Genomic-wide transcriptional profiling in primary myoblasts reveals Runx1-regulated genes in muscle regeneration

In response to muscle damage the muscle adult stem cells are activated and differentiate into myoblasts that regenerate the damaged tissue. We have recently showed that following myopathic damage the level of the Runx1 transcription factor (TF) is elevated and that during muscle regeneration this TF regulates the balance between myoblast proliferation and differentiation (Umansky et al.). We employed Runx1-dependent gene expression, Chromatin Immunoprecipitation sequencing (ChIP-seq), Assay for Transposase-Accessible Chromatin with high-throughput sequencing (ATAC-seq) and histone H3K4me1/H3K27ac modification analyses to identify a subset of Runx1-regulated genes that are co-occupied by the TFs MyoD and c-Jun and are involved in muscle regeneration (Umansky et al.). The data is available at the GEO database under the superseries accession number GSE56131

A template-proximal RNA paired element contributes to Saccharomyces cerevisiae telomerase activity

The ribonucleoprotein complex telomerase is critical for replenishing chromosome-end sequence during eukaryotic DNA replication. The template for the addition of telomeric repeats is provided by the RNA component of telomerase. However, in budding yeast, little is known about the structure and function of most of the remainder of the telomerase RNA. Here, we report the identification of a paired element located immediately 5′ of the template region in the Saccharomyces cerevisiae telomerase RNA. Mutations disrupting or replacing the helical element showed that this structure, but not its exact nucleotide sequence, is important for telomerase function in vivo and in vitro. Biochemical characterization of a paired element mutant showed that the mutant generated longer products and incorporated noncognate nucleotides. Sequencing of in vivo synthesized telomeres from this mutant showed that DNA synthesis proceeded beyond the normal template. Thus, the S. cerevisiae element resembles a similar element found in Kluyveromyces budding yeasts with respect to a function in template boundary specification. In addition, the in vitro activity of the paired element mutant indicates that the RNA element has additional functions in enzyme processivity and in directing template usage by telomerase

Runx1 Transcription Factor Is Required for Myoblasts Proliferation during Muscle Regeneration

<div><p>Following myonecrosis, muscle satellite cells proliferate, differentiate and fuse, creating new myofibers. The Runx1 transcription factor is not expressed in naïve developing muscle or in adult muscle tissue. However, it is highly expressed in muscles exposed to myopathic damage yet, the role of Runx1 in muscle regeneration is completely unknown. Our study of Runx1 function in the muscle’s response to myonecrosis reveals that this transcription factor is activated and cooperates with the MyoD and AP-1/c-Jun transcription factors to drive the transcription program of muscle regeneration. Mice lacking dystrophin and muscle Runx1 (<i>mdx</i>^<i>-</i><i>/Runx1</i>^<i>f/f</i>), exhibit impaired muscle regeneration leading to age-dependent muscle waste, gradual decrease in motor capabilities and a shortened lifespan. Runx1-deficient primary myoblasts are arrested at cell cycle G₁ and consequently differentiate. Such premature differentiation disrupts the myoblasts’ normal proliferation/differentiation balance, reduces the number and size of regenerating myofibers and impairs muscle regeneration. Our combined Runx1-dependent gene expression, ChIP-seq, ATAC-seq and histone H3K4me1/H3K27ac modification analyses revealed a subset of Runx1-regulated genes that are co-occupied by MyoD and c-Jun in <i>mdx</i>^<i>-</i><i>/Runx1</i>^<i>f/f</i> muscle. The data provide unique insights into the transcriptional program driving muscle regeneration and implicate Runx1 as an important participant in the pathology of muscle wasting diseases.</p></div

Runx1 expression in response to muscle damage.

<p>(A to D). IHC using anti- Runx1 Ab of gastrocnemius muscle from mice subjected to muscle stress. Runx1-positive cells show brown nuclear staining, scale bars, 50 μm. (A) Untreated WT mice. (B) 120 days-old <i>tg-mSOD1</i> mice. (C) CTX treated WT mice. (D) 2 month old <i>mdx</i> mice. (E) Runx1 and Pax7 IF analysis of CTX-treated WT muscle, scale bars, 50 μm. White arrowheads indicate Runx1⁺/Pax7⁺ cells. (F) IF analysis of cultured proliferating PM using anti- Runx1 and MyoD Abs. DAPI staining was used as a nuclear marker, and myoblasts were visualized by differential interface contrast (DIC) microscopy, scale bars, 50 μm. Results from one of four different experiments with similar findings are shown.</p

Validation of <i>in vivo</i> high confidence Runx1-regulated genes.

<p>(A) Volcano plot of differentially expressed genes in soleus muscle of 8 weeks old <i>mdx/Runx1</i>^<i>f/f</i> vs. <i>mdx</i> mice. Fold expression change against <i>p</i> value is plotted. Significant increased or decreased genes are indicated in red or blue, respectively. Filled triangles indicate Runx1-responsive genes that are known to participate in myoblast proliferation or differentiation. (B) Venn diagram summarizing the overlap between <i>mdx</i> Runx1- responsive (RNA-seq) and PM RMJ- regulated gene. These genes are defined as high confidence Runx1- regulated genes in <i>mdx</i> myoblasts. (C to E) UCSC genome browser screenshots showing ChIP-Seq performed in PM and <i>mdx/Runx1</i>^<i>f/f</i> vs. <i>mdx</i> mice RNA- seq tracing examples of high-confidence Runx1-regulated genes. Expression of these genes was quantified by RT-qPCR of cultured Runx1-deficient or-over expressing PM, and <i>in vivo</i> in <i>mdx/Runx1</i>^<i>f/f</i> vs. <i>mdx</i> muscles. Values are mean±SD (n = 3). (C) <i>Myog</i>, encoding Myogenin (**<i>p</i><0.001, *<i>p</i> <0.05). (D) <i>Dlk1</i>, encoding Delta-like 1 homolog (**<i>p</i> <0.001). (E) <i>Mstn</i>, encoding Myostatin (**<i>p</i> <0.001).</p

Runx1 attenuates PM proliferation.

<p>(A to F) <i>Runx1</i>^<i>L/L</i> and Runx1^<i>f/f</i> PM were purified and their proliferation properties were compared. (A) Average doubling time of <i>Runx1</i>^<i>L/L</i> and Runx1^<i>f/f</i> PM cultures. Values are mean±SD (n = 4, **<i>p</i> <0.001). (B and C) Cell cycle analysis of proliferating PM derived from <i>Runx1</i>^<i>L/L</i> (B), or <i>Runx1</i>^<i>f/f</i> (C) mice. Cell-cycle phases G1, S, and G2/M and the relative size (in %) of PI labeled populations out of total cells are indicated. Results from one of four <i>Runx1</i>^<i>L/L</i> or <i>Runx1</i>^<i>f/f</i> different cultures with similar findings are shown. Green and red arrows indicate increase in % of G1 and decrease in % of S and G2/M of <i>Runx1</i>^<i>f/f</i> vs. <i>Runx1</i>^<i>L/L</i> PM. (D) Histograms summarizing the distribution of cell populations as analyzed in C. Values are mean±SD (n = 4, *<i>p</i> <0.05). (E) IF analysis of proliferating PM from Runx1^<i>L/L</i> and <i>Runx1</i>^<i>f/f</i> mice using anti-Runx1 and MHC Abs. (<i>I-IV</i>) <i>Runx1</i>^<i>L/L</i> and (<i>V-VIII</i>) <i>Runx1</i>^<i>f/f</i> at x200 magnification, scale bars, 50 μm. Results from one of four <i>Runx1</i>^<i>L/L</i> or <i>Runx1</i>^<i>f/f</i> different cultures with similar findings are shown. (F) Average fusion index of proliferating PM. <i>Runx1</i>^<i>L/L</i> and <i>Runx1</i>^<i>f/f</i> proliferating PM cultures were stained with anti-MHC Ab and DAPI and the fractions (in %) of single (blue), double (red) and multinucleated (≥ 3, green) cells are shown. Values are mean±SE (n = 4, *<i>p</i> <0.05). (G to J) Proliferating WT PM were infected with either Ad5CMV-eGFP or Ad-Runx1 and then grown for 24 h in differentiation medium prior to analysis. (G) IF analysis of infected PM using anti- Runx1 and MHC Abs (scale bars, 50 μm and 20 μm for x200 or x630 magnification, respectively). DAPI was used as a nuclear marker. Results from one of four different experiments with similar findings are shown. (H) Histograms showing the average fusion index of differentiating PM analyzed in (G). The fractions (in %) of single (blue), double (red) and multinucleated (≥ 3, green) cells are shown. Values are mean±SE (n = 4, **<i>p</i> <0.001, *<i>p</i> <0.05). (I and J) RT-qPCR analysis of myogenic gene expression in proliferating PM (Pro) before or 72 h post differentiation induction (Diff). PM were grown and infected as indicated in (G), RNA was purified and analyzed by TaqMan assay. Values are mean±SD (n = 3, **<i>p</i> <0.001).</p

Runx1 is required for myoblast proliferation during muscle regeneration.

<p>Schematic diagram summarizing the scenario of Runx1-regulated myoblast proliferation during muscle regeneration: (A) Following myonecrosis of WT muscle, SC are activated, Runx1 is induced and promote proliferation and prevents premature differentiation. Once the critical mass of myoblasts is reached Runx1 is destined to degradation, myoblasts differentiate to produce normal size myofibers. (B) In <i>Runx</i>^<i>f/f</i> muscles, myoblasts lack Runx1 expression and therefore undergo premature differentiation. This leads to insufficient myoblast pool size, resulting in reduced number and size of myofibers and impaired muscle regeneration.</p

Loss of Runx1 in <i>mdx</i> mice decreases muscle mass, muscle strength and lifespan.

<p>(A) Scatter plot showing weight of <i>Runx1</i>^<i>L/L</i> (WT), <i>Runx1</i>^<i>f/f</i>, <i>mdx</i> and <i>mdx/Runx1</i>^<i>f/f</i> mice between 2–9 months of age (average ±SD, n = 9–28, **<i>P</i><0.01). (B) Representative image of <i>mdx</i> and <i>mdx/Runx1</i>^<i>f/f</i> littermates at 7 months of age. (C) Dot plot depicting the average lean weight (as % of total body weight) of <i>mdx</i> and <i>mdx/Runx1</i>^<i>f/f</i> mice between 4–9 months of age. <i>mdx</i> = open circles, and <i>mdx/Runx1</i>^<i>f/f</i> = open squares. Mean lean weight is indicated (n = 6–22, **<i>P</i><0.01, ***<i>P</i><0.0001). (D) Kaplan- Meyer survival curve of <i>Runx1</i>^<i>L/L</i> (n = 50, blue), <i>Runx1</i>^<i>f/f</i> (n = 50, red), <i>mdx</i> (n = 46, green) and <i>mdx/Runx1</i>^<i>f/f</i> (n = 55, purple) (***<i>P</i><0.0001). (E) Diaphragm muscle sections of <i>mdx</i> and <i>mdx/Runx1</i>^<i>f/f</i> mice stained with H&E (top) or Sirius Red (bottom) for collagen (Fibrosis), shown at x100 (left panels) or x400 (right panels). Scale bars, 200μm and 50μm for the X100 and X400 magnifications, respectively. (F) Histogram summarizing treadmill performance of mice between 2–7 months of age. <i>Runx1</i>^<i>L/L</i>, <i>Runx1</i>^<i>f/f</i>, <i>mdx</i> and <i>mdx/Runx1</i>^<i>f/f</i> mice were subjected to an exhaustion protocol (Average ±SD, n = 5–21, **<i>P</i><0.01, Bonferroni corrected post-hoc comparisons). (G) 4 months old <i>mdx</i> and <i>mdx/Runx1</i>^<i>f/f</i> mice were subjected to grip strength measurements. Left and right histograms show absolute and normalized (to body weight) force comparison respectively. Values are mean ±SEM (n = 9–14 *<i>P</i><0.05, **<i>P</i><0.01).</p

Analysis of PM high confidence Runx1-regulated genes.

<p>(A) Schematic representation of the selection procedures used to identify high-confidence Runx1-regulated genes. Each cylinder represents a gene subset, with the gene number given in brackets. I- Runx1-responsive genes were derived from <i>Runx1</i>^<i>L/L</i> vs. <i>Runx1</i>^<i>f/f</i> PM microarray expression data. II- Runx1-regulated genes were derived by cross analysis of the Runx1-responsive genes dataset and Runx1 ChIP-seq data. This gene subset represents Runx1-responsive genes that are also occupied by Runx1. III- RMJ-regulated genes are Runx1-responsive genes that are co-occupied by Runx1, MyoD and c-Jun. IV- High-confidence Runx1-regulated gene subset are RMJ-regulated genes that were also marked as having adjacent active regulatory elements by both anti histone modifications (H3K4me1 & H3K27ac) ChIP-seq and ATAC-seq. (B) Scatter plot of differentially expressed genes in WT vs. <i>Runx1</i>^<i>f/f-</i> PM. Gene expression level (log2 scale) in <i>Runx1</i>^<i>f/f</i> vs. WT PM is plotted. Significant increased or decreased genes are indicated in red or green, respectively. Filled circles indicate Runx1-responsive genes that are known to participate in myoblast proliferation or differentiation. (C) Pie chart depicting Runx1 binding sites distribution in relation to the nearest annotated TSS. Numbers represent % of bound regions. (D) Venn diagram summarizing the overlap between Runx1-ChIP-seq bound genes (ChIP) and Runx1-responsive genes, differentially expressed in <i>Runx1</i>^<i>f/f</i> vs. <i>Runx1</i>^<i>L/L</i>. Runx1-regulated genes are defined as Runx1-bound genes that were also Runx1-responsive. (E) Enriched TF motifs among Runx1-bound regions from PM ChIP-seq data. (F) Overrepresented TF modules in Runx1-bound regions from PM. Runx1 ChIP-seq data was analyzed using the module overrepresentation tool in Genomatix package (RegionMiner). The table presents the most highly enriched modules. (G) Venn diagram showing the overlap of regions bound by Runx1, MyoD and c-Jun and the common fraction of 11629 regions. (H) Cross analysis of all ChIP seq and ATAC-seq common loci with Runx1-responsive gene list (Fig 5B). Prominent genes are presented.</p