Journal: Heliyon

Article Title: Protein arginine methyltransferase 6 mediates cardiac hypertrophy by differential regulation of histone H3 arginine methylation

doi: 10.1016/j.heliyon.2020.e03864

Figure Lengend Snippet: PRMT6 expression is increased in the LV of DCM patients. (A Western blot analysis showing the increase in PRMT6 and H3R2Me2a expression in the heart of DCM patients (n = 5) compared to control hearts (n = 4). GATA-4 expression is shown in parallel. H3K4Me3 is decreased in DCM hearts. Total histone H3 and GAPDH were used as loading control. (B) shows the quantitative analysis of panel (A). (C) H3R2Me2a and H3K4Me3 levels were quantitated to show their reciprocal expression. Statistical significance was calculated by multiple unpaired t-tests assuming fewer assumptions without same scatter (SD) using GraphPad prism and data were graphically represented as mean ± SD. ∗p < 0.05. LV: left ventricle; DCM: dilated cardiomyopathy. The full blot images are available as supplementary material.

Article Snippet: H3R2Me2a (cat# MBS2520233) and H3K4Me3 (cat# MBS9385277) were from MyBioSource (San Diego, CA, USA).

Techniques: Expressing, Western Blot, Control

Journal: Heliyon

Article Title: Protein arginine methyltransferase 6 mediates cardiac hypertrophy by differential regulation of histone H3 arginine methylation

doi: 10.1016/j.heliyon.2020.e03864

Figure Lengend Snippet: PRMT6 expression is increased in the heart of mice subjected to pressure overload hypertrophy. Echocardiographic measurements were performed in C57BL/6 mice after 10, 21 and 42 days of sham or transverse aortic constriction (TAC) to induce pressure overload hypertrophy. (A) Interventricular septum thickness at diastole (IVS; d), (B) Left ventricular posterior wall thickness at end-diastole (LVPW; d), (C) Ejection fraction and (D) Fractional shortening. (E) At study end point, the animals were euthanized and the hearts were collected. The heart weight/body weight ratio was calculated for each animal. n = 5–17 for control mice, n = 5–17 for TAC mice according to the time-point. Data are represented as means ± SD. Statistical significance was determined using the Holm-Sidak method, alpha = 5.000%. Each time-point was analyzed individually, without assuming a consistent SD. ( F ) Whole heart lysates were prepared and PRMT6 and H3R2Me2a expression were analyzed by Western blot analysis. GAPDH and total histone H3 were used as loading controls. (G) Graph of the quantitative analysis of panel F done with ImageJ software. Data are represented as means ± SD. Statistical significance was determined using Student's t-test. ∗∗p < 0.01, ∗∗∗p < 0.001 were considered significant. The full blot images are available as supplementary material.

Article Snippet: H3R2Me2a (cat# MBS2520233) and H3K4Me3 (cat# MBS9385277) were from MyBioSource (San Diego, CA, USA).

Techniques: Expressing, Control, Western Blot, Software

Journal: Heliyon

Article Title: Protein arginine methyltransferase 6 mediates cardiac hypertrophy by differential regulation of histone H3 arginine methylation

doi: 10.1016/j.heliyon.2020.e03864

Figure Lengend Snippet: PRMT6 expression is increased in phenylephrine-treated NRVM. Monolayers of rat neonatal cardiomyocytes were treated with phenylephrine (PE, 100μM) for the indicated time periods. (A) Cell lysates were prepared and subjected to Western blot analysis to detect the expression of PRMT6, H3R2Me2a and H3K4Me3. GAPDH is shown as a loading control. (B-D) Quantitative analysis of PRMT6, H3R2Me2a and H3K3Me3 from 3 independent cardiomyocyte preparations. The full blot images are available as supplementary material.

Article Snippet: H3R2Me2a (cat# MBS2520233) and H3K4Me3 (cat# MBS9385277) were from MyBioSource (San Diego, CA, USA).

Techniques: Expressing, Western Blot, Control

Journal: Heliyon

Article Title: Protein arginine methyltransferase 6 mediates cardiac hypertrophy by differential regulation of histone H3 arginine methylation

doi: 10.1016/j.heliyon.2020.e03864

Figure Lengend Snippet: PRMT6 silencing blunts the effect of phenylephrine. NRVM were transfected with PRMT6 siRNA (siPRMT6) or control siRNA (siCTL) as mentioned in Materials and Methods. (A) after 48 h, total cell lysates were prepared and subjected to Western blot analysis to measure PRMT6 and ANP protein expression. GAPDH is shown as loading control. (B) Independent experiments performed to measure histone marks. Cell lysates were analyzed for H3R2Me2a and H3K4Me3 by Western blot. GAPDH was used as loading control. (C) Relationship between H3R2Me2a and H3K4Me3 in cells expressing normal or low PRMT6 maintained in serum-free condition or treated with PE. (D) NRVM were cultured in 6-well plates and transfected with control siRNA or PRMT6 siRNA. After transfection, cells were maintained in serum-free condition or challenged with 100 μM PE. After 24 h, the cells were stained with TRITC-labelled phalloidin for F-actin. Cells were visualized by fluorescence microscopy. ( E ) Cell surface area was measured in at least 5 different fields using a 40x objective using Adobe acrobat CS6. Data are expressed as mean ± SD. Statistical analysis was performed by One-way ANOVA (panel E) or two-way ANOVA (panel C) followed by Tukey's multiple comparison test, with ∗p < 0.05. The full blot images are available as supplementary material.

Article Snippet: H3R2Me2a (cat# MBS2520233) and H3K4Me3 (cat# MBS9385277) were from MyBioSource (San Diego, CA, USA).

Techniques: Transfection, Control, Western Blot, Expressing, Cell Culture, Staining, Fluorescence, Microscopy, Comparison

Journal: Heliyon

Article Title: Protein arginine methyltransferase 6 mediates cardiac hypertrophy by differential regulation of histone H3 arginine methylation

doi: 10.1016/j.heliyon.2020.e03864

Figure Lengend Snippet: Model depicting the role of PRMT6 in PE-induced cardiac hypertrophy. In response to PE stimulation, PRMT6 is upregulated in cardiac cells, which increases its signature histone methylation “mark” H3R2Me2a and reduces H3K4Me3. These histone posttranslational modifications may activate hypertrophic genes such as ANP directly or indirectly, resulting in cardiac hypertrophy. Over time, these changes have profound effects on cardiac beating rates and global electrical activity, indicating that PRMT6 is required for the PE-mediated hypertrophic effect and to maintain contractility as well as global electrical activity of cardiac cells.

Article Snippet: H3R2Me2a (cat# MBS2520233) and H3K4Me3 (cat# MBS9385277) were from MyBioSource (San Diego, CA, USA).

Techniques: Methylation, Activity Assay

Journal: bioRxiv

Article Title: A binary arginine methylation switch on histone H3 Arginine 2 regulates its interaction with WDR5

doi: 10.1101/2020.01.13.904581

Figure Lengend Snippet: 1D 1 H NMR spectra of H3 1-7aa peptides showing peak integrations of residues used to measure peptide concentration. a. H3 unmodified b. H3R2me1 c. H3R2me2s. d. H3R2me2a

Article Snippet: Histone H3 1-12aa unmodified, H3R2K, H3R8K, H3R2KR8K and H3 1-7aa unmodified, H3R2me1, H3R2me2s, and H3R2me2a peptides were obtained from GenScript USA.

Techniques: Concentration Assay

Journal: bioRxiv

Article Title: A binary arginine methylation switch on histone H3 Arginine 2 regulates its interaction with WDR5

doi: 10.1101/2020.01.13.904581

Figure Lengend Snippet: Replicate isothermal calorimetry assays showing WDR5 interacts with H3, H3R2me1, and H3R2me2s with similar heats and affinities but does not interact with H3R2me2a. a. Histone H3 1-7aa peptide sequence with methylarginine isoforms occurring at Arg2, underlined b. Raw power differentials (DP) of peptide titrations c. Integrated heats of binding; error bars represent the standard error between two replicate titrations d. Dissociation constants for replicate titrations determined by fitting integrated heats to a one-site binding model e and f. Thermodynamic parameters associated with WDR5 interacting peptides (n.d., not detected). Peptides that interacted with WDR5 bind with stoichiometries (N-values) between 0.9 and 1.

Article Snippet: Histone H3 1-12aa unmodified, H3R2K, H3R8K, H3R2KR8K and H3 1-7aa unmodified, H3R2me1, H3R2me2s, and H3R2me2a peptides were obtained from GenScript USA.

Techniques: Sequencing, Binding Assay

Journal: bioRxiv

Article Title: A binary arginine methylation switch on histone H3 Arginine 2 regulates its interaction with WDR5

doi: 10.1101/2020.01.13.904581

Figure Lengend Snippet: Replicate peptide pulldown assays showing WDR5 interacts with H3R2me0, me1, and me2s but not H3R2me2a. (-) negative control: no peptide, resin only. a. Histone H3 1-21aa peptide sequence with methylarginine isoforms ocurring at Arg2, underlined. b. and c. Replicate pulldowns 1 and 2

Article Snippet: Histone H3 1-12aa unmodified, H3R2K, H3R8K, H3R2KR8K and H3 1-7aa unmodified, H3R2me1, H3R2me2s, and H3R2me2a peptides were obtained from GenScript USA.

Techniques: Negative Control, Sequencing