Journal: PLoS ONE

Article Title: A Novel YY1-miR-1 Regulatory Circuit in Skeletal Myogenesis Revealed by Genome-Wide Prediction of YY1-miRNA Network

doi: 10.1371/journal.pone.0027596

Figure Lengend Snippet: (A) Two conserved enhancers (E1 and E2) were identified in the promoter region and intragenic region of miR-1-2/miR-133a-1 cluster, respectively. Three putative YY1 binding sites, A, B and C, were identified. (B) One conserved enhancer (E3) was identified in between miR-1-1 and miR-133a-2 with a putative YY1 binding site, D, identified. (C) One conserved enhancer (E4) was identified upstream of miR-206 and miR-133b cluster with a putative YY1 binding site, E, identified. Binding sites for MyoD, MEF2 and SRF were also shown. (D) C2C12 cells were transfected with 250 ng of E1, E2, E3 or E4 reporter plasmid along with Renilla and control vector (YY1 0 ng) or 50 ng, 200 ng, 500 ng YY1 expressing plasmid. Cells were then cultured for 48 h at which time luciferase activities were determined and normalized to Renilla protein. The data represent the average of three independent experiments ± S.D. (E) C2C12 cells were transfected with 0.25 µg of E1, E2, E3 or E4 reporter plasmid along with Renilla luciferase vector and siYY1 or siNC oligos. Luciferase activity was determined as in (D). (F) Chromatins were harvested from C2C12 myoblasts growing in growth medium (GM) or myotubes maintained in differentiation medium (DM) and subjected to ChIP-PCR analysis. Primers were designed to amplify regions encompassing putative YY1 binding sites A, B, C, D, or E. MyHC and Tnni2 are known YY1 targets and used as positive controls. A genomic region that contains no YY1 binding sites was included as a negative control (NC). (G) Site A (Mut A) or both A and B (Mut A+B) were mutated in E1 luciferase reporter plasmid and luciferase reporter assay was performed to measure the response of mutants to YY1 over-expression as in (D). Relative luciferase unit (RLU) is shown with respect to Vector transfection where luciferase activities were set to a value of 1. (H) ChIP-PCR for Ezh2 or H3K27me3 was performed as in (F). The p value was determined by Student's T-test: *p<0.05, **p<0.01, ***p<0.001.

Article Snippet: ChIP assays were performed as previously described (Tong Ihn Lee, 2006) using 5 μg of antibodies against YY1 (rabbit polyclonal from Santa Cruz Biotechnology, Cat# SC-1703), Ezh2 (mouse monoclonal from Cell Signaling, Cat# AC22), trimethyl-histone H3-K27 (rabbit polyclonal from Milipore, Cat# 07-449), or without any antibody as a negative control.

Techniques: Binding Assay, Transfection, Plasmid Preparation, Control, Expressing, Cell Culture, Luciferase, Activity Assay, Negative Control, Reporter Assay, Over Expression

Journal: Theranostics

Article Title: HOXB13 networking with ABCG1/EZH2/Slug mediates metastasis and confers resistance to cisplatin in lung adenocarcinoma patients

doi: 10.7150/thno.29463

Figure Lengend Snippet: HOXB13 targets to and upregulates EZH2. (A) Upregulation of EZH2 by HOXB13 in lung adenocarcinoma cells. H1299 and A549 cells were transiently transfected by Flag-HOXB13 or GFP-HOBX13 separately, controlled by Flag or GFP. Left panel: Cell lysates were prepared and were subjected to Western blot analysis using anti-EZH2 antibody. Right panel: Transcriptional detection of HOXB13-upregulated EZH2 by qPCR. (B) Enrichment of HOXB13 on the EZH2 promoter analyzed by ChIP-seq database from prostate cancer . (C) HOXB13 targets EZH2 in lung adenocarcinoma cells. Upper panel: Diagram of the EZH2 promoter with potential HOXB13 binding sites (double arrow). Lower panel: ChIP analysis was performed using either an anti-HOXB13 ChIP-grade antibody or control IgG in H1299 Flag-HOXB13 cells. Sites 3, 4, and 5 in EZH2 promoter are enriched in a qPCR analysis with known target genes of HOXB13 including ORM1, NKX3.1 as positive controls, and actin as a negative control. Insert is the gel picture of ChIP analysis for HOXB13 targeting on EZH2 promoter. (D) EZH2 promoter-luciferase reporter construct map. Lower panel: Luciferase reporter constructs were co-transfected with vector or HOXB13, towards the identification of 1062-1875bp upstream region critical for HOXB13-directed enhancement (Unpaired Student's t -test, **p < 0.01) in H1299 (left panel) and in A549 cells (right panel). (E) Levels of HOXB13 and EZH2 in patients' tumor specimens were detected by immunohistochemical analyses using HOXB13 and EZH2 antibodies separately. Patients 1-3: HOXB13 and EZH2 were low in cisplatin- and paclitaxel-sensitive lung adenocarcinoma patients. Patients 4-6: HOXB13 and EZH2 were high in cisplatin- and paclitaxel-resistant lung adenocarcinoma patients. (F) Quantification for the levels of HOXB13 and EZH2 in cisplatin- and paclitaxel-sensitive (n=6) or resistant (n=9) lung adenocarcinoma patients (Unpaired Student's t -test, ** p<0.01).

Article Snippet: Briefly, deparaffinization and hydration were performed followed by abolishing endogenous peroxidase activity using 0.3% hydrogen peroxide for 30 min, and then microwaved for antigen retrieval in 10 mM sodium citrate buffer (pH 6.0) for 20 min. HOXB13 antibody (Santa Cruz, SC-28333, USA) and EZH2 antibody (Cell Signaling Technology, 30233s, USA) were used at 2 μg/ml in all experiments, and incubated at 4 °C overnight followed by the PV-9000 2-step plus Poly-HRP anti-Mouse/Rabbit IgG Detection system (Zhong Shan Jin Qiao, China).

Techniques: Transfection, Western Blot, ChIP-sequencing, Binding Assay, Control, Negative Control, Luciferase, Construct, Plasmid Preparation, Immunohistochemical staining

Journal: Theranostics

Article Title: HOXB13 networking with ABCG1/EZH2/Slug mediates metastasis and confers resistance to cisplatin in lung adenocarcinoma patients

doi: 10.7150/thno.29463

Figure Lengend Snippet: Cisplatin induces expression of HOXB13. (A) HOXB13 and its target genes ABCG1and EZH2 were induced in cisplatin-resistant A549 cells (A549 DDP) at the protein (Upper) and transcriptional levels (Lower) determined by Western blot or qPCR analyses. All these drug resistance genes were significantly upregulated by cisplatin induction (**p<0.01). (B) HOXB13, EZH2, and ABCG1 were transiently induced in the presence of 5 μM or 10 μM cisplatin treatment at indicated time points in A549 and H1299 cells, as detected by Western blot analysis. (C) Quantification of the bands to show that cisplatin upregulates HOXB13 and its target protein expression. (Unpaired Student's t -test, *p<0.05, **p<0.01, ***p<0.001) (D) HOXB13 and EZH2 levels were detected in drug-sensitive and drug-resistant PDX samples with or without cisplatin treatment by IHC. Left were detected by HOXB13 antibody and right were detected by EZH2 antibody.

Article Snippet: Briefly, deparaffinization and hydration were performed followed by abolishing endogenous peroxidase activity using 0.3% hydrogen peroxide for 30 min, and then microwaved for antigen retrieval in 10 mM sodium citrate buffer (pH 6.0) for 20 min. HOXB13 antibody (Santa Cruz, SC-28333, USA) and EZH2 antibody (Cell Signaling Technology, 30233s, USA) were used at 2 μg/ml in all experiments, and incubated at 4 °C overnight followed by the PV-9000 2-step plus Poly-HRP anti-Mouse/Rabbit IgG Detection system (Zhong Shan Jin Qiao, China).

Techniques: Expressing, Western Blot

Journal: Theranostics

Article Title: HOXB13 networking with ABCG1/EZH2/Slug mediates metastasis and confers resistance to cisplatin in lung adenocarcinoma patients

doi: 10.7150/thno.29463

Figure Lengend Snippet: Combination use of HOXB13 with ABCG1 and EZH2 gives high precision in predicting lung adenocarcinoma patients' outcome. (A) Combination of HOXB13 with its target gene expressions to predict lung adenocarcinoma prognosis. (B) Working model: HOXB13 induced by cisplatin confers lung adenocarcinoma patients' drug resistance by direct targeting to the newly identified drug resistance gene ABCG1 and also known drug resistance gene EZH2. Further, HOXB13 mediates metastasis of lung adenocarcinoma patients by direct targeting to EZH2 and Slug. Combination of HOXB13, ABCG1, EZH2 presents a better strategy to predict outcome or resistance to chemotherapy in lung adenocarcinoma patients.

Article Snippet: Briefly, deparaffinization and hydration were performed followed by abolishing endogenous peroxidase activity using 0.3% hydrogen peroxide for 30 min, and then microwaved for antigen retrieval in 10 mM sodium citrate buffer (pH 6.0) for 20 min. HOXB13 antibody (Santa Cruz, SC-28333, USA) and EZH2 antibody (Cell Signaling Technology, 30233s, USA) were used at 2 μg/ml in all experiments, and incubated at 4 °C overnight followed by the PV-9000 2-step plus Poly-HRP anti-Mouse/Rabbit IgG Detection system (Zhong Shan Jin Qiao, China).

Techniques:

Journal: The FASEB Journal

Article Title: Maternal regulation of SATB2 in osteo‐progeniters impairs skeletal development in offspring

doi: 10.1096/fj.201901901r

Figure Lengend Snippet: FIGURE 1 Epigenetic regulation of skeletal development of fetuses from HFD (high fat diet) rat dams. A, Representative images from Alizarin red/Alcian blue staining of E18.5 embryos from dams fed either control diet or HFD. Cartilage and calcification stained dark blue; arrows indicate differences of skeletal ossification in the head. B, Western blots for H3K27me3, Ezh2, p-Ezh2 and CDK1 in proteins from EOCCs (embryonic rat osteogenic calvarial cells) either from six HFD obese dams or six control diet dams. M, male; F, female. C, Real-time PCR for Ezh2 mRNA expression in total RNA from EOCCs either from six HFD obese dams or six control diet dams. *P < .05 by t-test. D, and E, representing top genes and significantly H3K27me3 enriched or decreased gene body and gene promoter using Heat Map analysis of ChIP-seq data from EOCCs either from six HFD obese dams or six control diet dams (pooled)

Article Snippet: Ezh2 overexpression plasmid (Ezh2, #28060, Addgene) and shRNA Ezh2 (Ezh2-sh, #TG509722, Origene) were used.

Techniques: Staining, Control, Western Blot, Real-time Polymerase Chain Reaction, Expressing, ChIP-sequencing

Journal: The FASEB Journal

Article Title: Maternal regulation of SATB2 in osteo‐progeniters impairs skeletal development in offspring

doi: 10.1096/fj.201901901r

Figure Lengend Snippet: FIGURE 2 Identification of epigenetic target genes affected by maternal obesity during fetal skeletal development. A, SATB2 was significantly enriched for H3K27me3 within the gene body, especially in the promoter region based on the detection of a peak (black and red arrow head) of enrichment by Illumina DNA sequencing (male and female are mixed, and samples were pooled to three per group). B, Real-time PCR for SATB2 and ALP mRNA expression in total RNA from EOCCs either from six HFD obese dams or six control diet dams. *P < .05 by t-test. C, ChIP of rat SATB2 enhancer elements by specific anti H3K27me3 and Ezh2 antibodies. D, ChIP of enrichment of SATB2 after IP with H3K27me3 and Ezh2 antibodies. Fold enrichment relative to IgG. *P < .05 by t-test EOCCs from control vs EOCCs from HFD obese dams. E, Control EOCCs were treated with vehicle (control), NEFA mixture 400 µM with Palmitic and Oleic acid 2:1 mixture, Sigma-Aldrich), Palmitic acid (270 µM) or Oleic acid (130 µM) for 48 hours: Western blots for H3K27me3, Ezh2, p-Ezh2, SATB2 and Col 1 (collagen 1). n = 3/treatment

Article Snippet: Ezh2 overexpression plasmid (Ezh2, #28060, Addgene) and shRNA Ezh2 (Ezh2-sh, #TG509722, Origene) were used.

Techniques: DNA Sequencing, Real-time Polymerase Chain Reaction, Expressing, Control, Western Blot

Journal: The FASEB Journal

Article Title: Maternal regulation of SATB2 in osteo‐progeniters impairs skeletal development in offspring

doi: 10.1096/fj.201901901r

Figure Lengend Snippet: FIGURE 4 Increased Ezh2/H3K27me3 but decreased SATB2 expression in human UC MSCs from obese mothers. A, UC MSCs from either lean or obese mothers were cultured, passage 2 cells were immune-stained with anti-Ezh2 antibody (red, white arrows) or anti-SATB2 antibody (green, yellow arrows). B, and C, Real-time PCR (Box & Whiskers graphs) and Western blots (under Box & Whiskers graphs) of Ezh2 and SATB2 mRNA and protein expression in UC MSCs either from lean or obese mothers. *P < .05 vs lean, t-test. D, ChIP of human Ezh2, SATB2 and GAPDH enhancer elements by specific anti H3K27me3 antibody, and E, ChIP of enrichment of human Ezh2, SATB2 and GAPDH after IP with H3K27me3 antibody, fold enrichment relative to IgG. *P < .01 by t-test vs lean

Article Snippet: Ezh2 overexpression plasmid (Ezh2, #28060, Addgene) and shRNA Ezh2 (Ezh2-sh, #TG509722, Origene) were used.

Techniques: Expressing, Cell Culture, Staining, Real-time Polymerase Chain Reaction, Western Blot

Journal: The FASEB Journal

Article Title: Maternal regulation of SATB2 in osteo‐progeniters impairs skeletal development in offspring

doi: 10.1096/fj.201901901r

Figure Lengend Snippet: FIGURE 6 Increased trabecular bone mineral density in Ezh2 osteoblastic cell specific deletion male mice. A, Sagittal views of total, trabecular and cortical bone mineral density, and Strength Strain Index (SSI) from a represented mouse from cko and their respective control mice. B, Total bone mineral content in all female mice. B, Tibia pQCT parameters, TOT-BMC (total bone mineral content), P = .0499 by one-way ANOVA; TOT-BMD (total bone mineral density), P = .0365 by one-way ANOVA; TRAB-BMD (trabecular bone mineral density), P = .0188 by one-way ANOVA; CRT-BMD (cortical bone mineral density), P = .0620 by one-way ANOVA in male cko mice compared to their respective genotypic control mice, followed by Tukey's post hoc test comparing cko with its respective control genotype group, *means P < .05 significantly different, n = 4

Article Snippet: Ezh2 overexpression plasmid (Ezh2, #28060, Addgene) and shRNA Ezh2 (Ezh2-sh, #TG509722, Origene) were used.

Techniques: Control

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: PRC2 direct transfer from G-quadruplex RNA to dsDNA has implications for RNA-binding chromatin modifiers.

doi: 10.1073/pnas.2220528120

Figure Lengend Snippet: Fig. 1. Experiment and analysis strategy to measure direct transfer kinetics (FPCD Experiments). (1) The minimum amount of PRC2 required for saturated binding is mixed with a trace amount of fluorescently labeled nucleic acid (ligand), then incubated (at 4/25 °C) until thermal and reaction equilibrium. (2) Various concentrations of unlabeled nucleic acid (competitor) are added to the preformed complex to initiate reactions (at 25 °C). (3) The time-course reactions are immediately monitored by fluorescence polarization in a microplate reader (at 25 °C). Potential complexes with their polarization states are shown, and they are labeled with rate constants describing inter-complex transitions. Rate constants associated with a classic competition model are indicated by green boxes, and those additionally necessary for a direct transfer model are indicated by a purple box. The intercomplex transition solely associated with the direct transfer model has an implied unstable ternary complex intermediate. The system of differential equations describing these reactions is given by SI Appendix, Eq. S1. (4) Polarization signals are normalized to the range in polarization signal across all competitor concentrations to give proportion of initial complex remaining. Normalized polarization signals are plotted versus time and fit with one-phase exponential decay regression (SI Appendix, Eq. S3.1). (5) The regression initial slopes (koff obs; SI Appendix, Eq. S3.2) are plotted versus competitor concentration and regressed with custom equations describing the classic competition (SI Appendix, Eq. S4.2) and direct transfer (SI Appendix, Eq. S4.1) models to determine rate constant values. Model fits are compared with the Bayesian Information Criterion (BIC) to determine the appropriate model.

Article Snippet: The custom scripts referenced in these methods are available on GitHub (github.com/whemphil/PRC2_DirectTransfer_Manuscript) (60). pFastBac vectors for PRC2 expression have been deposited to AddGene (ID #125161-125165) by the Davidovich lab. All ligands and competitors are available from IDT via the sequences in SI Appendix, Table S2.

Techniques: Binding Assay, Labeling, Incubation, Fluorescence, Concentration Assay

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: PRC2 direct transfer from G-quadruplex RNA to dsDNA has implications for RNA-binding chromatin modifiers.

doi: 10.1073/pnas.2220528120

Figure Lengend Snippet: Fig. 2. PRC2 exhibits direct transfer kinetics for G4 RNA and dsDNA. FPCD experiments (Fig. 1) were performed with the Wang et al. and Long et al. ligand/ competitor over a range of competitor concentrations (panel A) and to measure direct transfer kinetics for every ligand–competitor combination of a G4 RNA and 60-bp dsDNA (panel B). Data are from representative experiments (of n ≥ 3), where error bars indicate mean ± SD for four technical replicates. Rate constant values from regression can be found in Table 1, additional nomenclature definitions are in SI Appendix, Table S1, and polynucleotide species definitions are in SI Appendix, Table S2. (A) Exponential regression fit lines from each condition (left plot), alongside the observed initial dissociation rates (koff obs, see SI Appendix, Eq. S3.2) as a function of competitor concentration (right plot). Solid line in right plot is a visual aid connecting data means. Raw data are shown in SI Appendix, Fig. S1. (B) Experiments were performed in BB10 buffer. Isotherm, carrier nucleic acid, and fluorophore controls for the RNA-RNA competition experiment (Top Left) can be found in SI Appendix, Fig. S2. Analogous studies with a 50-bp dsDNA can be found in SI Appendix, Fig. S3.

Article Snippet: The custom scripts referenced in these methods are available on GitHub (github.com/whemphil/PRC2_DirectTransfer_Manuscript) (60). pFastBac vectors for PRC2 expression have been deposited to AddGene (ID #125161-125165) by the Davidovich lab. All ligands and competitors are available from IDT via the sequences in SI Appendix, Table S2.

Techniques: Concentration Assay

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: PRC2 direct transfer from G-quadruplex RNA to dsDNA has implications for RNA-binding chromatin modifiers.

doi: 10.1073/pnas.2220528120

Figure Lengend Snippet: Fig. 3. Ionic interactions contribute to PRC2’s dsDNA but not G4 RNA affinities. FP-based equilibrium binding experiments (Materials and Methods) were carried out under various salt concentrations (BBX = X mM KCl) for a G4 RNA and 60-bp dsDNA ligand (no competitor present). Kinetic constant values from regression can be found in Table 1, additional nomenclature definitions are in SI Appendix, Table S1, and polynucleotide species definitions are in SI Appendix, Table S2. (A) Binding curves for indicated PRC2 ligands. Curves are composites of three experiments with four replicates each, where error bars indicate mean ± SD. Solid lines are visual aids connecting the data points. (B) Affinity versus ionic strength plots with Kd app values from regression of data in panel A. Data are composites of all experiments in panel A, where error bars indicate mean ± SD. Solid lines are from linear regression of data on the logarithmic axes shown. Regression values can be found in Materials and Methods or the corresponding text.

Article Snippet: The custom scripts referenced in these methods are available on GitHub (github.com/whemphil/PRC2_DirectTransfer_Manuscript) (60). pFastBac vectors for PRC2 expression have been deposited to AddGene (ID #125161-125165) by the Davidovich lab. All ligands and competitors are available from IDT via the sequences in SI Appendix, Table S2.

Techniques: Binding Assay

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: PRC2 direct transfer from G-quadruplex RNA to dsDNA has implications for RNA-binding chromatin modifiers.

doi: 10.1073/pnas.2220528120

Figure Lengend Snippet: Fig. 4. Direct transfer allows RNA to boost PRC2 HMTase activity. (A) Reaction scheme of PRC2-like protein (E) binding of RNA (R) and nucleosomes (N) with catalytic activity on nucleosomes (Nm), where conjugates are complexes of the respective reactants. Major protein states are shown in red, additional reactants in purple, and rate constants and tuning parameters in blue. For rate constants, k1 is for association, k−1 is for dissociation, kθ is for direct transfer, and kcat is for catalysis. The α tuning parameter is included for any protein complex transitions where RNA–nucleosome direct transfer is possible. It is an adjustment of effective molarity for direct transfer reactions, meant to account for the spatial proximity of nascent RNA and nucleosome DNA (e.g., nascent RNA), but it has a complex relationship with other factors that warrants qualitative interpretation (see corresponding text). Specific nomenclature definitions are in SI Appendix, Table S1. These reactions are described by the system of differential equations, SI Appendix, Eq. S5. Inter-complex transitions defined by the kθ rate constants are like those shown in Fig. 1, and their removal collapses this scheme to a classic model. (B) Reactions were simulated using SI Appendix, Eq. S5 for the scheme (panel A) to monitor rate of nucleosome methylation (H3K27me3) over time under varying RNA–nucleosome molar ratios (RNA:Nuc), direct transfer effective molarity adjustments (α), and protein concentrations. Black curves represent HMTase time-course reactions in the absence of RNA, and the colored lines represent the effect of increasing RNA concentrations. For simulations, k−1, kθ, and Kd values were taken from Table 1, kcat was taken from prior PRC2 literature, [NT] = 5 nM, [ET] = 0.1–2 × KdN, and other parameter values are indicated; explicit values are provided in Materials and Methods. Limited data from a single protein concentration (2 × KdN) are shown, but the full data set is provided in SI Appendix, Fig. S4. (C) HMTase activity rate data were used to calculate the relative initial rates (V0) for reactions with 8:1 versus 0:1 RNA–nucleosome molar ratios (R:N), across a range of α values. Data used were the same as for panel B. Dotted line is a visual aid for when RNA concentration has no effect on initial reaction rate.

Article Snippet: The custom scripts referenced in these methods are available on GitHub (github.com/whemphil/PRC2_DirectTransfer_Manuscript) (60). pFastBac vectors for PRC2 expression have been deposited to AddGene (ID #125161-125165) by the Davidovich lab. All ligands and competitors are available from IDT via the sequences in SI Appendix, Table S2.

Techniques: Activity Assay, Binding Assay, Methylation, Protein Concentration, Concentration Assay

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: PRC2 direct transfer from G-quadruplex RNA to dsDNA has implications for RNA-binding chromatin modifiers.

doi: 10.1073/pnas.2220528120

Figure Lengend Snippet: Fig. 5. Stable RNA and nucleosome cobinding could boost a protein’s activity. (A) Reaction scheme of a protein (E) binding RNA (R) and nucleosomes (N) independently, with the potential for catalytic activity on nucleosomes (Nm), where conjugates are complexes of the respective reactants. Major protein states are shown in red, additional reactants in purple, and rate constants and tuning parameters in blue. For rate constants, k1 is for association, k−1 for dissociation, and kcat is for catalysis. The α tuning parameter is included for any protein complex transitions where ternary complex formation from a bimolecular complex is possible. It is an adjustment of effective molarity for direct transfer reactions, meant to account for the spatial proximity of nascent RNA and nucleosome DNA (e.g., nascent RNA), but it has a complex relationship with other factors that warrants qualitative interpretation (see corresponding text). The β tuning parameter is effect of bound RNA on catalytic activity. The δ tuning parameters are the effects of bound RNA/nucleosome on ternary nucleosome/RNA binding or dissociation. Specific nomenclature definitions are in SI Appendix, Table. S1. (B) Reactions were simulated using SI Appendix, Eq. S6 for the scheme (panel A) to monitor rate of nucleosome methylation (H3K27me3) over time under varying RNA–nucleosome molar ratios (RNA:Nuc) and effective molarity adjustments (α). Black curves represent HMTase time-course reactions in the absence of RNA, and the colored lines represent the effect of increasing RNA concentrations. For α = 1, all lines overlap. For simulations, Kd values were taken from Sigova et al, kcat was set to the value from PRC2 literature, [NT] = 5 nM, [ET] = 0.1−2 × KdN, β = 0−1, δ = 1, and other parameter values are indicated; explicit values are provided in Materials and Methods. Limited data from a single protein concentration (0.125 × KdN) and β value (β = 1) are shown, but the full data set is provided in SI Appendix, Fig. S7. (C) HMTase activity rate data were used to calculate the relative initial rates (V0) for reactions with 8:1 versus 0:1 RNA–nucleosome molar ratios (R:N), across a range of α values. Data used were the same as for panel B. Dotted line indicates RNA concentration having no effect on initial reaction rate.

Article Snippet: The custom scripts referenced in these methods are available on GitHub (github.com/whemphil/PRC2_DirectTransfer_Manuscript) (60). pFastBac vectors for PRC2 expression have been deposited to AddGene (ID #125161-125165) by the Davidovich lab. All ligands and competitors are available from IDT via the sequences in SI Appendix, Table S2.

Techniques: Activity Assay, Binding Assay, RNA Binding Assay, Methylation, Protein Concentration, Concentration Assay

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: PRC2 direct transfer from G-quadruplex RNA to dsDNA has implications for RNA-binding chromatin modifiers.

doi: 10.1073/pnas.2220528120

Figure Lengend Snippet: Fig. 6. A direct transfer model of RNA regulation of PRC2 HMTase activity. (A) Proposed Steps for an RNA Recruitment Model of PRC2. (1) G4-containing nascent RNA at transcriptionally active PRC2 target genes (2) is bound by PRC2, (3) RNA-tethered PRC2 is transferred onto spatially proximal nucleosomes, then (4) PRC2 deposits its H3K27me3 mark. (B) Proposed Mechanisms for the Direct Transfer Step. PRC2 could have shared contacts for G4 RNA and nucleosome DNA binding but allow the ligands to occupy partially associated binding states that permit transient cobinding. Nucleosome DNA could give the appearance of actively disrupting a PRC2–RNA complex (Left) by forming a highly transient ternary intermediate where the PRC2-RNA interaction is destabilized (Middle Top). The unstable ternary intermediates may quickly dissociate to form a more stable PRC2-nucleosome (Right) or PRC2-RNA (Left) complex.

Article Snippet: The custom scripts referenced in these methods are available on GitHub (github.com/whemphil/PRC2_DirectTransfer_Manuscript) (60). pFastBac vectors for PRC2 expression have been deposited to AddGene (ID #125161-125165) by the Davidovich lab. All ligands and competitors are available from IDT via the sequences in SI Appendix, Table S2.

Techniques: Activity Assay, Binding Assay