The Histone Variant MacroH2A1 Regulates Key Genes for Myogenic Cell Fusion in a Splice-Isoform Dependent Manner

Abstract

MacroH2A histone variants have functions in differentiation, somatic cell reprogramming and cancer. However, at present, it is not clear how macroH2As affect gene regulation to exert these functions. We have parted from the initial observation that loss of total macroH2A1 led to a change in the morphology of murine myotubes differentiated ex vivo. The fusion of myoblasts to myotubes is a key process in embryonic myogenesis and highly relevant for muscle regeneration after acute or chronic injury. We have focused on this physiological process, to investigate the functions of the two splice isoforms of macroH2A1. Individual perturbation of the two isoforms in myotubes forming in vitro from myogenic C2C12 cells showed an opposing phenotype, with macroH2A1.1 enhancing, and macroH2A1.2 reducing, fusion. Differential regulation of a subset of fusion-related genes encoding components of the extracellular matrix and cell surface receptors for adhesion correlated with these phenotypes. We describe, for the first time, splice isoform-specific phenotypes for the histone variant macroH2A1 in a physiologic process and provide evidence for a novel underlying molecular mechanism of gene regulation.

Keywords: ADP ribose; PARP1; cell fusion; gene regulation; histone variants; macroH2A; myogenic differentiation; myotubes.

Figure 1

Myoblasts lacking macroH2A1 fail to form proper myotubes. (A) Immunoblot analysis of macroH2A1 (mH2A1), mH2A2 and Histone H3 of quadriceps from wild-type (WT) and knockout (KO) mice. (B) Immunostaining of differentiated primary mH2A1 WT and KO myotubes with anti-embryonic myosin heavy chain (eMHC). (C) Total nuclei, percent of differentiated eMHC positive cells and percent of differentiated eMHC positive cells with at least two nuclei after four days of differentiation. Data points are from sets of four to six photos obtained from three independent biological replicates, evaluating 100 and 200 myotubes and the same surface area per replicate. Lines indicate the median of replicates, * p < 0.05, Student’s t-test. (D) Violin graph of the nuclei distribution per primary mH2A1 WT and KO myoblasts. The data shown are the log2 of the median of 100–200 myotubes from three biological replicates, * p < 0.05, Wilcoxon test. (E) Bright field pictures of anti-eMHC immunostainings of primary macroH2A1 KO myoblasts transduced with Flag-mH2A1.1 or control vector after two days of differentiation. Arrows indicate large myotubes.

Figure 2

MacroH2A1 isoforms oppositely regulate myotube fusion. (A) A schematic representation of the used RNA interference protocol, and the resulting protein levels in C2C12 cells are shown. Immunoblotting was performed by using indicated antibodies. Differentiation was induced by changing growth medium (GM) to differentiation medium (DM), and samples were collected after four days (D4). (B) Differences in C2C12 myotube morphology are visible in phase contrast and by anti-eMHC immunofluorescence at D4. Nuclear DNA was counter-stained by DAPI. White arrows indicate particularly large myotubes. (C) The total nuclei number and the percentage of differentiated eMHC-positive cells were assessed at D4. Same areas with 600 myotubes were analyzed; data points are from four areas obtained from two independent biological replicates, * p < 0.05; Student’s t-test). (D) Violin graph of the nuclei distribution per myotubes. Data in D are the median of 600 myotubes from two biological replicates, * p < 0.05, Wilcoxon test. (E) Percent of myotube distribution between three groups: myotubes containing between 2 and 14, between 15 and 49, and more than 50 nuclei. Data points are the median of 600 myotubes, from four areas obtained from two independent biological replicates, * p < 0.05, Student’s t-test.

Figure 3

MacroH2A1 (mH2A1) isoforms affect genes related to extracellular matrix and adhesion in an opposing manner. (A) Volcano plot of -log10 of the adjusted p-value versus the log2 fold-change (FC). Dashed lines demarcate the chosen cutoffs of <0,01 and 0.8 for p-value and log2FC, respectively. Significant deregulated genes are highlighted in green (down) or orange (up). The analyzed data were obtained from three independent biological replicates. (B) Gene ontology analysis of downregulated genes with si mH2A1.1 versus upregulated with si mH2A1.2 (p < 0.05). The analyzed data were obtained from three independent biological replicates. (C) Venn diagram of deregulated genes identified in (A). (D) Scatterplot of deregulated genes (98) identified in (A) and shared in both comparisons, si mH2A1.1 versus control and si mH2A1.2. Five fusion genes of interest are highlighted. (E) Validation of selected genes by RT-qPCR during differentiation at days 0, 2 and 4 (left to right). Data is the mean of four independent experiments + SD; * p < 0.05; Student’s t-test.

Figure 4

Silencing of PARP1 does not affect cell fusion or expression of fusion genes. (A) Successful PARP1 silencing by siRNA is shown by immunoblotting of C2C12 cells after four days of differentiation. (B) Myotubes were analyzed by eMHC immunofluorescence in si Ctrl versus si PARP1 conditions at day 4. Nuclear DNA was stained by DAPI. (C) Percentage of myotube distribution divided into three groups, according to the range of nuclei per myotube. Data in C are the median of 600 myotubes from four distinct areas from three biological replicates, * p < 0.05, Student’s t-test. (D) Relative mRNA levels of five fusion genes (identified in Figure 3) in si Ctrl and si PARP1 conditions. Data in D are the mean of four independent experiments + SD; * p < 0.05; Student’s t-test.

Figure 5

MacroH2A1.1 (mH2A1.1) occupies fusion-related genes in myotubes. (A) Heatmaps and summary plots using normalized read coverages from mH2A1.1 ChIP-seq from myotubes after four days of differentiation. Signals over the body of all genes +/- 8 kb are shown. Heatmaps were divided into three categories, according to normalized read counts: not expressed, lowly expressed and highly expressed genes. Fusion genes of interest belong to the category of highly expressed genes. (B) Pathway analysis of the 46 genes of interest by Paintomics3 analysis. (C) Snapshots from the UCSC genome for macroH2A1.1, total macroH2A1 and the enhancer mark histone H3K4me2 in proliferating myoblasts in growth medium (GM) or myotubes in differentiation medium (DM) and input are shown. The exact coordinates are indicated in Table A1. (D) Levels of macroH2A1.1 enrichment by ChIP-qPCR on Fn1, Itga11 and Col1a1 in proliferative (GM) and differentiated (DM, four days) cells. As a background control, we used IgG. Data are mean of n = 3 independent experiments, normalized to the input signal, + SD; * p < 0.05; Student’s t-test. (E) Levels of macroH2A1.1 enrichment by ChIP-qPCR on three loci of Col1a1 genes, in si ctrl, si macroH2A1.1 and si macroH2A1.2 conditions at day 4. As a background control, we used IgG. Data are the mean of n = 4 independent experiments, normalized to the input signal, + SD; * p < 0.05; Student’s t-test.