This study characterizes the response of primary human endothelial cells (human umbilical vein endothelial cells, HUVECs) to the relative shear stress changes that occur during the initiation of arteriogenesis at the entrance regions to a collateral artery network. HUVECs were preconditioned to a baseline level of unidirectional shear of 15 dynes/cm2 for 24 hours. After 24 hours preconditioning, HUVECs were subjected to an arteriogenic stimulus that mimics the shear stress changes observed in the opposing entrance regions into a collateral artery network. The arteriogenic stimulus consisted of a 100% step wise increase in shear stress magnitude to a unidirectional 30 dynes/cm2 in either the same or opposite direction of the preconditioned shear stress. This simulates either the feeding entrance to the collateral artery circuit or the region that drains into the vasculature downstream of an obstruction in a major artery, respectively. In vivo analysis of collateral growth in the mouse hindlimb showed enhanced outward remodeling in the re-entrant (direction reversing) region that reconnects to the downstream arterial tree, suggesting reversal of shear stress direction as a key enhancer of arteriogenesis. Transcriptional profiling using microarray techniques identified that the reversal of shear stress direction, but not an increase in shear stress alone, yielded a broad-based enhancement of the mechanotransduction pathways necessary for the induction of arteriogenesis.
Mechanisms of Amplified Arteriogenesis in Collateral Artery Segments Exposed to Reversed Flow Direction.
Specimen partView Samples
NOTCH proteins regulate signaling pathways involved in cellular differentiation, proliferation and death. Overactive Notch signaling as been observed in numerous cancers and has been extensively studied in the context of T-cell acute lymphoblastic leukemia (T-ALL) where more than 50% of pateints harbour mutant NOTCH1. Small molecule modulators of these proteins would be important for understanding the role of NOTCH proteins in malignant and normal biological processes.
Direct inhibition of the NOTCH transcription factor complex.
Specimen part, Cell lineView Samples
Although transcriptional programs associated with T-cell specification and commitment have been described, the functional hierarchy and the roles of key regulators in structuring/ orchestrating these programs remain unclear. Activation of Notch signaling in uncommitted precursors by the thymic stroma initiates the T-cell differentiation program. One regulator first induced in these precursors is the DNA binding protein Tcf-1, a T-cell specific mediator of Wnt signaling. Yet the specific contribution of Tcf-1 to early T-cell development and the signals inducing it in these cells remain unclear. Here we assign functional significance to Tcf-1 as a gatekeeper of T-cell fate. We show that Tcf-1 is directly activated by Notch signals. Tcf-1 is required at the earliest phase of Tcell determination for progression beyond the early thymic progenitor (ETP) stage. The global expression profile of Tcf-1 deficient progenitors indicates that basic processes of DNA metabolism are downregulated in its absence and the blocked T-cell progenitors become abortive and die by apoptosis. Our data thus add an important functional relationship to the roadmap of T-cell development.
T-cell factor 1 is a gatekeeper for T-cell specification in response to Notch signaling.
Specimen partView Samples
Notch is normally activated by cleavage and nuclear translocation of its intracellular domain (ICN1), which turns on downstream target genes. Human T cell acute lymphoblastic leukemia (T-ALL), an aggressive immature T cell malignancy, is associated with Notch 1 gain-of-function mutations in more than 50% of the cases. Efforts to date to identify direct Notch1 targets have been confounded by the lack of a method to turn Notch1 on in a controlled fashion in T-ALL cells that are poised to respond to Notch signals. Of note, because Notch signaling activates transcriptional repressors that feedback to dampen the expression of many target genes (a process referred to as incoherent logic), it is likely that many direct targets are missed in Notch off analyses, which are further complicated by an inability to identify direct targets in a clear-cut fashion. We have overcome this limitation by developing a GSI washout method that results in the rapid translocation of activated Notch1 to the nucleus. We intend to use this method to study the assembly and loading of transcriptional complexes onto downstream targets, the kinetics of target activation. To date, our efforts have been devoted to comparing the gene expression signature of Notch-on and Notch-off in the human T-ALL cell line CUTLL. In addition to previously identified Notch1 target genes, we have also identified a series of novel genes upregulated by GSI washout in the presence of cycloheximide, suggesting that they are likely to be direct targets.
Genome-wide analysis reveals conserved and divergent features of Notch1/RBPJ binding in human and murine T-lymphoblastic leukemia cells.
Cell lineView Samples
Tristetraprolin is a vertebrate CCCH tandem zinc finger protein that can bind to and destabilize certain mRNAs containing AU-rich element binding sites. zfs1 is the single gene in the fission yeast, Schizosaccharomyces pombe, that encodes a protein containing the critical features of the tristetraprolin zinc finger domain. zfs1 has been linked to pheromone signal transduction control and to the coordination of mitosis, but no biological function has been ascribed to the zfs1 protein. Through a functional genomics approach we compared transcript levels in wild-type and zfs1-deficient S. pombe strains; those elevated in the zfs1-deficient strain were examined for the presence of potential tristetraprolin-like binding sites. One such potential target transcript was encoded by arz1, a gene encoding a protein of unknown function that contains armadillo repeats. arz1 mRNA decay was inhibited in the zfs1-deficient strain when it was expressed under the control of a thiamine-repressible promoter. Mutations within one AU-rich element present in the arz1 3-untranslated region protected this transcript from zfs1-promoted decay, whereas mutating another potential binding site had no effect. Binding assays confirmed a direct interaction between zfs1 and arz1 mRNA-based probes; this interaction was eliminated when key residues were mutated in either zfs1 zinc finger. zfs1 and its targets in S. pombe represent a useful model system for studies of zinc finger protein/AU-rich element interactions that result in mRNA decay.
Characterization of zfs1 as an mRNA-binding and -destabilizing protein in Schizosaccharomyces pombe.
No sample metadata fieldsView Samples
In order to study the effects of Kdm2b binding at CpG islands, Kdm2b was knocked down in mouse embryonic stem cells using shRNA and gene expression profiled using Affymetrix arrays
KDM2B links the Polycomb Repressive Complex 1 (PRC1) to recognition of CpG islands.
Specimen partView Samples
Tristetraprolin (TTP) is a tandem CCCH zinc finger protein that was identified through its rapid induction by mitogens in fibroblasts. Studies of TTP-deficient mice, and cells derived from them, showed that TTP could bind to certain AU-rich elements in mRNAs, leading to increases in the rates of mRNA deadenylation and destruction. Known physiological target
Novel mRNA targets for tristetraprolin (TTP) identified by global analysis of stabilized transcripts in TTP-deficient fibroblasts.
Cell lineView Samples
Purpose: 1. Bulk-RNA-Seq was performed to identify tancytye-enriched genes. 2. scRNA-Seq was performed to profile hypothalamic cells following leptin treatment Conclusions: Leptin receptor expression in tanycytes is either absent or undetectably low, that tanycytes do not directly regulate hypothalamic leptin signaling, and that leptin regulates gene expression in diverse hypothalamic cell types through both direct and indirect mechanisms. Overall design: Methods 1 (Bulk-RNA-Seq). Flow-sorted RNA samples from Rax-EGFP BAC transgenic mice were sent to the Deep Sequencing and Microarray Core (Johns Hopkins University) for library preparation and sequencing. Briefly, polyadenylated RNA was purified from the total RNA samples using Oligo dT conjugated magnetic beads and prepared for single-end sequencing according to the Illumina TruSeq RNA Sample Preparation Kit v2 (# RS-122-2001, Illumina). The libraries were sequenced for paired-end 75 cycles using the TruSeq SBS kit on NextSeq 500 system. Filtered sequencing reads were mapped to the mouse reference genome (mm10) using TopHat. FPKM value for each gene was estimated using Cufflink. Methods 2 (scRNA-Seq). Mice brain coronal slices (aCSF- or leptin-infused) were dissociated using Act-Seq protocol and re-suspended cells were loaded into V2 10x Genomics Chromium Single Cell system, and libraries were sequenced on Illumina NextSeq with ~150 million reads per library. Sequencing results were processed 10x Genomics pipeline. Seurat V2 was used to perform downstream analysis following the standard pipeline using cells with more than 500 genes and 1000 UMI counts.
Tanycyte-Independent Control of Hypothalamic Leptin Signaling.
Age, Specimen part, Cell line, SubjectView Samples
Arsenic is a potent environmental toxin and a cause of numerous health problems. Most studies have assumed that arsenic-induced changes in mRNA levels result from effects on gene transcription. The influence of arsenic on post-transcriptional regulation, another important locus of gene expression control, has remained largely unexplored.
Global analysis of posttranscriptional gene expression in response to sodium arsenite.
Cell lineView Samples