Journal: American Journal of Cancer Research

Article Title: Genome-wide target interactome profiling reveals a novel EEF1A1 epigenetic pathway for oncogenic lncRNA MALAT1 in breast cancer

doi:

Figure Lengend Snippet: Interactome of MALAT1 lncRNA by RAT-seq. A. RAT-seq assay. MALAT1 lncRNA was in situ reverse transcribed using MALAT1-specific complementary primers at 60°C with biotin-dCTP. The biotin-MALAT1 cDNA chromatin complex was isolated by streptavidin beads and cDNAs were isolated for Illumina library sequencing. RAT-seq will generate a genome-wide target interaction network for MALAT1 lncRNA in breast cancer cells. B. Gene ontology enrichment pathway analysis of the MALAT1 RAT-Seq data. GO enrichment was analyzed with Cytoscape software. C. The MALAT1 RAT-seq interactome. The MALAT1 interactome was drawn based on the enrichment fold of the top RAT-Seq pathway target genes.

Article Snippet: The biotinylated- MALAT1 -cDNA/chromatin DNA complex was pulled down with biotin-streptavidin magic beads (Invitrogen, CA).

Techniques: In Situ, Reverse Transcription, Isolation, Sequencing, Genome Wide, Software

Journal: American Journal of Cancer Research

Article Title: Genome-wide target interactome profiling reveals a novel EEF1A1 epigenetic pathway for oncogenic lncRNA MALAT1 in breast cancer

doi:

Figure Lengend Snippet: MALAT1 binds to the EEF1A1 promoter and epigenetically regulates its activity. A. The RAT-seq IGV binding of MALAT1 lncRNA at the EEF1A1 locus. MALAT1-RAT: the RAT-seq library created by the MALAT1-specific complementary primers; RC-RAT: the RAT-seq control library created by random oligonucleotide primers; pEEF1A1: EEF1A1 promoter; 3’-CT, 5’-CT: the 3’- and 5’-control sites; E1-E8: EEF1A1 exons. B. Quantitation of EEF1A1 binding in the MALAT1-specific RAT-seq products and the negative control RAT-seq products. C. EEF1A1 expression levels by Q-PCR in MALAT1-knockdown cells. β-Actin was used as an internal control. **P < 0.01 as compared with the control groups. D. Western blot of eEF1A1. Note the reduced expression of eEF1A1 in MALAT1-knockdown breast cancer cells. GAPDH was used as control.

Article Snippet: The biotinylated- MALAT1 -cDNA/chromatin DNA complex was pulled down with biotin-streptavidin magic beads (Invitrogen, CA).

Techniques: Activity Assay, Binding Assay, Control, Quantitation Assay, Negative Control, Expressing, Knockdown, Western Blot

Journal: American Journal of Cancer Research

Article Title: Genome-wide target interactome profiling reveals a novel EEF1A1 epigenetic pathway for oncogenic lncRNA MALAT1 in breast cancer

doi:

Figure Lengend Snippet: MALAT1 epigenetically regulates EF1A1. A. pEEF1A1-luciferase assay. The EEF1A1 promoter (pEEF1A1) sequence was cloned into the upstream of luciferase gene. Luciferase reporter assay was performed in CTL group, shNC group and shMALAT1 group by co-transfecting respectively with pGL3-Basic vector or luciferase reporter vector. Data were adjusted over the negative control (CTL) and were represented as means ± SD. **P < 0.01 as compared with the control groups. B. Quantitation of Histone 3-K4 (H3K4) trimethylation. All data are presented as the relative values after normalization over the input DNA. *P < 0.05 as compared with control.

Article Snippet: The biotinylated- MALAT1 -cDNA/chromatin DNA complex was pulled down with biotin-streptavidin magic beads (Invitrogen, CA).

Techniques: Luciferase, Sequencing, Clone Assay, Reporter Assay, Plasmid Preparation, Negative Control, Control, Quantitation Assay

Journal: American Journal of Cancer Research

Article Title: Genome-wide target interactome profiling reveals a novel EEF1A1 epigenetic pathway for oncogenic lncRNA MALAT1 in breast cancer

doi:

Figure Lengend Snippet: MALAT1 is dysregulated in breast cancer. A. Unsupervised hierarchical clustering analysis of the significantly differentially expressed genes in paracancer tissues and tumor tissues. Data from 64 mammary tissues was downloaded from TCGA database. In the heatmap, the normalized expression values are represented in shades of green and red, indicating the expression being above and below the median expression value across the samples. B. The normalized expression level of MALAT1 in 64 mammary tissues (32 paracancer tissues and 32 tumor tissues). ***P < 0.01 as compared with the paracancer group. C. Q-PCR quantitation of MALAT1 expression in breast cancer cell lines.

Article Snippet: The biotinylated- MALAT1 -cDNA/chromatin DNA complex was pulled down with biotin-streptavidin magic beads (Invitrogen, CA).

Techniques: Expressing, Quantitation Assay

Journal: American Journal of Cancer Research

Article Title: Genome-wide target interactome profiling reveals a novel EEF1A1 epigenetic pathway for oncogenic lncRNA MALAT1 in breast cancer

doi:

Figure Lengend Snippet: The role of MALAT1 in cell proliferation and cell cycle. A. MALAT1 shRNA knockdown in two breast cancer cell lines. MALAT1 expression was examined by Q-PCR in CTL (non-transfected control), shNC (random shRNA non-targeting control), shMALAT1 (MALAT1 shRNA-1 transfected cells), and shMALAT1-2 (MALAT1 shRNA-2 transfected cells). β-Actin was used as an internal control. **P < 0.01 as compared with CTL and shNC controls. B. Cell Proliferation. CCK-8 assay was used to determine cell growth viability at 0, 24, 48, 72 and 96 hour time points. C. Cell cycle. Flow cytometry was used to measure cell cycle profile with propidium iodide staining. Cell numbers were counted according to DNA content of G0/G1, S and G2/M phases. The statistical results are shown on the right panel. *P < 0.05 as compared with the control groups.

Article Snippet: The biotinylated- MALAT1 -cDNA/chromatin DNA complex was pulled down with biotin-streptavidin magic beads (Invitrogen, CA).

Techniques: shRNA, Knockdown, Expressing, Transfection, Control, CCK-8 Assay, Flow Cytometry, Staining

Journal: American Journal of Cancer Research

Article Title: Genome-wide target interactome profiling reveals a novel EEF1A1 epigenetic pathway for oncogenic lncRNA MALAT1 in breast cancer

doi:

Figure Lengend Snippet: MALAT1 knockdown inhibits cell invasion in breast cancer cells. Representative images of invading MDA-MB231 cells (A) and SKBR3 cells (B) are showed on the left panel. shNC: random shRNA control; shMALAT1: Cells that were transfected with MALAT1 shRNA-1. Quantitation of invaded cells is shown in the right panel, mean ± SD, **P < 0.01 as compared with the control groups.

Article Snippet: The biotinylated- MALAT1 -cDNA/chromatin DNA complex was pulled down with biotin-streptavidin magic beads (Invitrogen, CA).

Techniques: Knockdown, shRNA, Control, Transfection, Quantitation Assay

Journal: American Journal of Cancer Research

Article Title: Genome-wide target interactome profiling reveals a novel EEF1A1 epigenetic pathway for oncogenic lncRNA MALAT1 in breast cancer

doi:

Figure Lengend Snippet: EEF1A1 rescues the effect induced by MALAT1 knockdown. A. Overexpression of EEF1A1 in breast cancer cells. The expression of EEF1A1 was quantitated by Q-PCR. shNC: random shRNA control; shMALAT1: Cells that were transfected with MALAT1 shRNA-1. β-Actin was used as an internal control. ***P < 0.001 as compared with the control groups. Overexpression of eEF1A1 in breast cancer cells was measured by Western blot. B. Cell growth viability as measured by CCK-8 assay. C. Cell invasion as examined by Transwell assay. Quantitation of invaded cells was shown as mean ± SD, **P < 0.01 as compared with the control groups.

Article Snippet: The biotinylated- MALAT1 -cDNA/chromatin DNA complex was pulled down with biotin-streptavidin magic beads (Invitrogen, CA).

Techniques: Knockdown, Over Expression, Expressing, shRNA, Control, Transfection, Western Blot, CCK-8 Assay, Transwell Assay, Quantitation Assay

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: The EMBO Journal

Article Title: TET2 and TET3 regulate GlcNAcylation and H3K4 methylation through OGT and SET1/COMPASS

doi: 10.1038/emboj.2012.357

Figure Lengend Snippet: TET2 and TET3 associate with the O -GlcNAc transferase OGT and promote GlcNAcylation. ( A ) Silver stain gel of HaloTag-TET protein complex isolations and HaloTag alone control (Ctrl). Protein pulldowns were performed from HEK293T cells overexpressing the indicated HT constructs (see Materials and methods and for details). As not all of the indicated complex isolations were performed at the same time, two separate silver stain gels were run, as shown in this panel. ( B ) Table of transcriptional or chromatin protein interactors found in the various HaloTag-TET isolations. Spectral counts for each interacting protein are shown for biological replicates. TET1, but not TET2, as previously reported ( ; ), shows interaction with SIN3A. OGT interacts with all TET proteins, though it is most highly abundant with TET2 and TET3. ( C ) Detection of OGT by western blotting from HT-TET2 and HT-TET3 pulldowns from ( A ). The indicated pulldowns were probed with an anti-OGT antibody to detect the presence of OGT. OGT and beta-Actin shown as input loading controls. ( D ) TET2 and TET3 co-immunoprecipitate (CoIP) with endogenous OGT from untransfected HEK293T cells. Cell extracts were immunoprecipitated with anti-OGT or rabbit IgG and probed with antibodies against the indicated proteins. An IP control of OGT alone is shown to demonstrate specific capture and enrichment of OGT. Inputs loading controls are shown for all. Note that in this experiment very weak expression of TET2 relative to TET3 is observed. ( E ) The global level of hmC does not change after cell treatment with Alloxan or PUGNAc. Dot blot quantification of global hmC after the indicated treatments. The hmC content is normalized with respect to the input DNA and to mock-treated cells, where the ratio is set at 1.00. Error bars indicate s.d. of three independent experiments. As controls, western blots using anti- O -GlcNAc antibody show the expected decrease in GlcNAcylation with Alloxan and increase with PUGNAc. HDAC1 input loading controls are also shown. Vertical line indicates juxtaposition of lanes non-adjacent within the same blot, exposed for the same time. ( F ) Global decrease in GlcNAcylation is observed in TET2/3 knockdowns. Left: TET2 kd or TET3 kd show decreased GlcNAc activity. Nuclear extracts were prepared from HEK293T cells expressing RNAi Ctrl, RNAi TET2, or RNAi TET3, and UDP-[ 3 H]GlcNAc incorporation was measured. The amount incorporated into the control cells was set at 1. Error bars indicate s.d. of three independent experiments (* P <0.05). Right: Nuclear extracts were prepared from HEK293T cells expressing RNAi Ctrl or RNAi TET2/3 and global GlcNAcylation was visualized with an antibody against O -GlcNAc. HDAC1 input loading control is also shown.

Article Snippet: 2 μg of mouse monoclonal antibody for H3K4me3 (ab1012; Abcam), 6 μg of mouse monoclonal antibody for O -linked N-acetylglucosamine (ab2739; Abcam), 3 μg of rabbit polyclonal antibody for Tet2 (sc-136926; Santa Cruz), 5 μg of rabbit polyclonal for HCF1 (A301-399A-1; Bethyl Lab), or the respective amount of control antibody was incubated with chromatin overnight at 4°C.

Techniques: Silver Staining, Control, Construct, Western Blot, Immunoprecipitation, Expressing, Dot Blot, Activity Assay

Journal: The EMBO Journal

Article Title: TET2 and TET3 regulate GlcNAcylation and H3K4 methylation through OGT and SET1/COMPASS

doi: 10.1038/emboj.2012.357

Figure Lengend Snippet: TET2/3–OGT show genomic co-localization around TSSs and impact on H3K4me3 and transcriptional activation. ( A ) Left: Venn diagrams indicating significant overlap of TET2 and OGT bound regions (left part; P -value<10 −10 ) identified after HaloCHIP-Seq in HEK293T cells expressing HT-TET2, or HT-OGT. Right: TET2–OGT targets are primarily found at TSSs and CpG-rich sequences. Similar profiles were also observed for TET3–OGT . ( B ) An analysed subset of TET2–TET3–OGT targets show a lack of DNA methylation and hydroxymethylation, yet display GlcNAcylation. qPCR analysis of TET2–TET3–OGT binding and non-binding regions after MeDIP (top), hMeDIP (middle), or ChIP with an anti- O -GlcNAc antibody (bottom). ‘% Input' represents real-time qPCR values normalized with respect to the input chromatin. Known methylated and hydroxymethylated regions are shown as positive controls in MeDIP and hMeDIP panels.

Article Snippet: 2 μg of mouse monoclonal antibody for H3K4me3 (ab1012; Abcam), 6 μg of mouse monoclonal antibody for O -linked N-acetylglucosamine (ab2739; Abcam), 3 μg of rabbit polyclonal antibody for Tet2 (sc-136926; Santa Cruz), 5 μg of rabbit polyclonal for HCF1 (A301-399A-1; Bethyl Lab), or the respective amount of control antibody was incubated with chromatin overnight at 4°C.

Techniques: Activation Assay, Expressing, DNA Methylation Assay, Binding Assay, Methylated DNA Immunoprecipitation, Methylation

Journal: The EMBO Journal

Article Title: TET2 and TET3 regulate GlcNAcylation and H3K4 methylation through OGT and SET1/COMPASS

doi: 10.1038/emboj.2012.357

Figure Lengend Snippet: ( C ) TET2/3–OGT targets in HEK293T cells are enriched for H3K4me3 as depicted in a Venn diagram; P -value<10 −10 . ( D ) Examples of HaloCHIP-Seq OGT, TET2, TET3, and ChIP-Seq H3K4me3 profiles (UCSC tracks). ( E ) Decreased levels of H3K4me3 in TET2 kd cells. Upper-left: decrease in the normalized number of H3K4me3 reads in TET2/3–OGT-binding regions in TET2 kd cells versus control RNAi-treated cells. Upper-right: pie chart showing the percentage of TET2–TET3–OGT binding regions with a statistically significant reduction of the normalized number of H3K4me3 reads for TET2 kd versus control RNAi-treated cells. Lower-part: examples of H3K4me3 ChIP-Seq profiles (UCSC tracks) in TET2–TET3–OGT-binding regions for the RNAi control versus TET2 kd sample. ( F ) Western blot showing global decrease in H3K4me3 in a TET2/3 double kd cells. Lysates from mock HEK293T RNAi kd or TET2/3 kd cells were probed for H3K4me3 using an anti-H3K4 antibody in western blot. Tubulin is shown as a loading control. ( G ) OGT activity is important for H3K4me3. Cell extracts were prepared from HEK293T cells treated with or without the OGT inhibitor Alloxan, and then western blots for H3K4me3 were performed. HDAC1 and H3 are shown as loading controls and a western blot against O -GlcNAc was used to monitor specific GlcNAcylation inhibition by Alloxan. Vertical lines indicate juxtaposition of lanes non-adjacent within the same blot, exposed for the same time. ( H ) Decreases in transcription are observed in both TET2/3 knockdowns and an OGT knockdown. The indicated target genes (which showed decrease in H3K4me3 after TET2 kd; cf. E ) and negative controls (unbound TET2/3–OGT–H3K4me3 targets), were analysed by RT–qPCR in HEK293T cells subjected to the various listed RNAi treatments. Independent experiments were performed in duplicates.

Article Snippet: 2 μg of mouse monoclonal antibody for H3K4me3 (ab1012; Abcam), 6 μg of mouse monoclonal antibody for O -linked N-acetylglucosamine (ab2739; Abcam), 3 μg of rabbit polyclonal antibody for Tet2 (sc-136926; Santa Cruz), 5 μg of rabbit polyclonal for HCF1 (A301-399A-1; Bethyl Lab), or the respective amount of control antibody was incubated with chromatin overnight at 4°C.

Techniques: ChIP-sequencing, Binding Assay, Control, Western Blot, Activity Assay, Inhibition, Knockdown, Quantitative RT-PCR

Journal: The EMBO Journal

Article Title: TET2 and TET3 regulate GlcNAcylation and H3K4 methylation through OGT and SET1/COMPASS

doi: 10.1038/emboj.2012.357

Figure Lengend Snippet: TET2/3 promotes GlcNAcylation of HCF1, and both TET and OGT activity favour the integrity of SET1/COMPASS and SETD1A binding to chromatin. ( A ) Mass spectrometry reveals HCF1, a known target of OGT and component of SET1/COMPASS , as an interacting partner of HT-TET2 and HT-TET3. Biological duplicates and respective spectral counts (SpC) for HCF1 are shown. ( B ) Protein pulldowns of HT-OGT coupled with mass spectrometry identify HCF1, TET2, TET3, and all components of SET1/COMPASS as partners of OGT. Biological duplicates and SpC for each protein identified are shown for HT-OGT and Ctrl isolations. ( C ) The interaction of HCF1 and SET1/COMPASS components with HT-OGT depends on O -GlcNAc activity. Plot showing average SpCs for HCF1 and SET1/COMPASS components isolated from HT-OGT pulldowns of untreated (grey bars) and Alloxan-treated (green bars) HEK293T cells. Error bars represent s.d. of biological duplicates. Representative NSAF plots are shown in . ( D ) The interaction of HT-SETD1A with SET1/COMPASS components and OGT is reduced by a TET2/3 double kd. Plot showing average SpCs for SET1/COMPASS components and OGT isolated from HT-SETD1A pulldowns of control RNAi-treated (grey bars) and TET2/3 kd (blue bars) HEK293T cells. Error bars represent s.d. of biological duplicates. Representative NSAF plots are shown in . ( E ) A significant reduction in HCF1 GlcNAcylation is observed after TET2/3 double kd. Upper diagram shows a schematic representation of full-length HCF1 and its domains . The GlcNAcylated peptides identified by mass spectrometry from HT-SETD1A isolations from control RNAi-treated and TET2/3 kd cells are indicated below. The full-length HCF1 amino-acid sequence (NP_005325.2) shows the corresponding GlcNAcylated peptides highlighted in yellow with RNAi Ctrl on the left and RNAi TET2/3 kd on the right. ( F ) Bioluminescence resonance energy transfer (BRET) assays show reduction of SETD1A binding to histone H3.3 in the presence of an OGT inhibitor and in TET2/3 kd cells. Upper diagram showing the schematic of BRET energy transfer upon binding of a NanoLuc-SETD1A fusion donor and fluorescently labelled Histone H3.3-HaloTag fusion acceptor in live HEK293T cells (see Materials and methods for experimental details and calculation of BRET). Left: BRET measurements were calculated without treatment (grey) or with Alloxan treatment (green). Right: BRET measurement for RNAi control (grey) or RNAi TET2/3 (blue). Biological triplicates ±s.d. are shown.

Article Snippet: 2 μg of mouse monoclonal antibody for H3K4me3 (ab1012; Abcam), 6 μg of mouse monoclonal antibody for O -linked N-acetylglucosamine (ab2739; Abcam), 3 μg of rabbit polyclonal antibody for Tet2 (sc-136926; Santa Cruz), 5 μg of rabbit polyclonal for HCF1 (A301-399A-1; Bethyl Lab), or the respective amount of control antibody was incubated with chromatin overnight at 4°C.

Techniques: Activity Assay, Binding Assay, Mass Spectrometry, Isolation, Control, Sequencing, Bioluminescence Resonance Energy Transfer

Journal: The EMBO Journal

Article Title: TET2 and TET3 regulate GlcNAcylation and H3K4 methylation through OGT and SET1/COMPASS

doi: 10.1038/emboj.2012.357

Figure Lengend Snippet: Tet2 knockout mouse tissue shows that Tet2 is needed for global GlcNAcylation and H3K4me3 at target promoters. ( A ) Genome-wide co-localization of endogenous Tet2 with O -GlcNAc and H3K4me3 at promoters and CpG-rich regions. Venn diagrams are shown ( P -value overlap<10 −10 ) as well as the indicated genome-wide distribution. ( B ) Tet2, O -GlcNAc, and H3K4me3 are enriched at many active genes, mirroring the presence of RNA Pol II. Upper panel: Venn diagram showing the overlap of Tet2 and O -GlcNAc with RNA Pol II ( P -value overlap <10 −10 ); lower panel: Box plots showing the reads density at targets and non-targets (others) for ChIP-Seq RNA Pol II or RNA-Seq in mouse bone marrow. ( C ) Global decrease in GlcNAcylation is observed in Tet2 knockout mouse bone marrow. Mouse bone marrow tissues with or without a Tet2 knockout were analysed by western blot for O -GlcNAc levels using an anti- O -GlcNAc antibody. HDAC1 is shown as loading control. ( D ) ChIP-Seq for H3K4me3 in Tet2 knockout mouse tissues shows reduced global H3K4me3 at target promoters. Overall impact on H3K4me3 peak significance (−log( P -value) of the peaks) between wild-type and Tet2 knockout bone marrow is shown. ( E ) Table showing key haematopoietic genes with specifically reduced H3K4me3 in Tet2 knockout as compared to wild type. The location at CpG islands and the promoter class for each is listed. Lower part: example of H3K4me3 ChIP-Seq profiles (UCSC tracks) in wild type versus Tet2 knockout.

Article Snippet: 2 μg of mouse monoclonal antibody for H3K4me3 (ab1012; Abcam), 6 μg of mouse monoclonal antibody for O -linked N-acetylglucosamine (ab2739; Abcam), 3 μg of rabbit polyclonal antibody for Tet2 (sc-136926; Santa Cruz), 5 μg of rabbit polyclonal for HCF1 (A301-399A-1; Bethyl Lab), or the respective amount of control antibody was incubated with chromatin overnight at 4°C.

Techniques: Knock-Out, Genome Wide, ChIP-sequencing, RNA Sequencing, Western Blot, Control

Journal: The EMBO Journal

Article Title: TET2 and TET3 regulate GlcNAcylation and H3K4 methylation through OGT and SET1/COMPASS

doi: 10.1038/emboj.2012.357

Figure Lengend Snippet: Model connecting DNA modifying enzymes, TETs, a master cellular sensor protein, OGT, and a histone modifying complex, SET1/COMPASS. Based on our findings, a hierarchical model of the involved proteins, with the cascade of their respective activities, can be envisaged as followed: (1) The first sequence of events in the cascade is the formation of TET2/3–OGT interaction, which promotes OGT GlcNAcylation on numerous proteins, including HCF1; (2) In a TET-dependent manner, a GlcNAcylated HCF1 is important for the formation of the SET1/COMPASS; (3) In the last step, both TET proteins and OGT activity favour binding of SETD1A to chromatin, an event necessary for histone H3K4me3 and subsequent transcriptional activation.

Article Snippet: 2 μg of mouse monoclonal antibody for H3K4me3 (ab1012; Abcam), 6 μg of mouse monoclonal antibody for O -linked N-acetylglucosamine (ab2739; Abcam), 3 μg of rabbit polyclonal antibody for Tet2 (sc-136926; Santa Cruz), 5 μg of rabbit polyclonal for HCF1 (A301-399A-1; Bethyl Lab), or the respective amount of control antibody was incubated with chromatin overnight at 4°C.

Techniques: Sequencing, Activity Assay, Binding Assay, Activation Assay