distribution with n − 2 d.f. ( e ). The depicted genome-wide data represent an integration of three biological replicates.

Article Snippet: The antibodies used in this study are summarized below: rabbit monoclonal anti-H2AK119ub antibody (Cell Signaling Technology, 8240S, 1:1,000 for western blot, 1:50 for CUT&Tag); rabbit monoclonal anti-H3 antibody (Cell Signaling Technology, 4499S, 1:1,000); mouse monoclonal anti-TET2 antibody (MilliporeSigma, MABE462, 1:500); rabbit monoclonal anti-GAPDH antibody, HRP conjugate (Cell Signaling Technology, 8884S, 1:1,000); rabbit monoclonal anti-DDDDK tag antibody (Abcam, ab205606, 1:1,000 for western blot, 1:50 for immunoprecipitation); rabbit polyclonal anti-SNRP70/U1-70K antibody (Abcam, ab83306, 1:1,000); mouse monoclonal anti-5-methylcytosine antibody (Diagenode, C15200081-100, 1:1,000 for dot blot, 1:50 for meRIP); mouse monoclonal anti-hm 5 C antibody (Diagenode, C15200200-100, clone Mab-31HMC, 1:1,000 for dot blot); rabbit monoclonal anti-H3K27me3 antibody (Cell Signaling Technology, 9733S, only for CUT&Tag experiments, 1:50); mouse monoclonal anti-BAP1 antibody (Santa Cruz, sc-28383, 1:50 for CUT&Tag).

Techniques: Control, Methylation, Two Tailed Test, Genome Wide

Journal: Nature

Article Title: RNA m 5 C oxidation by TET2 regulates chromatin state and leukaemogenesis

doi: 10.1038/s41586-024-07969-x

Figure Lengend Snippet: a , Representative subcellular fractionation of K-562 cells. Cyt: cytosolic; Nuc: nuclear soluble; Chr: chromatin. b , Overall caRNA and whole-cell RNA abundance relative to spike-in in WT, TET2 -KO and TET2 -KO + MBD6 -KD K-562 cells. c , Metagene profile of the calibrated ATAC-seq signals of WT (siNC WT) and Tet2 KO (siNC Tet2 KO) K-562 cells, as well as in Mbd6 KD (siMbd6 Tet2 KO) Tet2 KO K-562 cells. d , Volcano plot comparing spike-in calibrated ATAC-seq peaks in Mbd6 -KD versus control in Tet2 -KO K-562 cells. e , Metagene profile of the spike-in calibrated H2AK119ub signals of WT (siNC WT) and Tet2 -KO (siNC Tet2 KO) K-562 cells, as well as in Mbd6 -KD (siMbd6 Tet2-KO) Tet2 -KO K-562 cells. f , Volcano plot comparing spike-in calibrated H2AK119ub peaks in Mbd6 -KD compared with control Tet2 -KO K-562 cells. g , Correlation between changes in ATAC and H2AK119ub signals, comparing Mbd6 -KD with control in Tet2 -KO K-562 cells. h , Venn diagram displaying the significantly up-regulated ATAC-seq peaks in Tet2 -KO compared with WT K-562 cells, and down-regulated ATAC-seq peaks in Nsun2 -KD or Mbd6 -KD compared with control Tet2 -KO K-562 cells. i , Overlap of differentially expressed genes upon TET2 KO in control K-562 cells, and upon MBD6 KD in TET2 -KO K-562 cells. Barplot depicting the odds ratio of upDEGs following TET2 KO, in comparison to both upDEGs and downDEGs upon MBD6 KD in TET2 KO K-562 cells. j , KEGG pathway enrichment analysis performed on genes that were upregulated in TET2 -KO in control K-562 cells, and downregulated in MBD6 -KD in TET2 -KO K-562 cells. Larger circles indicate greater significance. k , Correlation of gene expression changes between TET2 KO versus WT, and MBD6 KD versus control in TET2 KO K-562 cells. The genes are associated with various signalling pathways, including Th1 and Th2 cell differentiation, NF-kappa B signalling pathway, and C-type lectin receptor signalling pathways. Error band represents standard errors. Bar represents mean ( b ) and whiskers s.e.m. P values, Wald test ( d , f ), two-tailed Fisher’s Exact Test ( i ), one-sided Fisher’s Exact test ( j ), two-tailed t-distribution with n-2 degrees of freedom ( g ). n = 3 biological replicates ( a - c , e ). The depicted genome-wide data represent an integration of three biological replicates.

Article Snippet: The antibodies used in this study are summarized below: rabbit monoclonal anti-H2AK119ub antibody (Cell Signaling Technology, 8240S, 1:1,000 for western blot, 1:50 for CUT&Tag); rabbit monoclonal anti-H3 antibody (Cell Signaling Technology, 4499S, 1:1,000); mouse monoclonal anti-TET2 antibody (MilliporeSigma, MABE462, 1:500); rabbit monoclonal anti-GAPDH antibody, HRP conjugate (Cell Signaling Technology, 8884S, 1:1,000); rabbit monoclonal anti-DDDDK tag antibody (Abcam, ab205606, 1:1,000 for western blot, 1:50 for immunoprecipitation); rabbit polyclonal anti-SNRP70/U1-70K antibody (Abcam, ab83306, 1:1,000); mouse monoclonal anti-5-methylcytosine antibody (Diagenode, C15200081-100, 1:1,000 for dot blot, 1:50 for meRIP); mouse monoclonal anti-hm 5 C antibody (Diagenode, C15200200-100, clone Mab-31HMC, 1:1,000 for dot blot); rabbit monoclonal anti-H3K27me3 antibody (Cell Signaling Technology, 9733S, only for CUT&Tag experiments, 1:50); mouse monoclonal anti-BAP1 antibody (Santa Cruz, sc-28383, 1:50 for CUT&Tag).

Techniques: Fractionation, Control, Comparison, Gene Expression, Cell Differentiation, Two Tailed Test, Genome Wide

Journal: Nature Communications

Article Title: TRIM33 switches off Ifnb1 gene transcription during the late phase of macrophage activation

doi: 10.1038/ncomms9900

Figure Lengend Snippet: ( a ) ChIP-qPCR analysis of c-jun or p65 binding at ICE in WT and Trim33 −/− BMDM treated for the indicated times with LPS. Mean±s.e.m., n =2 to 4. ( b ) ChIP-qPCR analysis of RNA Pol II (upper left panel) and H3K4me3 (lower left panel) at ICE in WT and Trim33 −/− BMDM treated for the indicated times with LPS. Mean±s.e.m., n =3 to 5. (Right panel) UCSC genome browser images showing H3K4me3 ChIP-seq profiles at ICE in WT and Trim33 −/− BMDM treated for the indicated times with LPS. ( c ) ChIP-qPCR analysis of acetylated histone H3 (left panel) and CBP/p300 (right panel) at ICE in WT and Trim33 −/− BMDM treated for the indicated times with LPS. Mean±s.e.m., n =4. ** P <0.01, Mann–Whitney test. ( d ) DNA looping at the Ifnb1 locus was determined by 3C-seq performed before or 24 h after LPS activation of WT and Trim33 −/− BMDM using either ICE (upper panel) or Ifnb1 gene (lower panel) as viewpoints (shown by an eye and a yellow band). Data represent normalized reads per million (r.p.m.) per restriction fragment. The x axis shows the genomic coordinates of the Ifnb1 locus. The positions of the EcoRI restriction sites and the TRIM33 ChIP-seq profile in BMDM are indicated on the top.

Article Snippet: The following antibodies were used in western blot and ChIP: TRIM33 (3E9, Euromedex for WB (1:1,000); A301-059A, Bethyl for ChIP (1:100)), c-JUN (sc-1694X, Santa Cruz (1:200)), p65 (sc-372X, Santa Cruz (1:200)), RNA Pol II (sc-899X, Santa Cruz (1:200)), H3K4me3 (07-473, Millipore (1:500)), PU.1 (sc-352X, Santa Cruz (1:10,000 for western blot; 1:500 for ChIP)), p300 (sc-585X, Santa Cruz), AcH3 (06-599, Millipore (1:500)), H3K4me1 (ab-8895, Abcam (1:500)), Flag (M2 monoclonal, Sigma Aldrich (1:1,000)), CBP (A-22, sc-369, Santa Cruz (1:1,000)), β-actin (A5441, Sigma Aldrich (1:10,000)).

Techniques: ChIP-qPCR, Binding Assay, ChIP-sequencing, MANN-WHITNEY, 3C-Seq, Activation Assay

Journal: Nature Communications

Article Title: TRIM33 switches off Ifnb1 gene transcription during the late phase of macrophage activation

doi: 10.1038/ncomms9900

Figure Lengend Snippet: ( a ) ChIP-qPCR of p65 (left panel) and c-jun (right panel) at the Ifnb1 promoter in WT and Trim33 −/− BMDM treated with LPS. Mean±s.e.m., n =2 to 4. ( b , top) ChIP-qPCR of RNA Pol II at the Ifnb1 promoter (left panel) and at a region located +2.5 kb from TSS (middle panel) in WT and Trim33 −/− BMDM treated with LPS. Mean±s.e.m., n =3 to 5. (Bottom) ChIP-qPCR (left panel) and ChIP-seq (right panel) of H3K4me3 at the Ifnb1 promoter in WT and Trim33 −/− BMDM treated with LPS. Mean±s.e.m., n =3. ( c ) ChIP-qPCR of H3ac at the Ifnb1 promoter in WT and Trim33 −/− BMDM treated with LPS. Mean±s.e.m., n =4. ( d ) ChIP-qPCR of H3ac at the Ifnb1 promoter in Trim33 −/− iM expressing GFP (iM Trim33 −/− +GFP), full-length flag-TRIM33 and GFP (iM Trim33 −/− +flag-TRIM33), or flag-TRIM33 lacking the coiled-coil domain and GFP (iM Trim33 −/− +flag-ΔCC), and treated for 0 or 7 h with LPS. Mean±s.e.m., n =3. ( e , left) Relative expression of Ifnb1 mRNA in LPS-activated WT BMDM in presence or absence of Tricostatin A (TSA), added 4 h after LPS stimulation. Mean±s.e.m., n =3. (Right) ChIP-qPCR of CBP/p300 binding at the Ifnb1 promoter in WT and Trim33 −/− BMDM treated with LPS. Mean±s.e.m., n =4. ( f ) Western blot of CBP in WT and Trim33 −/− BMDM treated with LPS. β-actin is shown as control. n =4 independent experiments. ( g ) Kinetics of Ifnb1 mRNA levels in LPS-activated WT and Trim33 −/− BMDM, in the presence (dotted lines) or absence (continuous lines) of C646. C646 was added 2 h after LPS stimulation and Ifnb1 mRNA levels were determined. Mean±s.e.m., n =8. ( h ) ChIP-qPCR of H3ac at the Ifnb1 promoter in LPS-activated WT and Trim33 −/− BMDM, in the presence (hatched columns) or absence (solid columns) of C646. Data are presented relative to H3ac binding in untreated WT BMDM. Mean±s.e.m., n =2. ( i ) Schematic representation depicting the role of TRIM33 in the macrophage-restricted Ifnb1 gene transcription shut down. * P <0.05 and ** P <0.01, Mann–Whitney test.

Article Snippet: The following antibodies were used in western blot and ChIP: TRIM33 (3E9, Euromedex for WB (1:1,000); A301-059A, Bethyl for ChIP (1:100)), c-JUN (sc-1694X, Santa Cruz (1:200)), p65 (sc-372X, Santa Cruz (1:200)), RNA Pol II (sc-899X, Santa Cruz (1:200)), H3K4me3 (07-473, Millipore (1:500)), PU.1 (sc-352X, Santa Cruz (1:10,000 for western blot; 1:500 for ChIP)), p300 (sc-585X, Santa Cruz), AcH3 (06-599, Millipore (1:500)), H3K4me1 (ab-8895, Abcam (1:500)), Flag (M2 monoclonal, Sigma Aldrich (1:1,000)), CBP (A-22, sc-369, Santa Cruz (1:1,000)), β-actin (A5441, Sigma Aldrich (1:10,000)).

Techniques: ChIP-qPCR, ChIP-sequencing, Expressing, Binding Assay, Western Blot, Control, MANN-WHITNEY