Journal: iScience

Article Title: MyoD is essential in rhabdomyosarcoma by promoting survival through differentiation and CYLD

doi: 10.1016/j.isci.2025.113149

Figure Lengend Snippet: MyoD suppresses death genes through DNA methyltransferases (A) Volcano plot from transcriptomic analysis representing downregulated (blue, n = 1358) and upregulated genes (red, n = 1116) from RH30-SR MyoD Δ cells compared to RH30-SR cells (fold change >25%, FDR <0.05). (B) GO analysis of upregulated genes in RH30-SR MyoD Δ cells compared to RH30-SR cells. (C) Volcano plot from ATAC-seq analysis representing differentially accessible chromatin—closed (blue) and open (red)—in RH30-SR MyoD Δ cells compared to RH30-SR cells (fold change >25%, FDR <0.05). (D) Cell death was measured following the treatment of RH30-SR cells with decitabine (1 μM) and TNF (5 ng/mL) compared to RH30-SR control cells treated with DMSO and PBS. Significance was determined through two-way ANOVA analysis, with Sidak’s multiple comparisons, n = 3. (E) Expression levels of DNMT1, DNMT3A, and DNMT3B in RH30 MyoD Δ and RH30 Myf5 Δ compared to RH30-Vector control cells analyzed by one-way ANOVA with Dunnett’s multiple comparisons, n = 3. (F) Western blots of MyoD, DNMT1, DNMT3A, and DNMT3B in RH30-SR Vector or MyoD Δ cells, using α−tubulin as a loading control. Arrowheads point to respective DNMTs. (G) MyoD ChIP-seq data identifying enrichment peaks on the DNMT3A gene from FN (RD) and FP (RH4) RMS cells, graphed above representative ATAC-seq data revealing loss of chromatin accessibility signal in highlighted regions of interest in RH30 MyoD Δ (purple) compared to RH30 Vector cells (blue) with accompanying composite plot of ATAC-seq signals of both conditions, n = 3. Signal is represented as reads per million mapped reads (RPM). (H) Visualization of MyoD ChIP-seq data with enrichment peaks identified as S1-S3 on DNMT1, DNMT3A and DNMT3B genes in FN (RD) and FP (RH4) RMS cells. (I) MyoD ChIP analysis performed, as percent of input, on regions S1–S3 in DNMT1, DNMT3A and DNMT3B genes, n = 2. Data with error bars are depicted as mean ± SEM, ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001; ∗∗∗∗ p < 0.0001. See also .

Article Snippet: Antibodies used in this study included, IκBα (Cell Signaling Technology Cat# 9242, RRID: AB_331623 ), α-tubulin (Thermo Fisher Scientific Cat# A11126, RRID: AB_2534135 ), p65/RelA (Santa Cruz Biotechnology Cat# sc-109, RRID: AB_632039 ), MyoD (Santa Cruz Biotechnology Cat# sc-377460, RRID: AB_2813894 ), Myf5 (Santa Cruz Biotechnology Cat# sc-518039), myogenin (Santa Cruz Biotechnology Cat# sc-52903, RRID: AB_784707 ), MRF4 (Santa Cruz Biotechnology sc-514379), MEF2C (Abcam Cat# ab211493, RRID: AB_2864417 ), MEF2D (Santa Cruz Biotechnology Cat# sc-271153, RRID: AB_10614669 ), MYHC (Sigma-Aldrich Cat# M8421, RRID: AB_477248 ), TnnT (Sigma-Aldrich Cat# SAB4200717, RRID: AB_2892079 ), CYLD (Cell Signaling Technology Cat# 4495, RRID: AB_10557111 ), p -RIPK1 (Proteintech Cat# 66854-1-Ig, RRID: AB_2882194 ), RIPK1 (Thermo Fisher Scientific Cat# PA5-20811, RRID: AB_11154790 ), DNMT1 (Novus Cat# NB100-56519, RRID: AB_838131 ), DNMT3A (Santa Cruz Biotechnology Cat# sc-365769, RRID: AB_10844010 ), DNMT3B (Cell Signaling Technology Cat# 67259, RRID: AB_2799723 ), and HRP conjugated secondary antibodies anti-mouse IgG (Promega Cat# W4021, RRID: AB_430834 ) and anti-rabbit IgG (Promega Cat# W4011, RRID: AB_430833 ).

Techniques: Control, Expressing, Plasmid Preparation, Western Blot, ChIP-sequencing

Journal: Molecular Psychiatry

Article Title: Genome-wide methylation in alcohol use disorder subjects: implications for an epigenetic regulation of the cortico-limbic glucocorticoid receptors (NR3C1)

doi: 10.1038/s41380-019-0449-6

Figure Lengend Snippet: Schematic representation of chronic alcohol-induced stress response via an epigenetic regulation of glucocorticoid receptor expression. An adaptive response to stress is controlled by the glucocorticoid receptor (GR), the low-affinity receptor for cortisol. In unstressed control conditions (left panel) with low levels of cortisol, GRs are not translocated to the nucleus and their expression is regulated by transcription factors, such as specificity protein 1 (SP1) and nerve growth factor-induced protein A (EGR1). Alcohol consumption, similar to stress, leads to enhanced levels of cortisol, which enters the neurons and binds to the GR (right panel). The activation and nuclear translocation of the GR is crucial for the control of the stress response and is regulated by molecular chaperones (i.e., heat-shock protein 90 alpha family class A member 1, HSP90; BCL2- associated athanogene 1, BAG1; FK506 binding protein 4, FKBP4; and FK506 binding protein 51, FKBP5). Once bound to the chromatin, GR recruits chromatin-remodeling factors, including DNA methyltransferases (DNMT1 and –3A) and demethylating (ten–eleven translocases, TET1–3; growth arrest and DNA damage-inducible beta, GADD45B) enzymes, histone deacetylases (HDAC), and methyl CpG binding protein 2 (MECP2). By remodeling chromatin, excessive alcohol consumption results in the negative epigenetic regulation of the expression of glucocorticoid-sensitive genes, including the GR gene (nuclear receptor subfamily 3 group C member 1, NR3C1 ) itself, that may play an important role in the overall dysregulation of the stress response and pathophsiology of alcohol use disorders. This results in an altered promoter methylation (M: CpG methylation; H: CpG hydroxymethylation), leading to a maladaptive stress response in alcohol use disorder subjects

Article Snippet: Samples were incubated with an anti-MECP2 antibody (Diagenode, Denville, NJ; #C15410052) or an anti-DNMT1 antibody (Novus Biologicals, Centennial, CO; #NB100–56519).

Techniques: Expressing, Control, Activation Assay, Translocation Assay, Binding Assay, Methylation, CpG Methylation Assay