The traditional view of hematopoiesis has been that all the cells of the peripheral blood are the progeny of a unitary homogeneous pool of hematopoietic stem cells (HSCs). Recent evidence suggests that the hematopoietic system is actually maintained by a consortium of HSC subtypes with distinct functional characteristics. We show here that myeloid-biased HSCs (My-HSCs) and lymphoid-biased (Ly-HSCs) can be purified according to their capacity for Hoechst dye efflux in combination with canonical HSC markers.
Distinct hematopoietic stem cell subtypes are differentially regulated by TGF-beta1.
Sex, Specimen partView Samples
The goal of this study was to contrast genome-wide gene expression profiles of cultured human trabecular meshwork (HTM) cells, to that of control and primary open angle glaucoma (POAG) HTM tissues.
Genome-wide expression profile of human trabecular meshwork cultured cells, nonglaucomatous and primary open angle glaucoma tissue.
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Screening small molecules and drugs for activity to modulate alternative splicing, we found that amiloride, distinct from four other intracellular pH-affecting analogues, could normalize the splicing of BCL-X, HIPK3 and RON/MISTR1 transcripts in human hepatocellular carcinoma Huh-7 cells. To elucidate the underlying mechanisms, our proteomic analyses of amiloride-treated cells detected hypo-phosphorylation of splicing factor SF2/ASF and also decreased levels of SRp20 and two un-identified SR proteins. We further observed decreased phosphorylation of AKT, ERK1/2 and PP1, while increased phosphorylation of p38 and JNK, suggesting that amiloride treatment down-regulated kinases and up-regulated phosphatases in the signal pathways known to affect the splicing factor protein phosphorylation. The amiloride effects of splicing factor protein hypo-phosphorylation andnormalizedoncogenic RNA splicing were both abrogated by pre-treatment with a PP1 inhibitor. We then performed global exon array analysis of Huh-7 cells treated with amiloride for 24 hours. Using gene array chips (Affymetrix GeneChip Human Exon 1.0 ST Array of >518000 exons of 42974 genes) for exon array analysis (set parameters of correlation coefficient 0.7, splicing index -1.585 , and log2 ratio -1.585), we found that amiloride influenced the splicing patterns of 551 genes involving at least 584 exons, which included 495 known protein-coding genes involving 526 exons, many of which play key roles in functional networks of ion transport, extracellular matrix, cytoskeletons and genome maintenance. Cellular functional analyses revealed subsequent invasion and migration defects, cell cycle disruption, cytokinesis impairment, and lethal DNA degradation in amiloride-treated Huh-7 cells. This study thus provides mechanistic underpinnings for exploiting small molecule modulation of abnormal RNA splicing for cancer therapeutics.
Small molecule amiloride modulates oncogenic RNA alternative splicing to devitalize human cancer cells.
Cell lineView Samples
Alternative splicing is a mechanism for increasing the protein variety of a limited number of genes. Studies have shown that aberrant regulations of the alternative splicing of apoptotic gene transcripts may contribute to the development of cancer. In this study, we isolated 4ß-Hydroxywithanolide E (4bHWE) from the traditional herb Physalis peruviana, and analyzed its biological effects in cancer cells. The results demonstrated that 4bHWE modulates the alternative splicing of apoptotic genes (e.g., HIPK3, SMAC/DIABLO, and SURVIVIN), changes the expression level of splicing factors (e.g., hnRNP C1/C2, ASF/SF2, SRp20, and SRp55), and induces histone tail posttranslational modifications (e.g., H3K27me1, H3K27me2, H3K36me3, and H3K79me1). Pretreatment with okadaic acid to inhibit protein phosphatase-1 could partly relieve the effects of 4bHWE on the alternative splicing of HIPK3 and SMAC/DIABLO transcripts, as well as on the dephosphorylation of ASF/SF2. Genome-wide detection of alternative splicing further indicated that several other apoptosis-related genes are also regulated by 4bHWE, including APAF1, CARP-1, and RIPK1. Moreover, we extended our study to apoptosis-associated molecules, detecting an increasing level of CASPASE-3 activity and cleavage of poly ADP-ribose polymerase in 4bHWE-induced apoptosis. Furthermore, in vivo experiments showed that the treatment of tumor-bearing mice with 4bHWE resulted in a marked decrease of tumor size and weight. Taken together, this study is the first to show that 4bHWE affects alternative splicing through the modulations of splicing factors, providing a novel view of the antitumor mechanism of 4bHWE. Overall design: Examination of the global genes with altered alternative splicing in 4bHWE-treated Huh-7 cells.
4β-Hydroxywithanolide E Modulates Alternative Splicing of Apoptotic Genes in Human Hepatocellular Carcinoma Huh-7 Cells.
Specimen part, Treatment, SubjectView Samples
Cytosine methylation is an epigenetic mark usually associated with gene repression. Despite a requirement for de novo DNA methylation for differentiation of embryonic stem cells, its role in somatic stem cells is unknown. Using conditional ablation, we show that loss of either, or both, Dnmt3a or Dnmt3b, progressively impedes hematopoietic stem cell (HSC) differentiation during serial in vivo passage. Concomitantly, HSC self-renewal is immensely augmented in absence of either Dnmt3, particularly Dnmt3a. Dnmt3-KO HSCs show upregulation of HSC multipotency genes and downregulation of early differentiation factors, and the differentiated progeny of Dnmt3-KO HSCs exhibit hypomethylation and incomplete repression of HSC-specific genes. HSCs lacking Dnmt3a manifest hyper-methylation of CpG islands and hypo-methylation of genes which are highly correlated with human hematologic malignancies. These data establish that aberrant DNA methylation has direct pathologic consequences for somatic stem cell development, leading to inefficient differentiation and maintenance of a self-renewal program.
Dnmt3a is essential for hematopoietic stem cell differentiation.
Sex, Specimen partView Samples
Intestinal organoids are complex three-dimensional structures that mimic cell type composition and tissue organization of the intestine by recapitulating the self-organizing capacity of cell populations derived from a single stem cell. Crucial in this process is a first symmetry-breaking event, in which only a fraction of identical cells in a symmetrical cyst differentiate into Paneth cells, which in turn generates the stem cell niche and leads to asymmetric structures such as crypts and villi. We here combine a quantitative single-cell gene expression and imaging approach to characterize the development of intestinal organoids from a single cell. We show that intestinal organoid development follows a regeneration process driven by transient Yap1 activation. Cell-to-cell variability in Yap1, emerging in symmetrical cysts, initiates a Notch/Dll1 lateral inhibition event driving the symmetry-breaking event and the formation of the first Paneth cell. Our findings reveal how single cells exposed to a uniform growth-promoting environment have the intrinsic ability to generate emergent, self-organized behavior resulting in the formation of complex multicellular asymmetric structures. Overall design: Single cell RNA sequencing of single cells isolated from intestinal organoids day3 and intestinal organoids day 5
Self-organization and symmetry breaking in intestinal organoid development.
Age, Specimen part, Cell line, SubjectView Samples
Gene expression analysis of T-cell acute lymphoblastic leukemia blast cells from either control mice or Dnmt3a knockout mice carrying a Notch1 Intracellular Domain (NICD) retrovirus Overall design: Comparison of gene expression between control and Dnmt3a-KO NICD-driven T-ALL
Dnmt3a regulates T-cell development and suppresses T-ALL transformation.
Specimen part, Cell line, SubjectView Samples
Mice lacking the beta 2 subunit (Chrnb2) of the neuronal nicotinic acetylcholine receptor display altered retinal waves and disorganized projections of the retinal ganglion cells to the lateral geniculate nucleus (LGN). mRNA populations from retinas and LGN from Chrnb2-/-and wild type (C57BL/6J) mice were compared at 4 days postnatal, when RGC segregation to the LGN begins in WT mice. Retinal mRNAs were also compared at adulthood.
Mouse mutants for the nicotinic acetylcholine receptor ß2 subunit display changes in cell adhesion and neurodegeneration response genes.
Sex, Specimen partView Samples
The study demontrates differences in the transcriptome ( both of protein coding transcripts and long non-coding RNAs) in the unilateral ureteric obstruction model of renal fibrosis. Overall design: Renal tissue was studied from animals undergoing sham operation (as controls) or right ureteric ligation. Animals were sacrificed 2 and 8 days following ligation and the right kidney tissue was examined.
Whole-transcriptome analysis of UUO mouse model of renal fibrosis reveals new molecular players in kidney diseases.
Sex, Age, Specimen part, Cell line, SubjectView Samples
We demonstrate that GLUT4 up-regulation significantly increased cell migration and invasion in lower magligance head and neck cancer cell lines in vitro.
Glucose transporter 4 promotes head and neck squamous cell carcinoma metastasis through the TRIM24-DDX58 axis.
Specimen part, Cell lineView Samples