Pax3/7BP Is a Pax7- and Pax3-Binding Protein that Regulates the Proliferation of Muscle Precursor Cells by an Epigenetic Mechanism

SummaryIn mouse skeletal muscles, Pax7 uniquely marks muscle satellite cells and plays some important yet unknown functions at the perinatal stage. To elucidate its in vivo functions, we initiated a yeast two-hybrid screening to look for Pax7-interacting proteins and identified a previously uncharacterized Pax7- and Pax3-binding protein (Pax3/7BP). Pax3/7BP is a ubiquitously expressed nuclear protein, enriched in Pax7+ muscle precursor cells (MPCs), and serves as an indispensable adaptor for Pax7 to recruit the histone 3 lysine 4 (H3K4) methyltransferase (HMT) complex by bridging Pax7 and Wdr5. Knockdown of Pax3/7BP abolished the Pax3/7-associated H3K4 HMT activity and inhibited the proliferation of Pax7+ MPCs from young mice both in culture and in vivo. Id3 and Cdc20 were direct target genes of Pax7 and Pax3/7BP involved in the proliferation of Pax7+ MPCs. Collectively, our work establishes Pax3/7BP as an essential adaptor linking Pax3/7 with the H3K4 HMT to regulate the proliferation of MPCs

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

microRNAs (miRNAs) are non-coding RNAs that regulate gene expression post-transcriptionally, and mounting evidence supports the prevalence and functional significance of their interplay with transcription factors (TFs). Here we describe the identification of a regulatory circuit between muscle miRNAs (miR-1, miR-133 and miR-206) and Yin Yang 1 (YY1), an epigenetic repressor of skeletal myogenesis in mouse. Genome-wide identification of potential down-stream targets of YY1 by combining computational prediction with expression profiling data reveals a large number of putative miRNA targets of YY1 during skeletal myoblasts differentiation into myotubes with muscle miRs ranking on top of the list. The subsequent experimental results demonstrate that YY1 indeed represses muscle miRs expression in myoblasts and the repression is mediated through multiple enhancers and recruitment of Polycomb complex to several YY1 binding sites. YY1 regulating miR-1 is functionally important for both C2C12 myogenic differentiation and injury-induced muscle regeneration. Furthermore, we demonstrate that miR-1 in turn targets YY1, thus forming a negative feedback loop. Together, these results identify a novel regulatory circuit required for skeletal myogenesis and reinforce the idea that regulatory circuitries involving miRNAs and TFs are prevalent mechanisms

INFIMA leverages multi-omics model organism data to identify effector genes of human GWAS variants.

Genome-wide association studies reveal many non-coding variants associated with complex traits. However, model organism studies largely remain as an untapped resource for unveiling the effector genes of non-coding variants. We develop INFIMA, Integrative Fine-Mapping, to pinpoint causal SNPs for diversity outbred (DO) mice eQTL by integrating founder mice multi-omics data including ATAC-seq, RNA-seq, footprinting, and in silico mutation analysis. We demonstrate INFIMA\u27s superior performance compared to alternatives with human and mouse chromatin conformation capture datasets. We apply INFIMA to identify novel effector genes for GWAS variants associated with diabetes. The results of the application are available at http://www.statlab.wisc.edu/shiny/INFIMA/

Nf-κb Inhibition Rescues Cardiac Function By Remodeling Calcium Genes In A Duchenne Muscular Dystrophy Model

Duchenne muscular dystrophy (DMD) is a neuromuscular disorder causing progressive muscle degeneration. Although cardiomyopathy is a leading mortality cause in DMD patients, the mechanisms underlying heart failure are not well understood. Previously, we showed that NF-κB exacerbates DMD skeletal muscle pathology by promoting inflammation and impairing new muscle growth. Here, we show that NF-κB is activated in murine dystrophic (mdx) hearts, and that cardiomyocyte ablation of NF-κB rescues cardiac function. This physiological improvement is associated with a signature of upregulated calcium genes, coinciding with global enrichment of permissive H3K27 acetylation chromatin marks and depletion of the transcriptional repressors CCCTC-binding factor, SIN3 transcription regulator family member A, and histone deacetylase 1. In this respect, in DMD hearts, NF-κB acts differently from its established role as a transcriptional activator, instead promoting global changes in the chromatin landscape to regulate calcium genes and cardiac function

Inhibition of miR-29 by TGF-beta-Smad3 Signaling through Dual Mechanisms Promotes Transdifferentiation of Mouse Myoblasts into Myofibroblasts

MicroRNAs (miRNAs) are non-coding RNAs that regulate gene expression in post-transcriptional fashion, and emerging studies support their importance in regulating many biological processes, including myogenic differentiation and muscle development. miR-29 is a promoting factor during myogenesis but its full spectrum of impact on muscle cells has yet to be explored. Here we describe an analysis of miR-29 affected transcriptome in C2C12 muscle cells using a high throughput RNA-sequencing platform. The results reveal that miR-29 not only functions to promote myogenic differentiation but also suppresses the transdifferentiation of myoblasts into myofibroblasts. miR-29 inhibits the fibrogenic differentiation through down-regulating both extracellular matrix genes and cell adhesion genes. We further demonstrate that miR-29 is under negative regulation by TGF-beta (TGF-β)–Smad3 signaling via dual mechanisms of both inhibiting MyoD binding and enhancing Yin Yang 1 (YY1)-recruited Polycomb association. Together, these results identify miR-29 as a pleiotropic molecule in both myogenic and fibrogenic differentiation of muscle cells

Effects of liquid property on onset velocity of circulating fluidization in liquid-solid systems: A CFD-DEM simulation

Knowledge on regime transition from conventional to circulating fluidization is important to scale-up and operation of liquid-solid fluidized systems in industrial applications. Previous experimental studies reported that the onset velocity measured by bed empty time test is pertinent to the physical properties of particle and liquid, and independent of solid inventory, bed geometry, and configuration. The effect of liquid properties is, however, not clear yet since tap water is usually used as the working fluid in laboratory test for convenience. To address this problem, a Computational Fluid Dynamics-Discrete Element Method (CFD-DEM) model is applied to numerically measure the onset velocity of particles in different liquid media. The model is first validated in terms of the experimental data of bed expansion in literature. Then several kinds of particles and three kinds of liquid media of different densities and viscosities are further simulated. We find that the Reynolds number based on onset velocity and Archimedes number follows a power-law relationship. With the decrease of Archimedes number, the discrepancy of Reynolds number based on onset velocity and that on particle terminal velocity becomes highly significant. The ratio of the onset velocity to particle terminal velocity for various particles in one liquid medium is not a constant, and instead dependent on both the particle and liquid properties. (C) 2020 Elsevier B.V. All rights reserved

Genome-wide profiling of YY1 binding sites during skeletal myogenesis

Skeletal muscle differentiation is regulated by a network of transcription factors, epigenetic regulators and noncoding RNAs. We have recently performed ChIP-seq experiments to explore the genome-wide binding of transcription factor YY1 in skeletal muscle cells. Our results identified thousands of YY1 binding peaks, underscoring its multifaceted functions in muscle cells. In particular, we identified a very high proportion of YY1 binding peaks residing in the intergenic regions, which led to the discovery of some novel lincRNAs under YY1 regulation. Here we describe the details of the ChIP-seq experiments and data analysis procedures associated with the study published by Lu et al. in the EMBO Journal in 2013 [1]

Anti-Metatype Antibody Screening, Sandwich Immunoassay Development, and Structural Insights for β-Lactams Based on Penicillin Binding Protein

Theoretically, sandwich immunoassay is more sensitive and has a wider working range than that of competitive format. However, it has been thought that small molecules cannot be detected by the sandwich format due to their limited size. In the present study, we proposed a novel strategy for achieving sandwich immunoassay of β-lactams with low molecular weights. Firstly, five β-lactam antibiotics were selected to bind with penicillin binding protein (PBP)2x* to form complexes. Then, monoclonal and polyclonal antibodies against PBP2x*-β-lactams complexes were produced by animal immunization. Subsequently, the optimal pairing antibodies were utilized to establish sandwich immunoassay for detection of 18 PBP2x*-β-lactam complexes. Among them, ceftriaxone could be detected at as low as 1.65 ng/mL with working range of 1–1000 ng/mL in milk. To reveal the detection mechanism, computational chemistry and molecular recognition study were carried out. The results showed that β-lactams with a large size and complex structures maybe conducive to induce conformational changes of PBP2x*, and then exhibit greater possibility of being detected by sandwich immunoassay after combination with PBP2x*. This study provides insights for subsequent investigations of anti-metatype antibody screening and sandwich immunoassay establishment for small-molecule detection

miR-29 is anti-fibrogenic in C2C12 cells.

<p>(A) C2C12 cells were differentiated (DM) for 0, 1, 2 or 4 days, at which times total RNAs were isolated for qRT-PCR measurement of the expressions of Col 1A1, Col 1A2, Col 3A1, α-SMA or VIM as well as MyHC, α-Actin, Troponin and MyoG. Expression folds are shown with respect to 0 hr cells where normalized copy numbers were set to 1. Data are plotted as mean ± S.D. (B and C) C2C12 cells were transfected with negative control (NC) or miR-29 oligos and differentiated for 48 hrs, at which time total RNAs were isolated for qRT-PCR measurement of the expressions of Myogenin, Troponin, α-Actin or MyHC as well as Col 1A1, Col 1A2, Col 3A1, α-SMA or VIM. Expression folds are shown with respect to NC cells where normalized copy numbers were set to 1. Data are plotted as mean ± S.D. (D) C2C12 cells were transfected with negative control (Anti-NC) or Anti-miR-29 oligos and differentiated for 48 hrs, at which time total RNAs were isolated for qRT-PCR measurement of the expressions of Col 1A1, Col 1A2, and Col 3A1. Expression folds are shown with respect to Anti-NC cells where normalized copy numbers were set to 1. Data are plotted as mean ± S.D. (E) Wild type (WT) or Mutant Col 1A1, Col 1A2, or Col 3A1-3′UTR luciferase reporter constructs were transfected into C2C12 cells with indicated miRNA or negative control (NC) oligos. Luciferase activities were determined at 48 h post-transfection and normalized to β-Galactosidase protein. Relative luciferase unit (RLU) is shown with respect to NC cells where normalized luciferase values were set to 1. The data represent the average of three independent experiments ± S.D. (F) A schematic illustration of base pairing between mmu-miR-29c with 3078–3099 region on 3′UTR of mouse Lims1. (G) Lims1 protein expression was measured in NC or miR-29 stable C2C12 cells by Western blotting using GAPDH as a loading control. (H) NC or miR-29 stable cells were differentiated and Lims1 expression levels were measured at the indicated time points. (I) C2C12 cells were transfected with negative control (Anti-NC) or Anti-miR-29 oligos and differentiated for 48 hrs, at which time total RNAs were isolated for qRT-PCR measurement of the expressions of Lims1. Expression folds are shown with respect to Anti-NC cells where normalized copy numbers were set to 1. Data are plotted as mean ± S.D. (J) Wild type (WT) or Mutant Lims1-3′UTR luciferase reporter constructs were transfected into C2C12 cells with miR-29 or negative control (NC) oligos. Luciferase activities were determined at 48 h post-transfection and normalized to β-Galactosidase protein.</p

Smad3 suppresses miR-29 promoter through inhibiting MyoD binding and enhancing YY1 recruitment.

<p>(A) Schematic illustration of proximal promoter region of mmu-miR-29b/c primary transcript. The arrow denotes the Transcriptional Start Site (TSS). Predicted Smad3 (S), MyoD (M) and YY1 (Y) binding sites were displayed. The location of each site was indicated below. (B) C2C12 myoblasts were untreated or treated with TGF-β for 12 hrs at which time chromatins were collected for ChIP assays using antibodies against MyoD or IgG as controls. PCR assays were then used to measure the enrichment fold of MyoD on four putative binding sites, M1/M2, M3 and M4. (C, D and E) ChIP-PCR assays were performed as above to examine the binding of YY1, Ezh2 and H3K27me3 to putative YY1 binding sites, Y1, Y2, Y3 or Y6. Enrichment folds are shown with respect to IgG control where normalized PCR values were set to 1. Data are plotted as mean ± S.D. (F) Upper: 10T1/2 cells were transfected with 0.25 µg of miR-29-promoter-luc reporter plasmid along with 0.5 µg YY1 plasmid and Smad3 plasmid (0, 0.20, 0.50, 1.00, or 2.00 µg). 24 hr post-transfection, cells were treated with TGF-β for 48 hrs at which time luciferase activities were determined. (G) The transfections were performed as above with 0.5 µg of MyoD plasmid and Smad3 expression plasmid (0, 0.20, 0.50, 1.00, or 2.00 µg). (H) The transfections were performed as above with indicated plasmids (0.5 µg of MyoD, YY1 or Smad3 plasmids were used). (I) C2C12 cells were transfected with siRNA oligos knocking down Smad3, MyoD or YY1. The expression of Lims1 was examined by Western blotting using Tubulin as a loading control. *p<0.05. ** p<0.01.</p