Molecular Characterization of Pediatric Restrictive Cardiomyopathy from Integrative Genomics

Pediatric restrictive cardiomyopathy (RCM) is a genetically heterogeneous heart disease with limited therapeutic options. RCM cases are largely idiopathic; however, even within families with a known genetic cause for cardiomyopathy, there is striking variability in disease severity. Although accumulating evidence implicates both gene expression and alternative splicing in development of dilated cardiomyopathy (DCM), there have been no detailed molecular characterizations of underlying pathways dysregulated in RCM. RNA-Seq on a cohort of pediatric RCM patients compared to other forms of adult cardiomyopathy and controls identified transcriptional differences highly common to the cardiomyopathies, as well as those unique to RCM. Transcripts selectively induced in RCM include many known and novel G-protein coupled receptors linked to calcium handling and contractile regulation. In-depth comparisons of alternative splicing revealed splicing events shared among cardiomyopathy subtypes, as well as those linked solely to RCM. Genes identified with altered alternative splicing implicate RBM20, a DCM splicing factor, as a potential mediator of alternative splicing in RCM. We present the first comprehensive report on molecular pathways dysregulated in pediatric RCM including unique/shared pathways identified compared to other cardiomyopathy subtypes and demonstrate that disruption of alternative splicing patterns in pediatric RCM occurs in the inverse direction as DCM

Gi-Coupled GPCR Signaling Controls the Formation and Organization of Human Pluripotent Colonies

BACKGROUND:Reprogramming adult human somatic cells to create human induced pluripotent stem (hiPS) cell colonies involves a dramatic morphological and organizational transition. These colonies are morphologically indistinguishable from those of pluripotent human embryonic stem (hES) cells. G protein-coupled receptors (GPCRs) are required in diverse developmental processes, but their role in pluripotent colony morphology and organization is unknown. We tested the hypothesis that G(i)-coupled GPCR signaling contributes to the characteristic morphology and organization of human pluripotent colonies. METHODOLOGY/PRINCIPAL FINDINGS:Specific and irreversible inhibition of G(i)-coupled GPCR signaling by pertussis toxin markedly altered pluripotent colony morphology. Wild-type hES and hiPS cells formed monolayer colonies, but colonies treated with pertussis toxin retracted inward, adopting a dense, multi-layered conformation. The treated colonies were unable to reform after a scratch wound insult, whereas control colonies healed completely within 48 h. In contrast, activation of an alternative GPCR pathway, G(s)-coupled signaling, with cholera toxin did not affect colony morphology or the healing response. Pertussis toxin did not alter the proliferation, apoptosis or pluripotency of pluripotent stem cells. CONCLUSIONS/SIGNIFICANCE:Experiments with pertussis toxin suggest that G(i) signaling plays a critical role in the morphology and organization of pluripotent colonies. These results may be explained by a G(i)-mediated density-sensing mechanism that propels the cells radially outward. GPCRs are a promising target for modulating the formation and organization of hiPS and hES cell colonies and may be important for understanding somatic cell reprogramming and for engineering pluripotent stem cells for therapeutic applications

MAPPFinder: using Gene Ontology and GenMAPP to create a global gene-expression profile from microarray data

MAPPFinder is a tool that creates a global gene-expression profile across all areas of biology by integrating the annotations of the Gene Ontology (GO) Project with the free software package GenMAPP . The results are displayed in a searchable browser, allowing the user to rapidly identify GO terms with over-represented numbers of gene-expression changes. Clicking on GO terms generates GenMAPP graphical files where gene relationships can be explored, annotated, and files can be freely exchanged

Identifying genetic networks underlying myometrial transition to labor

BACKGROUND: Early transition to labor remains a major cause of infant mortality, yet the causes are largely unknown. Although several marker genes have been identified, little is known about the underlying global gene expression patterns and pathways that orchestrate these striking changes. RESULTS: We performed a detailed time-course study of over 9,000 genes in mouse myometrium at defined physiological states: non-pregnant, mid-gestation, late gestation, and postpartum. This dataset allowed us to identify distinct patterns of gene expression that correspond to phases of myometrial 'quiescence', 'term activation', and 'postpartum involution'. Using recently developed functional mapping tools (HOPACH (hierarchical ordered partitioning and collapsing hybrid) and GenMAPP 2.0), we have identified new potential transcriptional regulatory gene networks mediating the transition from quiescence to term activation. CONCLUSIONS: These results implicate the myometrium as an essential regulator of endocrine hormone (cortisol and progesterone synthesis) and signaling pathways (cyclic AMP and cyclic GMP stimulation) that direct quiescence via the transcripitional upregulation of both novel and previously associated regulators. With term activation, we observe the upregulation of cytoskeletal remodeling mediators (intermediate filaments), cell junctions, transcriptional regulators, and the coordinate downregulation of negative control checkpoints of smooth muscle contractile signaling. This analysis provides new evidence of multiple parallel mechanisms of uterine contractile regulation and presents new putative targets for regulating myometrial transformation and contraction

Intragraft antiviral-specific gene expression as a distinctive transcriptional signature for studies in polyomavirus-associated nephropathy

Background: polyomavirus nephropathy (PVAN) is a common cause of kidney allograft dysfunction and loss. To identify PVAN-specific gene expression and underlying molecular mechanisms, we analyzed kidney biopsies with and without PVAN. Methods: the study included 168 posttransplant renal allograft biopsies (T cell-mediated rejection [TCMR] = 26, PVAN = 10, normal functioning graft = 73, and interstitial fibrosis/tubular atrophy = 59) from 168 unique kidney allograft recipients. We performed gene expression assays and bioinformatics analysis to identify a set of PVAN-specific genes. Validity and relevance of a subset of these genes are validated by quantitative polymerase chain reaction and immunohistochemistry. Results: unsupervised hierarchical clustering analysis of all the biopsies revealed high similarity between PVAN and TCMR gene expression. Increased statistical stringency identified 158 and 252 unique PVAN and TCMR injury-specific gene transcripts respectively. Although TCMR-specific genes were overwhelmingly involved in immune response costimulation and TCR signaling, PVAN-specific genes were mainly related to DNA replication process, RNA polymerase assembly, and pathogen recognition receptors. A principal component analysis (PCA) using these genes further confirmed the most optimal separation between the 3 different clinical phenotypes. Validation of 4 PVAN-specific genes (RPS15, complement factor D, lactotransferrin, and nitric oxide synthase interacting protein) by quantitative polymerase chain reaction and confirmation by immunohistochemistry of 2 PVAN-specific proteins with antiviral function (lactotransferrin and IFN-inducible transmembrane 1) was done. Conclusions: in conclusion, even though PVAN and TCMR kidney allografts share great similarities on gene perturbation, PVAN-specific genes were identified with well-known antiviral properties that provide tools for discerning PVAN and AR as well as attractive targets for rational drug design

miR-196b target screen reveals mechanisms maintaining leukemia stemness with therapeutic potential.

We have shown that antagomiR inhibition of miRNA miR-21 and miR-196b activity is sufficient to ablate MLL-AF9 leukemia stem cells (LSC) in vivo. Here, we used an shRNA screening approach to mimic miRNA activity on experimentally verified miR-196b targets to identify functionally important and therapeutically relevant pathways downstream of oncogenic miRNA in MLL-r AML. We found Cdkn1b (p27Kip1) is a direct miR-196b target whose repression enhanced an embryonic stem cell–like signature associated with decreased leukemia latency and increased numbers of leukemia stem cells in vivo. Conversely, elevation of p27Kip1 significantly reduced MLL-r leukemia self-renewal, promoted monocytic differentiation of leukemic blasts, and induced cell death. Antagonism of miR-196b activity or pharmacologic inhibition of the Cks1-Skp2–containing SCF E3-ubiquitin ligase complex increased p27Kip1 and inhibited human AML growth. This work illustrates that understanding oncogenic miRNA target pathways can identify actionable targets in leukemia

AltAnalyze and DomainGraph: analyzing and visualizing exon expression data

Alternative splicing is an important mechanism for increasing protein diversity. However, its functional effects are largely unknown. Here, we present our new software workflow composed of the open-source application AltAnalyze and the Cytoscape plugin DomainGraph. Both programs provide an intuitive and comprehensive end-to-end solution for the analysis and visualization of alternative splicing data from Affymetrix Exon and Gene Arrays at the level of proteins, domains, microRNA binding sites, molecular interactions and pathways. Our software tools include easy-to-use graphical user interfaces, rigorous statistical methods (FIRMA, MiDAS and DABG filtering) and do not require prior knowledge of exon array analysis or programming. They provide new methods for automatic interpretation and visualization of the effects of alternative exon inclusion on protein domain composition and microRNA binding sites. These data can be visualized together with affected pathways and gene or protein interaction networks, allowing a straightforward identification of potential biological effects due to alternative splicing at different levels of granularity. Our programs are available at http://www.altanalyze.org and http://www.domaingraph.de. These websites also include extensive documentation, tutorials and sample data

Time- and exercise-dependent gene regulation in human skeletal muscle

BACKGROUND: Skeletal muscle remodeling is a critical component of an organism's response to environmental changes. Exercise causes structural changes in muscle and can induce phase shifts in circadian rhythms, fluctuations in physiology and behavior with a period of around 24 hours that are maintained by a core clock mechanism. Both exercise-induced remodeling and circadian rhythms rely on the transcriptional regulation of key genes. RESULTS: We used DNA microarrays to determine the effects of resistance exercise (RE) on gene regulation in biopsy samples of human quadriceps muscle obtained 6 and 18 hours after an acute bout of isotonic exercise with one leg. We also profiled diurnal gene regulation at the same time points (2000 and 0800 hours) in the non-exercised leg. Comparison of our results with published circadian gene profiles in mice identified 44 putative genes that were regulated in a circadian fashion. We then used quantitative PCR to validate the circadian expression of selected gene orthologs in mouse skeletal muscle. CONCLUSIONS: The coordinated regulation of the circadian clock genes Cry1, Per2, and Bmal1 6 hours after RE and diurnal genes 18 hours after RE in the exercised leg suggest that RE may directly modulate circadian rhythms in human skeletal muscle