Systems biology surveillance decrypts pathological transcriptome remodeling
© Faustino et al. 2015
Received: 28 August 2014
Accepted: 5 June 2015
Published: 17 July 2015
Pathological cardiac development is precipitated by dysregulation of calreticulin, an endoplasmic reticulum (ER)-resident calcium binding chaperone and critical contributor to cardiogenesis and embryonic viability. However, pleiotropic phenotype derangements induced by calreticulin deficiency challenge the identification of specific downstream transcriptome elements that direct proper cardiac formation. Here, differential transcriptome navigation was used to diagnose high priority calreticulin domain-specific gene expression changes and decrypt complex cardiac-specific molecular responses elicited by discrete functional regions of calreticulin.
Wild type (WT), calreticulin-deficient (CALR−/−), and calreticulin truncation variant (CALR−/−-NP and CALR−/−-PC) pluripotent stem cells were used to investigate molecular remodeling underlying a model of cardiopathology. Bioinformatic deconvolution of isolated transcriptomes was performed to identify predominant expression trends, gene ontology prioritizations, and molecular network features characteristic of discrete cell types.
Stem cell lines with wild type (WT), calreticulin-deficient (CALR−/−) genomes, as well as calreticulin truncation variants exclusively expressing either the chaperoning (CALR−/−-NP) or the calcium binding (CALR−/−-PC) domain exhibited characteristic molecular signatures determined by unsupervised agglomerative clustering. Kohonen mapping of RNA expression changes identified transcriptome dynamics that segregated into 12 discrete gene expression meta-profiles which were enriched for regulation of Eukaryotic Initiation Factor 2 (EIF2) signaling. Focused examination of domain-specific gene ontology remodeling revealed a general enrichment of Cardiovascular Development in the truncation variants, with unique prioritization of “Cardiovascular Disease” exclusive to the cohort of down regulated genes of the PC truncation variant. Molecular cartography of genes that comprised this cardiopathological category revealed uncharacterized and novel gene relationships, with identification of Pitx2 as a critical hub within the topology of a CALR−/− compromised network.
Diagnostic surveillance, through an algorithm that integrates pluripotent stem cell transcriptomes with advanced high throughput assays and computational bioinformatics, revealed collective gene expression network changes that underlie differential phenotype development. Stem cell transcriptomes provide a deep collective molecular index that reflects ad hoc robustness of the pluripotent gene network. Remodeling events such as monogenic lesions provide a background by which high priority candidate disease effectors and regulators can be identified, demonstrated here by a molecular profiling algorithm that decrypts pluripotent wild type versus disrupted genomes.
A functional myocardium is the culmination of intricate dynamic signaling orchestrated by regulated processes [1, 2], and disruptions of cardiogenesis that lead to congenital cardiac defects occur in as high as 8 % of prenatal cases [3, 4]. Discrete molecular elements that give rise to inborn cardiac errors are numerous and precipitate developmental cardiopathology through multiple mechanisms including inhibition of cardiomyocyte proliferation and differentiation , abrogation of syncytial/electrical function , and disruption of nascent myocardial anatomy [7, 8]. Precise models of cardiac development that report phenotype derangement specific for a targeted gene have enhanced resolution of signaling axes that drive clinical pathology, yet these platforms are of limited use for anticipatory diagnosis of cardiopathology in advance of symptomatic presentation. Present methods to detect congenital heart defects are limited to time frames proximal to phenotype onset, which critically restricts opportunities for curative or palliative interventions.
Pre-manifestation in silico extrapolation of cardiac defects is feasible with the use of large volume, multidimensional biodata, as high throughput techniques generate profound datasets which could serve as the foundation for incisive dissection of complex molecular patterns . Indeed, continuous development of bioinformatic tools expands the range of quantifiable properties of “omic” data, and provides the basis for advanced systems biology algorithms of deconvolution . Resolution of the intricate molecular framework underlying phenotype dynamics using this interdisciplinary methodology has been successful [9, 11], yet parsing diverse and complex datasets remains an extant challenge.
Embryonic stem (ES) cells harbor a pluripotent genome with an engaged pre-differentiation molecular landscape [12, 13], and the naive transcriptome of an ES cell provides a unique source of material for exploration of the primordial gene network that supports normal development [14–17]. Disruptions within this principal transcriptome may ultimately drive pathological onset and disease progression, thus elucidation of primary transcriptome dynamics under normal and diseased conditions is required for advanced refinement of disease prediction [9, 18]. Case in point, ES cells that lack a functional calreticulin gene, i.e. calr −/− , abrogates calreticulin (CALR) expression, and provides a robust model for interrogation of a discrete mechanism of pathological cardiogenesis [19, 20]. Systems biology dissection of prioritized gene ontology remodeling in calr −/− ES cell lines revealed the underlying molecular network that drove ultrastructural, functional, and anatomical derangements in cardiac phenotype . However, CALR is a Ca2+-handling chaperone [21, 22], and specific contributions of its particular functional domains, i.e. calcium binding versus protein folding, to final phenotype has remained uncharacterized.
Dissection of the potential contributions of each CALR domain to global phenotype generation would identify differential gene ontology remodeling specified by distinct regions within a multifunctional protein. This would refine and increase resolution of the primordial CALR-driven molecular atlas preceding organogenesis. Here, using an algorithm of bioinformatic deconvolution to unravel the intricate transcriptome networks harbored by calreticulin truncation variants, the contributions of the protein chaperoning domain (NP) versus the calcium handling (PC) region were investigated. Truncation variants, generated by transfection of CALR−/− cell lines with constructs that exclusively expressed the protein chaperoning (CALR−/−-NP) or the calcium handling (CALR−/−-PC) domain, were used to determine differential Gene Ontology enrichments coded for by each CALR functional region. To build upon previous work, dysfunctional transcriptome recalibration preceding cardiogenesis was prioritized. The classification of Cardiovascular Development was enriched for by both truncation variants, though the down regulated cohort of genes in the transcriptome of the CALR−/−-PC truncation variant exhibited specific prioritization of genes that promote Cardiovascular Disease. To determine functional interconnectivity of genes within this pathological category, molecular cartography was used to reveal a local scale-free network with significant prioritization of genes previously uncharacterized as part of CALR-governed cardiogenesis. Advanced systems biology-derived algorithms, combined with the molecular dynamics of pluripotent ES cells, thus facilitates decryption of covert gene network features and functional enrichment remodeling actuated by discrete regions within CALR that drive phenotype manifestation.
Dataset generation and global transcriptome profiling
Domain-specific transcriptome dissection and gene ontology enrichment
To investigate functional themes recruited by these specialized regions of calr, focused examination of NP and PC truncation variant cell lines versus wild type counterparts was performed. The contribution of the NP and PC variants, which exclusively express the chaperoning or calcium binding domains, respectively, may differentially contribute unique gene ontology composition to overall molecular signature. Pairwise comparison of truncation mutants to wild type cells was performed by volcano plot analysis to streamline gene profiles to transcripts changing by more than 2-fold when compared to WT, and these lists were analyzed by Ingenuity to extract significant functional annotations and gene ontology enrichments.
Categorical enrichment of network functions, disease classifications and/or dysregulated biological themes in the NP truncation variant
Hematological Disease, Cell Death and Survival, Cellular Development
Hematological Disease, Organismal Injury and Abnormalities, Cellular Compromise
Cellular Development, Hematological System Development and Function, Hematopoiesis
Cellular Compromise, DNA Replication, Recombination and Repair, Cell Death and Survival
RNA Post-Transcriptional Modification, Molecular Transport, RNA Trafficking
Cellular Assembly and Organization, Cellular Function and Maintenance, Endocrine System Development and Function
Cellular Assembly and Organization, Molecular Transport, RNA Trafficking
Organismal Development, DNA Replication, Recombination and Repair, Reproductive System Development and Function
Lipid Metabolism, Small Molecule Biochemistry, Embryonic Development
Cellular Movement, Immune Cell Trafficking, Cellular Growth and Proliferation
Top Diseases and Bio Functions
Organismal Injury and Abnormalities
Reproductive System Disease
Categorical enrichment of network functions, disease classifications and/or dysregulated biological themes in the PC truncation variant
Cellular Assembly and Organization, Molecular Transport, RNA Trafficking
Cellular Assembly and Organization, Cellular Function and Maintenance, Hematological System Development and Function
RNA Post-Transcriptional Modification, Embryonic Development, Organismal Development
Cell Signaling, Cellular Assembly and Organization, DNA Replication, Recombination, and Repair
Cell Cycle, Cellular Development, Cell Morphology
Lipid Metabolism, Molecular Transport, Small Molecule Biochemistry
Cellular Function and Maintenance, Cellular Movement, Nervous System Development and Function
Cell Death and Survival, Hematological System Development and Function, Immune Cell Trafficking
Nervous System Development and Function, Cell Cycle, Digestive System Development and Function
Cell Morphology, Nervous System Development and Function, Cell-to-cell Signaling and Interaction
Top Diseases and Bio Functions
Organismal Injury and Abnormalities
Dermatological Diseases and Conditions
Network cartography, topological deconvolution, and signature conservation
As an enriched cardiopathologic functional theme was uniquely and discretely identified in down regulated genes of the PC truncation line, the contribution of down regulated transcripts within the original calr-deficient phenotype was selected for investigation. Venn diagram intersection of down regulated PC genes with all down regulated transcripts in calr −/− cells revealed common decreased expression of Ccnd2, Mest, Mid1, Ndnf, Nosip, Pitx2, Prmt3, and Vim in both transcriptomes (Fig. 5b). Network cartography of these genes revealed a streamlined 31 node sub-network (Fig. 5c). Topological analysis identified features typical of a scale-free biological network , where Pitx2 possessed the highest degree, or number of connections to other nodes in the network (Fig. 5c and d).
Transcriptome meta-analysis uncovers precise molecular signatures , and DAVID was employed to investigate conservation of gene ontology categories identified by Ingenuity Pathways Analysis. Heatmaps of transcripts down regulated in PC versus wild type revealed a functional cluster that contained Pitx2 (Fig. 5e and Additional file 1), corroborating its priority as an identified network hub.
Anticipatory diagnosis of disease prior to symptomatic presentation is complicated by gene expression diversity that underlies pathology. To address this, stem cell transcriptomes were utilized as ad hoc multidimensional indices to interrogate molecular statuses of calreticulin-disrupted cell types, which identified novel molecular dynamics that enforced cardiopathological phenotype. Identification of prioritized gene expression network hubs and altered prioritization of global molecular functions reveals intricate systems biology phenomena that comprise pathological genome remodeling, critical for advanced understanding of disease etiology.
Snapshot overviews of mRNA expression changes that underlie cellular phenotypes define transcript expression differences, gene ontology re-organization, and gene network remodeling events that can be mapped in silico to extract novel molecular information . Here, a broad bioinformatics overview identified multiple riboproteins within Eukaryotic Initiation Factor 2 (EIF2) Signaling as priority-regulated in gene lesion variant transcriptomes. This supports reported prevalence of altered translational machinery in CALR-disrupted transcriptomes and phenotypes . CALR deletion leads to impaired system wide quality control of newly synthesized glycoproteins, as well as global accumulation of conformationally compromised substrates and general abrogation of calcium-dependent protein folding processes [25–27]. Accumulation of unfolded or misfolded proteins is salvaged in part by the unfolded protein response (UPR) of the endoplasmic reticulum , which enacts translational re-calibration in the event of stress stimuli. As part of this response, EIF pathways are targeted to facilitate restoration of protein homeostasis through prevention of misfolded protein accumulation that occurs under conditions of physiological and/or environmental duress, while concomitantly stimulating cells that contain conformationally defective proteins to initiate apoptosis [29–32]. Thus, global prioritization of EIF2 signaling components, in addition to enrichment within the down regulated transcript cohort of the PC variant, may reflect a cellular strategy of pre-emptive transcriptome recruitment to compensate for catastrophic system stress.
Global gene ontology remodeling precipitated by calr deficiency may reflect net effects of discrete contributions made by specific functional CALR regions. To assess this, CALR-truncated variants that exclusively expressed either the protein chaperoning or calcium binding domain were examined for transcriptome changes. Overall, multiple physiological categories were impacted, in line with previous observations reported for diverse gene ontology enrichments in the calr −/− condition . Broad cellular processes were targeted, with the most enriched disease category for both truncation variants being Cancer. While curation bias is responsible for “Cancer” prioritization in multiple databases , functional distribution of CALR outside the endoplasmic reticulum, coupled with roles in cellular adhesion and migration, support the prioritized role of oncological development and progression in these CALR truncation mutants [33, 34].
Targeted focus on cardiac related processes for both CALR truncation variants revealed significant impacts on Cardiac Development and Cardiac Physiology themes, with Cardiovascular Disease uniquely enriched within down regulated genes of the PC variant, which lacks the chaperoning function of full length CALR. Jeopardized expression and/or function of genes within this CALR-truncated transcriptome in a cardiac context identified prioritized molecules that may underlie cardiac pathogenesis precipitated by CALR derangement. The present study identified Pitx2 as a prioritized gene network hub, i.e. specific promotion of Pitx2 in Cardiovascular Disease development was observed under conditions of CALR truncation/deficiency. This result suggests a critical role for Pitx2 within the CALR-regulated cardiogenic network. In the CALR-compromised sub-network, a high betweenness score indicates load and importance of Pitx2 to the sub-network, while high radiality demonstrates functional relevance and control that Pitx2 exerts on neighboring nodes [24, 35].
Specific promotion of Pitx2 in cardiac disease development under conditions of CALR deficiency suggests its critical involvement within the CALR-regulated cardiogenic network. Pitx2 has been reported to precipitate arrhythmogenesis upon dysregulated expression . Previous studies have identified sinus bradycardia, complete heart block and sudden death associated with dysregulated CALR expression [37, 38]. Here, CALR-dependent remodeling of gene networks that contain Pitx2, and gene ontology prioritization of “Cardiac Arrhythmia”, provide bioinformatic evidence that suggest the arrhythmogenic phenotype generated by CALR dysregulation could be mediated by a CALR-PITX2 signaling pathway.
Further data for the critical cardiogenic role of Pitx2 have been reported in models that express hypomorphic Pitx2, which caused disorganization of the atrial septum, increased deposition of extracellular matrix in the atrial chambers, and elongation of the atrioventricular canal . Importantly, Pitx2 determines right-left heart patterning [40, 41], thus complete loss of Pitx2 function causes embryonic lethality with associated catastrophic cardiovascular defects . Interestingly, non-cardiac Pitx2 knockout phenotypes, such as omphalocele, are also present in calreticulin-deficient experimental animal models [42–44]. Together, these observations further support an integrated role for Pitx2 in CALR-defective phenotypes.
Pleiotropic responses to single gene lesions recruit a diversity of transcriptome elements that dictate developmental progress, and such dynamic expression changes creates a complex network that make identification of inconspicuous gene relationships challenging. Here, application of a systems biology-derived algorithm of bioinformatic dissection identified discrete gene ontology remodeling effects ascribed to either chaperone or calcium binding regions of CALR. Furthermore, expression changes of a Pitx2-inclusive cardiogenic sub-transcriptome were revealed downstream of calr deletion, providing novel bioinformatic support for cardiogenic disruption subsequent to calr derangement. This paradigm of systems deconvolution thus demonstrates that bioinformatic navigation of an intricate gene expression landscape identifies specific sub-transcriptome remodeling which enhances molecular resolution to expose novel elements of global pathways underlying development and disease processes.
Cell culture, transcriptome isolation and microarray profiling
Independent biological replicates of WT (n = 2), calr −/− (n = 5), and calr truncation mutants NP (n = 3) and PC (n = 3) were cultured on 0.1 % gelatin-coated 10 cm dishes in 7.5 % FBS in DMEM supplemented with L-glutamine, NEAA, penicillin/streptomycin, LIF, and BME. Media was changed regularly every 24 or 48 h. Cells reaching ~80 % confluency were passaged 1:10 to maintain proliferative undifferentiated state of all cell lines, as previously performed . Total RNA isolation from embryonic stem cells was performed using the Micro-to-Midi Total RNA Purification System (Invitrogen, Carlsbad, CA) for analysis of transcriptome dynamics. Double stranded complementary cDNA and labeled complementary cRNA were obtained from isolated total RNA, with the latter hybridized against the Mouse 430 2.0 GeneChip (Affymetrix). Arrays were scanned using an argon-ion laser, and visualized using MAS 5.0 Affymetrix software to assess quality of hybridization. Data were deposited to the Gene Expression Omnibus (GEO) repository under accession number GSE13805, with relevant updates.
Transcript expression analysis
Gene expression data (GEO ID: GSE13805) for calreticulin-deficient embryonic stem cells versus wild type counterparts were analyzed as described previously . To assess the transcript profile changes in calreticulin-truncated mutants, samples were analyzed using Genespring GX to visualize expression dynamics and extract significant differences in each of the truncation mutants compared to wild type control embryonic stem cells. Metaprofile segregation was performed via SOM clustering using Euclidean distance to identify 12 prevalent expression trends among the 4 transcriptomes sampled. Metaprofiles represented by a trendline were plotted in a hexagonal U-Matrix that provides information on each cluster as well as relationship between clusters.
Equation 1. Summation notation for Pearson coefficient used to establish molecular fingerprint of calr −/− stem cells.
In Equation 1, (A) and (B) are respective sample means for genes A i and B i for sample (i) out of the total number of samples (n), with standard deviation terms for A i and B i used as denominator. Condition clustering was performed to determine sample similarity using Euclidean distance as a measure of sample “nearness” , and plotted as gene heatmaps. Gene expression changes were profiled using volcano plot analysis, which calculates significance (p-value) and fold-change for each quality-filtered transcript, and visualizes these changes in a plot of –log (p-value) versus log (fold change).
Functional category extraction and network cartography
To extract the gene expression network framing the transcriptome signature in WT versus calr mutant embryonic stem cells, quality filtered transcripts were analyzed using Ingenuity Pathways Analysis to identify prioritized signaling pathways and molecular themes enriched by transcriptome dynamics. Curated molecular interactions among differentially expressed genes were mapped for CALR-variant cell lines versus WT. Global functional categories associated with quality filtered genes were identified in the Ingenuity Pathways Knowledge Base and ranked with a right-tailed Fisher’s exact test. P-values for enriched developmental categories were transformed into their –log values and used to generate significance plots, to visualize prioritization in CALR-variant transcriptomes. Cardiac-specific transcriptome meta-analysis was investigated using DAVID (http://david.abcc.ncifcrf.gov/) for independent corroboration of gene prioritizations and correlated Gene Ontology enrichments identified in Ingenuity Pathways Analysis.
Topographical properties of selected networks were examined using the NetworkAnalyzer tool in Cytoscape v3.1.0 to identify degree, degree distribution, betweenness centrality, and radiality. Degree – the number of connections (called edges) within a network. Degree distribution – plot of nodes versus degree that identifies which nodes have the greatest number of connections within a network. Betweenness centrality – metric that reports which node has the greatest influence on informational flow within a network, i.e. – how connected a node is. Radiality – metric which measures the probability of a node being central to the regulation of its network neighbors and indicates functional relevancy.
This work was supported by grants from the National Institutes of Health (HL85208), the Canadian Institutes of Health Research (MOP53050), and the Ted Nash Long Life Foundation.
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