DataSheet2_Sumoylation-deficient phosphoglycerate mutase 2 impairs myogenic differentiation.pdf

Phosphoglycerate mutase 2 (PGAM2) is a critical glycolytic enzyme that is highly expressed in skeletal muscle. In humans, naturally occurring mutations in Phosphoglycerate mutase 2 have been etiologically linked to glycogen storage disease X (GSDX). Phosphoglycerate mutase 2 activity is regulated by several posttranslational modifications such as ubiquitination and acetylation. Here, we report that Phosphoglycerate mutase 2 activity is regulated by sumoylation—a covalent conjugation involved in a wide spectrum of cellular events. We found that Phosphoglycerate mutase 2 contains two primary SUMO acceptor sites, lysine (K)49 and K176, and that the mutation of either K to arginine (R) abolished Phosphoglycerate mutase 2 sumoylation. Given that K176 is more highly evolutionarily conserved across paralogs and orthologs than K49 is, we used the CRISPR-mediated homologous recombination technique in myogenic C2C12 cells to generate homozygous K176R knock-in cells (PGAM2K176R/K176R). Compared with wild-type (WT) C2C12 cells, PGAM2K176R/K176R C2C12 cells exhibited impaired myogenic differentiation, as indicated by decreased differentiation and fusion indexes. Furthermore, the results of glycolytic and mitochondrial stress assays with the XF96 Extracellular Flux analyzer revealed a reduced proton efflux rate (PER), glycolytic PER (glycoPER), extracellular acidification rate (ECAR), and oxygen consumption rate (OCR) in PGAM2K176R/K176R C2C12 cells, both at baseline and in response to stress. Impaired mitochondrial function was also observed in PGAM2K176R/K176R P19 cells, a carcinoma cell line. These findings indicate that the PGAM2-K176R mutation impaired glycolysis and mitochondrial function. Gene ontology term analysis of RNA sequencing data further revealed that several downregulated genes in PGAM2K176R/K176R C2C12 cells were associated with muscle differentiation/development/contraction programs. Finally, PGAM2 with either of two naturally occurring missense mutations linked to GSDX, E89A (conversion of glutamic acid 89 to alanine) or R90W (conversion of arginine 90 to tryptophan), exhibited reduced Phosphoglycerate mutase 2 sumoylation. Thus, sumoylation is an important mechanism that mediates Phosphoglycerate mutase 2 activity and is potentially implicated in Phosphoglycerate mutase 2 mutation-linked disease in humans.</p

DataSheet1_Sumoylation-deficient phosphoglycerate mutase 2 impairs myogenic differentiation.docx

Phosphoglycerate mutase 2 (PGAM2) is a critical glycolytic enzyme that is highly expressed in skeletal muscle. In humans, naturally occurring mutations in Phosphoglycerate mutase 2 have been etiologically linked to glycogen storage disease X (GSDX). Phosphoglycerate mutase 2 activity is regulated by several posttranslational modifications such as ubiquitination and acetylation. Here, we report that Phosphoglycerate mutase 2 activity is regulated by sumoylation—a covalent conjugation involved in a wide spectrum of cellular events. We found that Phosphoglycerate mutase 2 contains two primary SUMO acceptor sites, lysine (K)49 and K176, and that the mutation of either K to arginine (R) abolished Phosphoglycerate mutase 2 sumoylation. Given that K176 is more highly evolutionarily conserved across paralogs and orthologs than K49 is, we used the CRISPR-mediated homologous recombination technique in myogenic C2C12 cells to generate homozygous K176R knock-in cells (PGAM2K176R/K176R). Compared with wild-type (WT) C2C12 cells, PGAM2K176R/K176R C2C12 cells exhibited impaired myogenic differentiation, as indicated by decreased differentiation and fusion indexes. Furthermore, the results of glycolytic and mitochondrial stress assays with the XF96 Extracellular Flux analyzer revealed a reduced proton efflux rate (PER), glycolytic PER (glycoPER), extracellular acidification rate (ECAR), and oxygen consumption rate (OCR) in PGAM2K176R/K176R C2C12 cells, both at baseline and in response to stress. Impaired mitochondrial function was also observed in PGAM2K176R/K176R P19 cells, a carcinoma cell line. These findings indicate that the PGAM2-K176R mutation impaired glycolysis and mitochondrial function. Gene ontology term analysis of RNA sequencing data further revealed that several downregulated genes in PGAM2K176R/K176R C2C12 cells were associated with muscle differentiation/development/contraction programs. Finally, PGAM2 with either of two naturally occurring missense mutations linked to GSDX, E89A (conversion of glutamic acid 89 to alanine) or R90W (conversion of arginine 90 to tryptophan), exhibited reduced Phosphoglycerate mutase 2 sumoylation. Thus, sumoylation is an important mechanism that mediates Phosphoglycerate mutase 2 activity and is potentially implicated in Phosphoglycerate mutase 2 mutation-linked disease in humans.</p

The putative SRE motif located in the intragenic enhancer of <i>miR-1-1/133a-2</i> was verified by the chromatin immunoprecipitation (ChIP) assay with sequencing assessment <i>in vivo</i>.

<p>The cell chromatins were extracted from C2C12 cells transfected with either SRF expression vector or siRNA specific for SRF, and the SRF-SRE complexes were pulled down by the anti-SRF antibody. <b>A</b>. The oligonucleotides containing the SRF-binding site were detected (left panel). The PCR product was sequenced and the sequence matched with the enhancer (right panel). <b>B</b>. Quantitative PCR showed that SRF knockdown was achieved. <b>C</b>. Quantitative ChIP assays showed that loss of SRF resulted in decreases in the SRF-SRE complexes, and non-specific PCR primers failed to detect the SRE motif. SRE-P: specific primers for SRE; NS-P: non-specific primers upstream and downstream of the SRE site.</p

<i>Ezh2^fl/fl:Nkx2.5-cre+</i> mice exhibited hypoplastic endocardial cushions.

<p>(<b>A–B′</b>) H&E staining revealed a decrease in the cell population in the EC region of <i>Ezh2</i> null hearts compared with that of control (arrows) at E10.5. Also, <i>Ezh2</i> mutant heart displayed defective trabeculation (arrowheads). <b>A′</b> and <b>B′</b> are the amplification of the boxed region of <b>A</b> and <b>B</b>, respectively. <b>A & B</b>, 20× magnification; <b>A′ & B′</b>, 40× magnification. (<b>C–D″ & G</b>) Double-immunofluorescence with anti-Ki67 antibody (red) and anti-cardiac Troponin T antibody (cTnT, green) revealed a reduced number of Ki67⁺ cells in both the compact myocardium and the AV cushion of <i>Ezh2-cKO</i> hearts at E10.5, compared with that of littermate controls. <b>C′</b> and <b>D′</b> are the amplification of the bigger boxed region of <b>C</b> and <b>D</b>; <b>C″</b> and <b>D″</b> are the amplification of the smaller boxed region of <b>C</b> and <b>D</b>. <b>C & D</b>, 20× magnification; <b>C′–D″</b>, 40× magnification. Scale bar, 100 µm. <b>G</b> shows that the number of Ki67⁺ cells was significantly decreased in <i>Ezh2-cKO</i> hearts relative to the control. The number of Ki67⁺ cells was counted from 10 randomly selected fields in the ECs. Data are presented as mean ± SD. *, p<0.05. n = 3 per group. (<b>E–F′ & H</b>) Double immunofluorescence of TUNEL staining (red) and anti-cTnT (green) revealed an increased number of TUNEL⁺ cells in both the AV cushion of <i>Ezh2-cKO</i> hearts at E10.5, compared with that of littermate controls. Nuclei were counterstained with DAPI, as shown in blue. <b>E′</b> and <b>F′</b> are the amplification of the boxed region of <b>E</b> and <b>F</b> respectively. <b>E & F</b>, 20× magnification; <b>E′& F′</b>, 40× magnification. Scale bar: 100 µm. <b>H</b> shows that the number of TUNEL⁺ cells was significantly increased in <i>Ezh2-cKO</i> hearts relative to the control. The number of TUNEL⁺ cells was counted from 10 randomly selected fields. Data are presented as mean ± SD. *, p<0.05. n = 3 per group. OFT, outflow tract; A, common atrium; V, common ventricle.</p

Analysis of <i>miR-1-1/133a-2</i> intragenic enhancer region containing the SRE motif by luciferase assay.

<p>Empty bar represents reporter vector activity directed by the enhancer with the wild-type SRE motif. Solid bar represents reporter vector activity directed by the enhancer with the mutant SRE motif. SRF increased the luciferase reporter gene activity, and the addition of NKx2.5 facilitated SRF functional activity. Mutation of the SRF binding site resulted in a reduction of the reporter gene activity. The data in each group represents the averages of three experiments with a total of six measurements. Statistical significance was determined by unpaired, two-tailed student’s <i>t</i> test. Data are means ± S.E.</p

Dysregulation of cardiac genes in K51R-Tg mice.

<p>RT-qPCR revealed altered transcription of some cardiac genes examined such as BNP and myosin light chain (MLC)-2v in K51R-Tg hearts (n = 6) compared with those of control hearts (n = 4) at P50. RT-qPCR was performed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0020803#s2" target="_blank">Materials and Methods</a>. Unpaired student's <i>t</i> test was used for statistically significant analysis.</p

Characterization of cardiac phenotypes in <i>Ezh2-cKO</i> mice.

<p>(<b>A–D</b>) H&E staining of the coronal sections of the control mouse hearts at P1 showed normal structure of heart and great vessels. (<b>E–J</b>) H&E staining of the coronal sections of the <i>Ezh2-cKO</i> (<i>Ezh2^fl/fl:Nkx2.5-cre+</i>) hearts at P1 showed a variety of cardiovascular defects: <b>E</b>, double outlet right ventricle (DORV) and perimembranous ventricular septal defect (VSD, arrow); <b>F</b> & <b>G</b>, two serial sections showing the transition of persistent truncus arteriosus (PTA). <b>F′</b> and <b>G′</b> are the amplification of the boxed region in <b>F</b> and <b>G</b>, respectively. Arrows in <b>G</b> and <b>G′</b> indicate the septal opening; <b>H</b>, muscular VSD (arrow); <b>I</b>, atrial septal defect (ASD, arrow); <b>J</b>, atrioventricular canal defects (AVCD, upper arrow) and membranous VSD (lower arrow). (<b>K</b>) The penetrance of cardiac defects in the mutant hearts was 100%, with variable incidence of different CHDs, as indicated. RV: right ventricle; LV, left ventricle; RA, right atrium; LA, left atrium; Ao, Aorta; PT, pulmonary trunk. n = 10. Scale bar: 100 µm.</p

The assessment of <i>miR-1-1/133a-2</i> intragenic enhancer using the transgenic mice expressing the reporter gene, <i>LacZ</i>, and driven by the intragenic enhancer.

<p>β-Galactosidase staining (blue) was performed on embryos (<b>A</b>-<b>D</b>) and the embryos were sectioned for detailed analysis (<b>E</b>-<b>L</b>). The enhancer containing wild-type SRE directed the <i>LacZ</i> gene expression in heart and somite areas (<b>A</b>, <b>E</b> and <b>I</b>). Close-up of β-Galactosidase staining showed the cardiac-specific expression of lacZ. (<b>B</b>, <b>F</b> and <b>J</b>) The enhancer containing the mutant SRE directed the <i>LacZ</i> gene expression in somite areas but not in the heart (<b>C</b>, <b>G</b> and <b>K</b>). Close-up of β-Galactosidase staining displayed the abolishment of lacZ expression in heart (<b>D</b>, <b>H</b> and <b>L</b>). Embryos were at E10.5 (<b>A</b>-<b>H</b>) and E11.5 (<b>I</b>-<b>L</b>). HR: heart. lv: left ventricle. rv: right ventricle, la: left atrium. ra: right atrium, m: somite myotomes. White arrows indicate somite myotomes.</p

Cardiac functional analysis in the progenies obtained from cross-breeding between K51R-Tg and Nkx2.5^+/− mouse lines.

<p>Echocardiography was performed on animals of 4 months age as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0020803#s2" target="_blank">Materials and Methods</a>. Note that although there was no statistically significant difference reached in all parameters shown between compound K51R-Tg/Nkx2.5^+/− group and the other three groups due to small size of the sample examined, there was a trend of increase in diastolic and systolic volumes, and a trend of decrease in cardiac function (%EF and %FS) in the compound K51R-Tg/Nkx2.5^+/− mice compared with the other group animals. EF, ejection fraction; FS, fractional shortening.</p

Conditional knockout of <i>Ezh2</i> gene in murine hearts caused perinatal lethality.

<p>(<b>A</b>) Schematic illustration showing the annealing sites of primer pairs of mouse <i>Ezh2</i> and <i>Ezh1</i> (a homolog of <i>Ezh2</i>). (<b>B</b>) Ezh2 expression in the murine embryonic hearts. Semi-quantitative RT-PCR with two different numbers of amplification cycles (30 and 35) was performed on RNA samples purified from WT hearts of E9.5, E10.5, and E11.5. 1 µl or 0.5 µl cDNA from reverse transcription reaction was used for detection of Ezh2 or GAPDH (as a control), respectively. (<b>C</b>) qRT-PCR analysis revealed ∼70% decrease in <i>Ezh2</i> mRNA of <i>Ezh2</i>-cKO heart (n = 3, p<0.001), but <i>Ezh1</i> mRNA was not affected, compared with that of the control littermate hearts. Data are presented as mean ± SD of 3 replicate samples for each strain/gene analyzed. (<b>D</b>) The level of H3K27Me3 was reduced in the E18.5 <i>Ezh2-cKO</i> hearts, compared to that of control. The total Histone H3 and GAPDH both serve as controls. The asterisks indicate H3K27Me3. NS, non-specific. (<b>E</b>) The neonatal mortality rate (%) and (<b>F</b>) the P1 frequency of four different genotypes resulting from the crossbreeding between <i>Ezh2^fl/+:Nkx2.5-cre+</i> and <i>Ezh2^fl/fl</i> mice. Data in panel E & F represent the average of 12 litters comprising a total of 99 animals. (<b>G</b>) The number and recovery rate (in parenthesis) of Ezh2^fl/+, Ezh2^fl/+:Nkx2.5-cre+, Ezh2^fl/fl, and <i>Ezh2^fl/fl:Nkx2.5-cre+</i> embryos collected at various embryonic stages are shown. Offspring from intercrossing between genotypes of <i>Ezh2^fl/fl</i> and <i>Ezh2^fl/+:Nkx2.5-cre+</i> mice. Note that the expected Mendelian rate of embryo recovery for each of these four genotypes is 25%. (<b>H</b>) Embryonic demise of Ezh2 mutant mice at a later gestational stage. The representative images of E16.5 WT and dead Ezh2 mutant embryos are shown.</p