Knockdown of Death-Associated Protein Expression Induces Global Transcriptome Changes in Proliferating and Differentiating Muscle Satellite Cells

Abstract

Death-associated protein (DAP) undergoes substantial changes in expression during turkey skeletal muscle development, decreasing from the 18 day embryonic stage to 1 day posthatch, and again from 1 day posthatch to 16 weeks of age. These changes suggest that DAP plays an important role at critical stages of the developmental process. The objective of this study was to elucidate the role of DAP in muscle development by examining the effect of reduced DAP expression on global gene expression in proliferating and differentiating turkey pectoralis major muscle satellite cells. Small interfering RNA was used to knock down expression of DAP and the transcriptome was subsequently profiled using a turkey skeletal muscle long oligonucleotide microarray. Microarray data were corroborated using quantitative real-time PCR. In proliferating cells, 458 loci, resulting in 378 uniquely annotated genes, showed differential expression (false discovery rate, FDR < 0.05). Pathway analysis highlighted altered eukaryotic translational initiation factors (eIFs) signaling, protein ubiquitination, sirtuin signaling, and mechanistic target of rapamycin (mTOR) signaling as the primary pathways affected in the knockdown proliferating cells. The findings underpinned the potential DAP involvement in cell proliferation of turkey satellite cells through the coordination between protein synthesis and cell cycle. In differentiating cells, 270 loci, accounting for 189 unique genes, showed differential expression (FDR < 0.05). Decreased expression of genes encoding various myofibrillar proteins and proteins involved in sarcoplasmic reticulum calcium flux suggests that DAP may affect regulation of calcium homeostasis and cytoskeleton signaling. This study provides the first evidence that reduced expression of DAP significantly alters the transcriptome profile of pectoralis major muscle satellite cells, thereby reducing proliferation and differentiation.

Keywords: death-associated protein; global gene expression; microarray; muscle; muscle development; satellite cells; turkey.

FIGURE 1

Timeline for plating, transfection, proliferation, and differentiation of satellite cells. Following transfection or mock transfection, cells underwent proliferation for 72 h and were frozen for subsequent analysis. Alternatively, the medium was changed at 72 h to induce differentiation. Following 48 h of differentiation, cells were frozen for subsequent analysis. Experimental details are provided by Velleman et al. (2012).

FIGURE 2

Distribution of differentially expressed genes in the turkey satellite cells. For each comparison, the number of significant genes (FDR p-value < 0.05) shared or unique to each treatment are indicated in the Venn diagram. Circle size is proportional to the number of genes and direction of expression change (↑ or ↓) is given for each group.

FIGURE 3

Microarray and qPCR data show similar trends of gene expression in proliferation experiments. Fold change is defined as the change in gene expression in the DAP knockdown samples relative to control samples. Bars below the origin indicate lower expression (down-regulation) of the gene in the DAP knockdown samples; bars above the origin indicate higher expression (up-regulation). *P < 0.05, **P < 0.01, ^†FDR < 0.05, and ^††FDR < 0.01. AKT1, Akt1; ANXA6, Annexin A6; BHMT, Betaine-homosysteine S-methyltransferase; DAP, death-associated protein; ENO1, enolase 1; FTH1, Ferritin heavy polypeptide 1.

FIGURE 4

Microarray and qPCR data show similar trends of expression differences in differentiation experiments. Fold change is described as change in gene expression in the DAP knockdown samples relative to controls. Bars below the origin indicate lower expression (down-regulation) of the gene in the DAP knockdown samples; bars above the origin indicate higher expression (up-regulation). *P < 0.05, **P < 0.01, ***P < 0.001, ^†FDR < 0.05, ^††FDR < 0.01, and ^†††FDR < 0.001. ACTA1, actin alpha 1 skeletal; ATP2A2, sarcoplasmic/endoplasmic reticulum ATPase 2 cardiac; CACNG1, voltage dependent calcium channel gamma 1; DAP, death-associated protein; FTH1, ferritin heavy polypeptide 1; PCOLCE2, procollagen C-endopeptidase enhancer; TNNC2, Troponin C type 2 fast skeletal; TNNT2, Troponin T type 2 cardiac; TPM1, Tropomyosin 1.

FIGURE 5

Knockdown of DAP alters gene expression of the mTOR signaling pathway during proliferation of turkey satellite cells. Intermolecular connections between genes are as defined by IPA analysis. *FDR < 0.05. 4EBP, 4E binding protein; ATG13, autophagy related 13; eIF3, eukaryotic translation initiation factor 3; eIF4A, eukaryotic translation initiation factor 4A; eIF4B, eukaryotic translation initiation factor 4B; eIF4G, eukaryotic translation initiation factor 4G; FKBP1, FK506 binding protein 1A; GBL, G protein beta subunit like; HIF1α = hypoxia inducible factor 1 alpha subunit; mTOR, mechanistic target of rapamycin; mTORC1, mechanistic target of rapamycin complex 1; mTORC2, mechanistic target of rapamycin complex 2; p70S6K, p70S6 kinase; PI3K, phosphoinositide 3-kinase; PKC, protein kinase C; PP2A, protein phosphatase 2; catalytic subunit, alpha isozyme; PRAS40, proline-rich Akt substrate 40; RAC, Ras-related C3 botulinum toxin substrate; Rheb, Ras homolog enriched in brain; Rho, Rhodopsin; RPS6, ribosomal protein S6; TSC1, tuberous sclerosis protein 1; TSC2, tuberous sclerosis protein 2; VEGF, vascular endothelial growth factor. Red highlighting indicates up-regulated genes; green highlighting indicates down-regulated genes. This pathway was generated through the use of Ingenuitay Pathway Analysis (QIAGEN, Inc. https://digitalinsights.qiagen.com/products-overview/discovery-insights-portfolio/analysis-and-visualization/qiagen-ipa/).

FIGURE 6

DAP knockdown alters expression of calcium signaling genes during differentiation of turkey satellite cells. Intermolecular connections between genes are as defined by IPA analysis. *FDR < 0.05, **FDR < 0.01, and ***FDR < 0.001. ASPH, aspartate beta-hydroxylase; CASQ, calsequestrin; DHPR, dihydropyridine receptor; nACHR, nicotinic cholinergic receptor gamma subunit; RYR, ryanodine receptor; RRYR, RYR interaction network; SERCA, sarcoplasmic/endoplasmic reticulum Ca²⁺-ATPase; TRDN, Triadin. Symbols are defined as in Figure 4. Red highlighting indicates up-regulated genes; green highlighting indicates down-regulated genes. This pathway was generated through the use of Ingenuitay Pathway Analysis (QIAGEN, Inc. https://digitalinsights.qiagen.com/products- overview/discovery-insights-portfolio/analysis-and-visualization/qiagen-ipa/).