Functional characterization of AtWRKY72 using Arabidopsis T-DNA insertion lines showed that this gene is important for basal defense to root-knot nematode (RKN) and Hyaloperonospora parasitica arabidopsis (Hpa), but not several tested R gene-mediated defenses.To profile transcriptional reprogramming associated with AtWRKY72-dependent basal defense we used Affymetrix ATH1 GeneChips representing ~24,000 Arabidopsis genes. Three independent biological replicates were performed with Col-0, wrky72-1 and wrky72-2 plants at 96 hpt with HpaNoco2 or mock treatment. Using a false discovery rate of less than 0.05 we identified for each of these three lines genes that showed significant transcriptional changes in response to HpaNoco2 compared to the mock-treated controls. Identification of downstream targets of WRKY72 in Arabidopsis by this microarray suggests that WRKY72 uses a unique signaling pathway that involves AP2/ERF TFs independent of the ethylene signaling pathway.
WRKY72-type transcription factors contribute to basal immunity in tomato and Arabidopsis as well as gene-for-gene resistance mediated by the tomato R gene Mi-1.
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Biologic characterization of SB-559457 (SB), a non-peptidyl hydrazone class of thrombopoietin receptor (Mpl) agonist, revealed toxicity towards human leukemia cells. Anti-proliferative effects followed by significant, non-apoptotic, cell death within 72 hours occurred in 24/26 AML, 0/6 ALL, and 3/6 CML patient samples exposed to SB, but not recombinant human thrombopoietin (rhTpo), in liquid suspension culture. Further investigation revealed increased phosphorylation of p70S6/S6 kinases in SB, but not in rhTpo, treated cells. Expression profiling of cells exposed to SB vs rhTpo revealed statistically significant, ~2-fold changes in GAPDH and REDD1 gene expression, confirmed by QRT-PCR. These genes, induced in energy or hypoxia stressed cells, have been implicated in cell death pathways, and may provide important clues to the mechanism of SB induced, leukemic cell death. These results suggest that nonpeptidyl, hydrazone class Mpl agonists may be clinically useful anti-leukemic agents by virtue of their combined thrombopoietic and anti-leukemic effects.
A prototype nonpeptidyl, hydrazone class, thrombopoietin receptor agonist, SB-559457, is toxic to primary human myeloid leukemia cells.
Specimen part, Disease, TreatmentView Samples
Multiple signaling pathways, structural proteins and transcription factors are involved in regulation of endothelial barrier function. The Forkhead protein FOXF1 is a key transcriptional regulator of lung embryonic development, and we use a conditional knockout approach to examine the role of FOXF1 in adult lung homeostasis and lung injury and repair. Tamoxifen-regulated deletion of both Foxf1 alleles in endothelial cells of adult mice (Pdgfb-iCreER/Foxf1 caused lung inflammation and edema, leading to respiratory insuffency and uniform mortality. Deletion of a single foxf1 allele was sufficient to increase susceptibility of heterozygous mice to acute lung injury. FOXF1 abundance was decreased in pulmonary endothelial cells of human patients with acute lung injury. Gene expression analysis of pulmonary endothelial cells of FOXF1 deletion indicated reduced expression for genes critical for maintance and regulation of adherens junctions. FOXF1 knockdown in vitro and in vivo disrupted adherens junctions, increased lung endothelial permeability, and the abundance of mRNA and protein for sphingosine 1 phosphate receptor 1 (S1PR1), a key regulator of endothelial barrier function. Chromatin immunoprecipitation and luciferase reporter assay demonstrated that FOXF1 directly bound to and induced the tanscriptional activity of the S1pr1 promoter. Pharmacological administratiion of S1P to injured pdgfb-iCreER/Foxf1 mice restored endothelial barrier function, decreased lung edema and improved survival. Thus, FOXF1 promotes normal lung homeostasis and lung repair, at least in part, by enhancing endothelial barrier function through transcriptional activation of the S1P/S1PR1/ signaling pathway. Overall design: RNA was isolated and pooled from the lungs of multiple mice with either the Foxf1 floxed alleles alone or Pdgfb-iCreER Foxf1 floxed mice.
FOXF1 maintains endothelial barrier function and prevents edema after lung injury.
Specimen part, SubjectView Samples
Myotonic dystrophy type 1 (DM1) is a dominantly inherited disease that affects multiple organ systems. Cardiac dysfunction is the second leading cause of death in DM1. We quantified gene expression in heart tissue from a heart-specific DM1 mouse model (EpA960/MCM) which inducibly expresses human DMPK exon 15 containing 960 CUG expanded repeats and that reproduced Celf1 up regulation. To assess if, in addition to splicing and miRNA defects, CUGexp RNA also perturbed the steady state mRNA levels of genes, we carried out a microarray study on wildtype E14, adult, MCM controls and DM1 mouse hearts. As anticipated we noted a large number of genes to be developmentally regulated in wildtype hearts, however, within 72h of induction of CUGexp RNA there appeared to be a coordinate adult-to-embryonic shift in steady state levels of many genes.
The Mef2 transcription network is disrupted in myotonic dystrophy heart tissue, dramatically altering miRNA and mRNA expression.
Specimen part, TimeView Samples
In this data set we include expression data from human CD4+ T cells isolated on day 0, 6, 11 and 24 follow anti-CD3/anti-CD28 magnetic bead stimulation and chimeric antigen receptor transduction.
Identification of chimeric antigen receptors that mediate constitutive or inducible proliferation of T cells.
Specimen partView Samples
The Mesio-Temporal Lobe Epilepsy (MTLE) syndrome is the most common form of intractable epilepsies. It is characterized by the recurrence of focal seizures occurring in mesio-temporal limbic structures and is often associated with hippocampal sclerosis and drug resistance.
Glial responses during epileptogenesis in Mus musculus point to potential therapeutic targets.
Sex, Specimen partView Samples
Hepatic fibrosis is the common end stage to a variety of chronic liver injuries and is characterized by an excessive deposition of extracellular matrix (ECM), which disrupts the liver architecture and impairs liver function. The fibrous lesions are produced by myofibroblasts, which differentiate from hepatic stellate cells (HSC). The myofibroblasts transcriptional networks remain poorly characterized. Previous studies have shown that the Forkhead box F1 (FOXF1) transcription factor is expressed in HSCs and stimulates their activation during acute liver injury; however, the role of FOXF1 in the progression of hepatic fibrosis is unknown. In the present study, we generated aSMACreER;Foxf1fl/fl mice to conditionally inactivate Foxf1 in myofibroblasts during carbon tetrachloride-mediated liver fibrosis. Foxf1 deletion increased collagen depositions and disrupted liver architecture. Timp2 expression was significantly increased in Foxf1-deficient mice while MMP9 activity was reduced. RNA sequencing of purified liver myofibroblasts demonstrated that FOXF1 inhibits expression of pro-fibrotic genes, Col1a2, Col5a2, and Mmp2 in fibrotic livers and binds to active repressors located in promotors and introns of these genes. Overexpression of FOXF1 inhibits Col1a2, Col5a2, and MMP2 in primary murine HSCs in vitro. Altogether, FOXF1 prevents aberrant ECM depositions during hepatic fibrosis by repressing pro-fibrotic gene transcription in myofibroblasts and HSCs. Overall design: RNAseq on isolated hepatic stromal cells from Foxf1 fl/fl and aSMACreER;Foxf1 fl/fl mice after 5 weeks of carbon tetrachloride-induced liver injury.
The Forkhead box F1 transcription factor inhibits collagen deposition and accumulation of myofibroblasts during liver fibrosis.
Specimen part, Cell line, SubjectView Samples
Alternative cleavage and polyadenylation (APA) results in mRNA isoforms containing different 3' untranslated regions (3'UTRs) and/or coding sequences. How core cleavage and polyadenylation (C/P) factors regulate APA is not well understood. Using siRNA knockdown coupled with deep sequencing, we found that several C/P factors can play significant roles in 3'UTR-APA. Whereas Pcf11 and Fip1 enhance usage of proximal poly(A) sites (pAs), CFI-25/68, PABPN1, and PABPC1 promote usage of distal pAs. Strong cis element biases were found for pAs regulated by CFI or Fip1, and the distance between pAs plays an important role in APA regulation. In addition, intronic pAs are substantially regulated by splicing factors, with U1 mostly influencing C/P events in 5' introns and U2 impacting those in efficiently spliced introns. Furthermore, PABPN1 regulates expression of transcripts with pAs near the transcription start site, a property possibly related to its role in RNA degradation. Finally, we found that groups of APA events regulated by C/P factors are also modulated in cell differentiation and development with distinct trends. Together, our results indicate that the abundance of different C/P factors and splicing factors plays diverse roles in APA, and is relevant to APA regulation in biological conditions. Overall design: knockdown experiments of 23 C/P factors, 3 splicing factors and U1D in mouse C2C12 myoblast cells
Systematic profiling of poly(A)+ transcripts modulated by core 3' end processing and splicing factors reveals regulatory rules of alternative cleavage and polyadenylation.
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Transcription factor FoxM1 is expressed in proliferating cells, and its expression is critical for cell proliferation in embryos and tumors. FoxM1 regulates a multi-gene transcriptional network for cell cycle regulation.
Forkhead box M1 transcriptional factor is required for smooth muscle cells during embryonic development of blood vessels and esophagus.
Specimen partView Samples
RNAseq analysis of human bone marrow derived stromal cells (MSCs) treated for 24 hours with or wihout 10ng/ml Fibroblast Growth Factor 2 (FGF2) MSCs were derived from 4 different healthy donors. Cells were expanded to passage 3-4. Then cells were treated with FGF-2. 24 hours later, total RNA was extracted (total 8 samples). Overall design: RNA was submitted to BGI Americas for RNAseq. Here, QC was performed using Agilent 2100. All samples had a RIN above 8.0. For preparation for library, mRNA was enriched by using the oligo (dT) magnetic beads. mRNA was enriched by using the oligo (dT) magnetic beads. mRNA was fragmented into short fragments (about 200bp) using a fragmentation buffer. Then the first strand of cDNA was synthesized by random hexamer-primer using the mRNA fragments as templates. Buffer, dNTSPs, RNase H and DNA polymerase I were added to synthesize the second strand. The double strand cDNA was purified with QiaQuick PCR extraction kit and washed with EB buffer for end repair and base A addition. Finally, sequencing adapters were ligated to the fragments. The fragments are purified by Agarose gel electrophoresis and enriched by PCR amplification. The library products are ready for sequencing analysis via 2 sE50 lanes in Illumina HiSeqâ„¢ 2000.
FGF2 Induces Migration of Human Bone Marrow Stromal Cells by Increasing Core Fucosylations on N-Glycans of Integrins.
Specimen part, Treatment, SubjectView Samples