Journal: Oncogene

Article Title: H19 lncRNA identified as a master regulator of genes that drive uterine leiomyomas

doi: 10.1038/s41388-019-0808-4

Figure Lengend Snippet: TET3 affects DNA methylation and histone modifications of the MED12, TGFBR2, and TSP1 promoters. a UtLM cells were transfected with siCon or siTET3 for 48 h, followed by ChIP-qPCR analysis. Data are presented as mean relative TET3 enrichment over input. n = 3. Red numbers indicate nucleotide positions relative to the transcriptional start sites, with PCR products depicted as red-stripped bars. b Sequences of critical transcription regulatory regions (CTRR) of MED12 , TGFBR2 , and TSP1 . The differentially methylated cytosine residues are marked in red. The red numbers mark the positions of the indicated nucleotides relative to the transcriptional start sites. c UtLM cells were transfected with siCon or siTET3 for 48 h, followed by QMSP analysis. n = 3. d UtLM cells were transfected with siCon or siTET3 for 48 h, followed by ChIP-qPCR analysis. Data are presented as mean relative enrichment over input. n = 3. All data are representative of at least two independent experiments and are presented as mean ± SEM. * p < 0.05, ** p < 0.01

Article Snippet: Antibodies for TET3 (GeneTex, GTX121453; used at a dilution of 1/500), TGFBR2 (Abcam, ab184948; used at a dilution of 1/1000), TSP1 (Abcam, ab85762; used at a dilution of 1/500), MED12 (Novus Biological, NB100–2357; used at a dilution of 1/500), HMGA2 (Proteintech, 20795–1-AP; used at a dilution of 1/500), GRAF1 (Cell Signaling, 8802; used at a dilution of 1/500), SPARC (Cell Signaling, 8725; used at a dilution of 1/500), COL3A1 (LS-Bio, LS-C159386; used at a dilution of 1/1000), COL4A1 (LS-Bio, LS-C100552; used at a dilution of 1/500), COL5A2 (Origene, TA809611; used at a dilution of 1/500), and GAPDH (Abcam, ab128915; used at a dilution of 1/10000) were purchased.

Techniques: DNA Methylation Assay, Transfection, ChIP-qPCR, Methylation

Journal: Oncogene

Article Title: H19 lncRNA identified as a master regulator of genes that drive uterine leiomyomas

doi: 10.1038/s41388-019-0808-4

Figure Lengend Snippet: H19 and TET3 co-express with fibroid-promoting genes in vivo. a , c RT-qPCR analyses were performed on RNAs extracted from human fibroids and matched myometrium tissues. Spearman’s correlation showed positive correlations between expression of H19 and TET3 ( a , left panel), as well as TET3 and its target genes MED12 , TGFBR2 , and TSP1 ( c ) in a statistically significant manner. No correlation between expression of H19 and HMGA2 at the RNA level was detected ( a , right panel). Spearman’s correlation coefficient, p -values, and sample numbers are presented. b Results of western blotting analysis of HMGA2 in human fibroids and matched myometrium. n = 3. Data are representative of two independent experiments and are presented as mean ± SEM

Article Snippet: Antibodies for TET3 (GeneTex, GTX121453; used at a dilution of 1/500), TGFBR2 (Abcam, ab184948; used at a dilution of 1/1000), TSP1 (Abcam, ab85762; used at a dilution of 1/500), MED12 (Novus Biological, NB100–2357; used at a dilution of 1/500), HMGA2 (Proteintech, 20795–1-AP; used at a dilution of 1/500), GRAF1 (Cell Signaling, 8802; used at a dilution of 1/500), SPARC (Cell Signaling, 8725; used at a dilution of 1/500), COL3A1 (LS-Bio, LS-C159386; used at a dilution of 1/1000), COL4A1 (LS-Bio, LS-C100552; used at a dilution of 1/500), COL5A2 (Origene, TA809611; used at a dilution of 1/500), and GAPDH (Abcam, ab128915; used at a dilution of 1/10000) were purchased.

Techniques: In Vivo, Quantitative RT-PCR, Expressing, Western Blot

Journal: Epigenetics & Chromatin

Article Title: Epigenetic regulation of MED12: a key contributor to the leukemic chromatin landscape and transcriptional dysregulation

doi: 10.1186/s13072-025-00610-9

Figure Lengend Snippet: MED12 expression is subtype-specific in AML and independent of somatic mutation burden. ( A ) Violin plot showing MED12 expression (log₂ TPM) across adult AML subgroups from the BEAT AML cohort. Significant upregulation is observed in PML–RARα ( p = 6.0 × 10⁻⁵), CN–CEBPA^mut ( p = 2.6 × 10⁻³), CN–NPM1^mut ( p = 1.3 × 10⁻³), and CN ( p = 9.8 × 10⁻³) cases compared to normal bone marrow mononuclear cells (BM–MNC), based on Kruskal–Wallis one-way ANOVA with Dunn’s post-hoc test. No significant difference was found in KMT2A–MLLT3 ( p > 0.99), CBFB–MYH11 ( p = 0.094), KMT2A-rearranged ( p = 0.014), or RUNX1–RUNXT1 ( p = 0.044) after correction. ( B ) Violin plot showing MED12 expression across pediatric AML subgroups in the TARGET pAML cohort. Significant upregulation is observed in CBFA2T3–GLIS2 ( p < 1 × 10⁻⁶), CBFB–MYH11 ( p = 2 × 10⁻⁶), RUNX1–RUNXT1 ( p = 7 × 10⁻⁶), and CN AML ( p = 6 × 10⁻⁶) relative to BM–MNC, while KMT2A-ELL, -MLLT3, -MLLT4, and -MLLT10 subtypes do not show statistically elevated expression (all p > 0.48). ( C ) Immunoblot analysis of MED12 protein in AML cell lines and umbilical cord blood (UCB)-derived CD34⁺ and CD41⁺ hematopoietic cells or megakaryocyte progenitors respectively. Strong MED12 expression is observed in ME–1 (CBFB–MYH11⁺), M07e (CBFA2T3–GLIS2⁺), and KG–1 A (FGFR1-fusion⁺), while minimal expression is seen in KMT2A-r lines (MOLM–13, MV4–11), HL–60, AML–193, and normal controls. β-actin serves as loading control. ( D ) Scatter plot showing the correlation between MED12 expression (log₂ TPM) and total somatic mutation count in BEAT AML. No significant correlation is observed (Spearman’s r = − 0.06), suggesting MED12 expression is not associated with overall mutational burden. ( E ) MED12 expression in the three BEAT AML cases harboring MED12 mutations, highlighted by sample ID and cytogenetic context. One KMT2A–MLLT3 + case (BA2025) shows expression above the cohort median (dashed line), while two CN cases (BA2850, BA2993) show lower-than-median expression, indicating potential context-dependent effects

Article Snippet: Membranes were blocked with 5% milk in TBST for 1 h, followed by overnight incubation at 4 °C with primary antibodies against MED12 (#14360) and β-actin (#4967) (Cell Signaling Technology) diluted in 1% milk.

Techniques: Expressing, Mutagenesis, Western Blot, Derivative Assay, Control

Journal: Epigenetics & Chromatin

Article Title: Epigenetic regulation of MED12: a key contributor to the leukemic chromatin landscape and transcriptional dysregulation

doi: 10.1186/s13072-025-00610-9

Figure Lengend Snippet: The MED12 locus exhibits AML-specific chromatin accessibility and transcription factor motif enrichment. ( A ) DNase I hypersensitivity (DHS) tracks showing chromatin accessibility across (DHS peaks) the MED12 locus in hematopoietic stem progenitor cells (HSC; ENCODE) and AML blasts (BLUEPRINT). Two AML-specific DHS regions—DHS-I and DHS-II—are highlighted, spanning the TSS proximal regulatory region (TPRR) region (E1–E2) and internal coding exons (E25–E26), respectively. ( B-C ) Box plots quantifying the span of accessible regions (in base pairs) for DHS-I and DHS-II. DHS-I is significantly broader in AML compared to HSCs ( p < 0.01, Mann–Whitney U test), while DHS-II also shows increased accessibility. ( D-F ) Motif enrichment analysis of the AML-specific DHS-I region identifies significant overrepresentation of binding motifs for ELK1 ( p = 2.25 × 10⁻¹²), HSF4 ( p = 1.03 × 10⁻¹¹), and HIC2 ( p = 4.60 × 10⁻¹⁴), based on in-silico prediction. These findings are computational and intended to provide a hypothesis-generating framework for potential regulatory interactions at the MED12 locus in AML

Article Snippet: Membranes were blocked with 5% milk in TBST for 1 h, followed by overnight incubation at 4 °C with primary antibodies against MED12 (#14360) and β-actin (#4967) (Cell Signaling Technology) diluted in 1% milk.

Techniques: MANN-WHITNEY, Binding Assay, In Silico

Journal: Epigenetics & Chromatin

Article Title: Epigenetic regulation of MED12: a key contributor to the leukemic chromatin landscape and transcriptional dysregulation

doi: 10.1186/s13072-025-00610-9

Figure Lengend Snippet: Epigenetic landscape of the MED12 regulatory locus in AML reveals active histone features and focal TPRR hypermethylation. ( A ) Integrated epigenomic view of the MED12 regulatory locus (chrX:71,118,596–71,142,450, hg38), including 5 kb upstream of the transcription start site (TSS), visualizing chromatin accessibility, histone modifications, and DNA methylation in AML blasts from bone marrow (BM) and venous blood (VB). Two DHS-enriched regions overlapping exon 1 (E1) and exon 2 (E2) are co-marked by active histone modifications H3K4me3 and H3K27ac. H3K36me3 spans the gene body, while H3K4me1 and H3K27me3 show more limited and variable deposition. ( B-C ) Bar plots showing the median enrichment score for each histone modification (H3K4me3, H3K27ac, H3K27me3, H3K36me3, H3K4me1) in BM ( B ) and VB ( C ) AML samples. Enrichment scores were derived from pre-processed BED files and correspond to peaks overlapping the MED12 gene body or upstream TPRR region. ( D ) DNA methylation profile across the MED12 regulatory locus (chrX:71,118,596–71,142,450), with CpG sites represented as hypermethylated (≥ 80% DNAm, dark orange) or hypomethylated (≤ 20% DNAm, light orange). Focal hypomethylation occurs at DHS-adjacent CpG island near the TSS, while the upstream TPRR and gene body exhibit widespread hypermethylation. ( E ) Box plots comparing the number of hyper- and hypomethylated DMRs across all BM samples. Hyper-DMRs were significantly more frequent than hypo-DMRs (64 vs. 23 DMRs; p < 0.01, Mann–Whitney U test). ( F ) Box plot comparing DNAm levels of hypermethylated DMRs in BM vs. VB. BM samples showed significantly higher DNAm at hyper-DMRs than VB samples ( p < 0.01, Mann–Whitney U test). ( G ) Box plot comparing the number of hyper- and hypomethylated DMRs in VB samples. Hyper-DMRs were more abundant than hypo-DMRs (137 vs. 65 DMRs; p < 0.01, Mann–Whitney U test). ( H ) Box plot of DNAm levels at hypomethylated DMRs in BM vs. VB. No significant difference was observed ( p = 0.24, Mann–Whitney U test), indicating conserved focal hypomethylation at DHS-associated elements

Article Snippet: Membranes were blocked with 5% milk in TBST for 1 h, followed by overnight incubation at 4 °C with primary antibodies against MED12 (#14360) and β-actin (#4967) (Cell Signaling Technology) diluted in 1% milk.

Techniques: DNA Methylation Assay, Modification, Derivative Assay, MANN-WHITNEY

Journal: Epigenetics & Chromatin

Article Title: Epigenetic regulation of MED12: a key contributor to the leukemic chromatin landscape and transcriptional dysregulation

doi: 10.1186/s13072-025-00610-9

Figure Lengend Snippet: DNA Methylation Analysis of the MED12 TPRR and Gene Body in Various Cell Lines. ( A-B ) Methylation profiling of MED12 promoter region using Illumina 850 K array in TARGET AML cohort. ( A ) CBFA2T3–GLIS2-positive patients ( n = 24) exhibit significantly higher DNA methylation at probe cg04768312 in the promoter compared to NBM controls ( n = 10) ( p = 0.0011, Mann–Whitney U test). ( B ) KMT2A-rearranged cases ( n = 4) show a trend toward lower methylation (median = 0.4501) than NBM (median = 0.5237), though not statistically significant ( p = 0.2). ( C ) Schematic of the MED12 locus with four regions (Regions 1–4) selected for pyrosequencing analysis. Regions 1–3 span the transcriptional promoter regulatory region (TPRR) and overlap a CpG island encompassing the TSS, TPRR, and exon 1. Sequencing primers specific to each region are labeled as S1–S4 in the diagram. ( D ) DNA methylation levels (% mC) across Regions 1–3 in a panel of cell lines. Cells with high MED12 expression (red) display increased methylation compared to those with low expression (blue). ( E ) Schematic of the MED12 locus with Region–4, selected for pyrosequencing analysis. Region 4 is located in the gene body and overlaps a DNase I hypersensitive site (DHS-II), and was sequenced using primer S4. ( F ) DNA methylation levels (% mC) across Region 4 in a panel of cell lines. Cells with high MED12 expression (red) display increased methylation compared to those with low expression (blue). ( G-J ) Quantitative comparison of methylation percentages between MED12 low and MED12 high expressing cell lines across the four regions. Statistical significance is indicated: ( G ) Region-1 shows significantly higher methylation in MED12 high cells ( p < 0.001). ( H ) Region-2 exhibits moderate but significant methylation differences ( p < 0.05). ( I ) Region-3 does not show a statistically significant difference ( p = 0.22, ns). ( J ) Region-4 displays significantly increased methylation in MED12 high cells ( p < 0.01)

Article Snippet: Membranes were blocked with 5% milk in TBST for 1 h, followed by overnight incubation at 4 °C with primary antibodies against MED12 (#14360) and β-actin (#4967) (Cell Signaling Technology) diluted in 1% milk.

Techniques: DNA Methylation Assay, Methylation, MANN-WHITNEY, Sequencing, Labeling, Expressing, Comparison

Journal: Epigenetics & Chromatin

Article Title: Epigenetic regulation of MED12: a key contributor to the leukemic chromatin landscape and transcriptional dysregulation

doi: 10.1186/s13072-025-00610-9

Figure Lengend Snippet: Correlation Between MED12 Expression, DNA Methylation, and DNMTs, and the Effects of 5-Azacytidine Treatment. ( A – C ) Correlation of MED12 expression with DNA methyltransferases in the BEAT AML cohort ( n = 624). DNMT1 shows moderate correlation ( r = 0.38, A ), while DNMT3A ( r = 0.68, B ) and DNMT3B ( r = 0.55, C ) exhibit moderate positive correlations with MED12 , implicating de novo methyltransferases in regulating promoter methylation. ( D ) Dose–response curves for 5-azacytidine (5-AZA) in AML cell lines. IC₅₀ values: M07e (1.5 µM), KG1A (3.5 µM), MOLM-13 (3.5 µM), and MV4-11 (4.6 µM), showing comparable sensitivity independent of baseline methylation. ( E , F ) Methylation analysis of 13 CpGs in Region-1 and 10 CpGs in Region-2 of the MED12 TPRR in M07e cells (control: red; 5-AZA: blue). Significant demethylation occurs at CpG-1 and − 13 in Region-1 ( E ), and CpG-2, -4, -7, and − 9 in Region-2 ( F ) after 5-AZA treatment ( p -values shown). ( G ) qRT-PCR analysis confirms that MED12 expression is significantly reduced in M07e cells after 5-AZA treatment ( p < 0.001), supporting a noncanonical, methylation-dependent activation mechanism. (H–I) In MOLM-13 cells, 5-AZA induces modest but significant demethylation at CpG-9 and − 13 in Region-1 ( H ) and at CpG-4 in Region-2 ( I ). (J) In contrast to M07e, MED12 expression increases following 5-AZA treatment in MOLM-13 ( p < 0.05), consistent with canonical repression being relieved by promoter demethylation

Article Snippet: Membranes were blocked with 5% milk in TBST for 1 h, followed by overnight incubation at 4 °C with primary antibodies against MED12 (#14360) and β-actin (#4967) (Cell Signaling Technology) diluted in 1% milk.

Techniques: Expressing, DNA Methylation Assay, Methylation, Control, Quantitative RT-PCR, Activation Assay

Journal: bioRxiv

Article Title: CDK8 Inhibition Releases the Muscle Differentiation Block in Fusion-driven Alveolar Rhabdomyosarcoma

doi: 10.1101/2025.07.14.663986

Figure Lengend Snippet: A. Volcano plot of ssGSEA on genome-wide differential effect size of CORUM complexes comparing aRMS to other non-RMS tumor cell lines. Red indicates Mediator complex. B. Distribution of CDK8 gene effect score across different cancer cell lines from the Broad Institute’s CRISPR Dependency Map (24Q2). C. Dot plot of kinase dependencies in the Broad Institute’s CRISPR Dependency Map comparing fusion-positive RMS to all other cancer cell lines. CDK8 is highlighted in red. D. Violin plots showing distribution of CCNC , MED13 , and MED12 gene effect score from the Broad Institute’s CRISPR Dependency Map (24Q2) comparing the fusion-positive aRMS and fusion-negative eRMS with all other indicated cancer cell lines. aRMS is highlighted in red and eRMS is highlighted in blue. E. shRNA-mediated suppression of CDK8 by two different shRNAs impairs Rh30 and Rh28 aRMS cell growth in vitro . Cell numbers were determined by trypan blue live cell counting. Data are presented as mean ± SEM (*: p <=5.0e-02, **: p <=1.0e-02, ***: p <= 1.0e-03, ****: p <=1.0e-04). F. Line graph showing mean subcutaneous tumor volume (mm3) formed by Rh28 cells after treatment with inducible knock down of CDK8 using shRNA. Data are presented as mean ± SEM (*: p <=5.0e-02, **: p <=1.0e-02, ***: p <= 1.0e-03, ****: p <=1.0e-04). G. CRISPR-mediated knockout of CDK8 by two different gRNAs impairs Rh30 and Rh4 aRMS cell growth in vitro . Relative growth was assessed by CellTiter-Glo after CRISPR knockout. Data are presented as mean ± SEM (*: p <=5.0e-02, **: p <=1.0e-02, ***: p <= 1.0e-03, ****: p <=1.0e-04).

Article Snippet: Primary antibodies used for CUT7RUN in this study includes: CDK8 (ProteinTech, #22067), SIX4 (Santa Cruz Biotechnology, #SC-390779), HA (Cell Signaling Technology, #C29F4-3724), TADA2B (ProteinTech, #17367), CCNC (ProteinTech, #26464), MED13 (ProteinTech, #26464), MED12 (ProteinTech, #20028), H3K4me3 (EpiCypher, #13-0041), H3K27ac (Cell Signaling Technology, #8173S).

Techniques: Genome Wide, CRISPR, shRNA, In Vitro, Cell Counting, Knockdown, Knock-Out

Journal: bioRxiv

Article Title: CDK8 Inhibition Releases the Muscle Differentiation Block in Fusion-driven Alveolar Rhabdomyosarcoma

doi: 10.1101/2025.07.14.663986

Figure Lengend Snippet: A. Box plots showing construct-level Z-score averages for individual genes in the Mediator complex from a genome-wide CRISPR-Cas9 screen in Rh30 cells treated with DMSO (gray/black) or BI-1347 (blue/red) for 14 days (gray and blue) or 21 days (black and red). Genes are grouped by Mediator functional modules. B. Live cell proliferation assessed by Incucyte for BI-1347+/-sgCDK8 (red) and BI-1347+/-sgCCNC (blue). C. MA plot showing changes of CDK8 binding site assessed by CUT&RUN after 24 hrs of BI-1347 treatment. Significantly increased CDK8 peaks are highlighted in red; significantly decreased CDK8 peaks are highlighted in blue (padj<0.05, fold change>1.5 or <-1.5). D. Motif analysis of the regions with increased CDK8 DNA binding peaks from CUT&RUN analysis in Rh30 cells. E. Heatmaps showing chromatin occupancy of CDK8, CCNC, MED12, and MED13 at regions with upregulated SIX4 binding at 24 hrs of DMSO or BI-1347 treatment. F. IGV gene tracks showing the PRO-seq, CDK8, CCNC, MED12, and MED13 binding at the RUNX1 gene body and enhancer loci at indicated time points after BI-1347 treatment. G. Heatmaps of CDK8, CCNC, MED12, and MED13 CUT&RUN signal around PAX3::FOXO1-regulated enhancers before and after 24 hrs of BI-1347 treatment. H. IGV gene tracks showing the binding of CDK8, CCNC, MED12, and MED13 at a RUNX2 super enhancer cluster at indicated time points after BI-1347 treatment.

Article Snippet: Primary antibodies used for CUT7RUN in this study includes: CDK8 (ProteinTech, #22067), SIX4 (Santa Cruz Biotechnology, #SC-390779), HA (Cell Signaling Technology, #C29F4-3724), TADA2B (ProteinTech, #17367), CCNC (ProteinTech, #26464), MED13 (ProteinTech, #26464), MED12 (ProteinTech, #20028), H3K4me3 (EpiCypher, #13-0041), H3K27ac (Cell Signaling Technology, #8173S).

Techniques: Construct, Genome Wide, CRISPR, Functional Assay, Binding Assay