Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

Transcription factors MYOCD, SRF, Mesp1 and SMARCD3 significantly enhance the cardio-inducing effect of GATA4, TBX5, and MEF2C during direct cellular reprogramming

ABSTRACT: Transient over-expression of defined combinations of master regulator genes can effectively induce cellular reprogramming: the acquisition of an alternative predicted phenotype from a differentiated cell lineage. This can be of particular importance in cardiac regenerative medicine wherein the heart lacks the capacity to heal itself, but simultaneously contains a large pool of fibroblasts. In this study we determined the cardio-inducing capacity of ten transcription factors to actuate cellular reprogramming of mouse embryonic fibroblasts into cardiomyocyte-like cells. Over-expression of transcription factors MYOCD and SRF alone or in conjunction with Mesp1 and SMARCD3 significantly enhanced the basal but necessary cardio-inducing effect of the previously reported GATA4, TBX5, and MEF2C. In particular, combinations of five or seven transcription factors significantly enhanced the activation of cardiac reporter vectors, and induced an upregulation of cardiac-specific genes. Global gene expression analysis also demonstrated a significantly greater cardio-inducing effect when the transcription factors MYOCD and SRF were used. Detection of cross-striated cells was highly dependent on the cell culture conditions and was enhanced by the addition of valproic acid and JAK inhibitor. Although we detected Ca2+ transient oscillations in the reprogrammed cells, we did not detect significant changes in resting membrane potential or spontaneously contracting cells. This study further elucidates the cardio-inducing effect of the transcriptional networks involved in cardiac cellular reprogramming, contributing to the ongoing rational design of a robust protocol required for cardiac regenerative therapies. Mouse embryonic fibroblasts were transduced lentiviruses allowing the inducible overexpression of three unique sets of transcription factors and negative control. A total of four experimental groups which included three biological replicates in each.

ORGANISM(S): Mus musculus

SUBMITTER: Nicolas Christoforou

PROVIDER: E-GEOD-44401 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

ACCESS DATA

Similar Datasets

Project description:Background: Cardiac transcription factors are master regulators during heart development. Recently, some were shown to transdifferentiate noncardiac mesoderm cells and cardiac fibroblasts into cardiomyocytes. However, the individual roles of each transcription factors in activating cardiac gene program have not been elucidated. We examined cardiac-specific and genome-wide gene expression in fibroblasts induced with cardiac transcription factors Nkx2.5 (N), Tbx5 (T), Gata4 (G), Myocardin (M) alone or different combinations. Methodology/Principal Findings: We applied different combinations of human Nkx2.5 (N), Tbx5 (T), Gata4 (G) and Myocardin (M) lentiviruses into 10T1/2 fibroblasts. Immunostaining and quantitative reverse transcription polymerase chain reaction (qRT-PCR) showed that N, T, G or M alone did not induce expression of cardiac marker genes M-NM-1-myosin heavy chain (M-NM-1MHC) and cardiac troponin T (cTnT). Only T+M and T+G+M combinations induced M-NM-1MHC and cTnT expression. Microarray-based gene ontology analysis revealed that T alone inhibited most genes involved in cardiac-related processes and activated genes involved in Wnt receptor signaling pathway and in aberrant processes. M alone inhibited genes involved in Wnt receptor signaling pathway and activated genes involved in cardiac-related processes and in aberrant processes. G alone inhibited genes involved in ectoderm development. T+G+M combination was the most effective activator of genes associated with cardiac-related processes including muscle cell differentiation, sarcomere, striated muscle contraction, regulation of heart contraction, and glucose metabolism and fatty acid oxidation (two significant forms of cardiomyocyte energy metabolism). And unlike T, M, G alone or T+M, T+G+M did not activate genes associated with aberrant processes. Conclusions: Tbx5, Gata4 and Myocardin play different roles in activating cardiac gene program and in avoiding aberrant gene program activation. The combination of T+G+M activated cardiac gene program and avoided aberrant gene program activation. Two weeks after doxycline induction, total RNA was isolated from 10T1/2-tTA cells infected with different combinations of Tbx5, Gata4, and Myocardin lentiviruses. Biological triplicated.

Project description:The mTOR (mammalian Target of Rapamycin) pathway is constitutively activated in Diffuse Large B-Cell Lymphoma (DLBCL). mTOR inhibition has been shown to have clinical activity in patients with DLBCL, although overall response rates remain low. We therefore evaluated differences in the transcriptome between DLBCL cell lines with differential sensitivity to the mTOR inhibitor Rapamycin, to (A) identify gene-expression patterns(GEP) capable of identifying sensitivity to Rapamycin, (B) understand the underlying mechanisms of resistance to Rapamycin in DLBCL and (C) identify bioactive molecules likely to synergize with mTOR inhibitors. Using Affymetrix HuGene ST 1.0 microarrays, we were able to identify a gene expression signature capable of accurately predicting sensitivity and resistance to Rapamycin in DLBCL cell lines. Pathway analysis identified the serine/threonine kinase Akt as central to the differentially-expressed gene network. Connectivity mapping of our datasets identified compounds targeting the AKT pathway with a high likelihood of reversing the GEP associated with resistance to Rapamycin. Specifically, we evaluated the HIV protease inhibitor (PI) Nelfinavir, which is known to have anti-cancer and Akt-inhibitory properties, as well as the small molecule Akt inhibitor MK-2206, for their potential to synergize with to Rapamycin in DLBCL. Nelfinavir and MK-2206 caused profound inhibition of cell viability in combination with Rapamycin in DLBCL cell lines. Low nanomolar concentrations of Rapamycin inhibited phosphorylation of Akt and also downstream targets of activated mTOR when used in combination with these Akt inhibitors. These findings have the potential to significantly improve patient selection for mTOR inhibitor therapy, and to improve rates and depths of response. More broadly, they support the use of global RNA expression and connectivity mapping to improve patient selection and identify synergistic drug combinations for cancer therapy. DLBCL cell lines were tested for Rapamycin sensitivity and classified as "sensitive" or "resistant." Genome-wide analysis of all cell lines were performed using the Affymetrix HuGene ST 1.0 Array Platform. Genes with differential expression between sensitive and resistant cell lines were analyzed using Statistical Analysis of Microarrays (SAM) software, and a signature of genes determnined. This signature was found to accurately predict sensitivity or resistance of other DLBCL cell lines, and to identify the protein kinase Akt as central to resistance.

Project description:Chinese medicine is a complex system guided by traditional Chinese medicine (TCM) theories, which has proven to be especially effective in treating chronic and complex diseases. However, the underlying modes of action (MOA) are not always systematically investigated. Herein, a systematic study was designed to elucidate the multi-compound, multi-target and multi-pathway MOA of a Chinese medicine ,QSYQ, on myocardial infarction. Male Sprague Dawley rat model of myocardial infarction were administered QSYQ intragastrically for 7 days while the control group was not treated. The differentially expressed genes (DEGs) were identified from myocardial infarction rat model treated with QSYQ, followed by constructing a cardiovascular disease (CVD)-related multilevel compound-target-pathway network connecting main compounds to those DEGs supported by literature evidences and the pathways that are functionally enriched. Three conditions were compared with three replicates each: (1) sham, i.e. without left anterior descending coronary artery (LAD) ligation; (2) model, with LAD ligation; (3) QSYQ, with LAD ligation and treated with QSYQ intragastrically for 7 days, the dosage was 105.6 mg/kg once a day. Rats were sacrificed after 7 days of i.g. administration under 10% chloral hydrate anesthesia (300mg/kg). Three tissue samples on the border between infarct and non-infarct area were dissected from left ventricles of each group. The tissue samples were stored at -80M-bM-^DM-^C refrigerator. Total RNA was extracted using TRIZol Reagent (Invitrogen) and purified using RNeasy Mini kit (QIAGEN), following manufacturersM-bM-^@M-^Y protocols. RNA quality was evaluated using an Agilent 2100 Bioanalyzer and electrophoresis in 2% (w/v) agarose gels. Only RNA with RNA integrity numbers (RINs) greater than 7.0 and 28SrRNA/18S rRNA ratio greater than 0.7 was used for microarray analyses. Whole genome microarray analysis was performed using Affymetrix rat Genome 230 2.0 chips.

Dataset Information

Transcription factors MYOCD, SRF, Mesp1 and SMARCD3 significantly enhance the cardio-inducing effect of GATA4, TBX5, and MEF2C during direct cellular reprogramming

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure