ABSTRACT
Deconvolution of mouse transcriptomic data is challenged by the fact that mouse models carry various genetic and physiological perturbations, making it questionable to assume fixed cell types and cell type marker genes for different data set scenarios. We developed a Semi-Supervised Mouse data Deconvolution (SSMD) method to study the mouse tissue microenvironment. SSMD is featured by (i) a novel nonparametric method to discover data set-specific cell type signature genes; (ii) a community detection approach for fixing cell types and their marker genes; (iii) a constrained matrix decomposition method to solve cell type relative proportions that is robust to diverse experimental platforms. In summary, SSMD addressed several key challenges in the deconvolution of mouse tissue data, including: (i) varied cell types and marker genes caused by highly divergent genotypic and phenotypic conditions of mouse experiment; (ii) diverse experimental platforms of mouse transcriptomics data; (iii) small sample size and limited training data source and (iv) capable to estimate the proportion of 35 cell types in blood, inflammatory, central nervous or hematopoietic systems. In silico and experimental validation of SSMD demonstrated its high sensitivity and accuracy in identifying (sub) cell types and predicting cell proportions comparing with state-of-the-arts methods. A user-friendly R package and a web server of SSMD are released via https://github.com/xiaoyulu95/SSMD.
Subject(s)
Antigens, Differentiation , Cellular Microenvironment , Computational Biology , Databases, Genetic , Gene Expression Profiling , Transcriptome , Animals , Antigens, Differentiation/biosynthesis , Antigens, Differentiation/genetics , Mice , Organ SpecificityABSTRACT
WNK [with no lysine (K)] protein kinases are found in all sequenced multicellular and many unicellular organisms. WNKs influence ion balance. Two WNK family members are associated with a single gene form of hypertension. RNA interference screens have implicated WNKs in survival and growth, and WNK1 is essential for viability of mice. We found that the majority of WNK1 is localized on cytoplasmic puncta in resting cells. During cell division, WNK1 localizes to mitotic spindles. Therefore, we analyzed mitotic phenotypes in WNK1 knockdown cells. A large percentage of WNK1 knockdown cells fail to complete cell division, displaying defects in mitotic spindles and also in abscission and cell survival. One of the best-characterized WNK1 targets is the protein kinase OSR1 (oxidative stress responsive 1). OSR1 regulates ion cotransporters, is activated in response to osmotic stress by WNK family members, and is largely associated with WNK1. In resting cells, the majority of OSR1, like WNK1, is on cytoplasmic puncta. OSR1 is also in nuclei. In contrast to WNK1, however, OSR1 does not concentrate around spindles during mitosis and does not show a WNK1-like localization pattern in mitotic cells. Knockdown of OSR1 has only a modest effect on cell survival and does not lead to spindle defects. We conclude that decreased cell survival associated with loss of WNK1 is attributable to defects in chromosome segregation and abscission and is independent of the effector kinase OSR1.
Subject(s)
Cytokinesis/physiology , Mitosis/physiology , Protein Serine-Threonine Kinases/metabolism , Spindle Apparatus/metabolism , Analysis of Variance , Animals , Cell Line, Tumor , Cell Survival/genetics , Cell Survival/physiology , Cytokinesis/genetics , Cytoplasm/metabolism , Fluorescent Antibody Technique , Genetic Complementation Test , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , HT29 Cells , HeLa Cells , Humans , Immunoblotting , Intracellular Signaling Peptides and Proteins , Minor Histocompatibility Antigens , Mitosis/genetics , Protein Serine-Threonine Kinases/genetics , RNA Interference , Rats , WNK Lysine-Deficient Protein Kinase 1ABSTRACT
Two of the four WNK (with no lysine (K)) protein kinases are associated with a heritable form of ion imbalance culminating in hypertension. WNK1 affects ion transport in part through activation of the closely related Ste20 family protein kinases oxidative stress-responsive 1 (OSR1) and STE20/SPS1-related proline-, alanine-rich kinase (SPAK). Once activated by WNK1, OSR1 and SPAK phosphorylate and stimulate the sodium, potassium, two chloride co-transporters, NKCC1 and NKCC2, and also affect other related ion co-transporters. We find that WNK1 and OSR1 co-localize on cytoplasmic puncta in HeLa and other cell types. We show that the C-terminal region of WNK1 including a coiled coil is sufficient to localize the fragment in a manner similar to the full-length protein, but some other fragments lacking this region are mislocalized. Photobleaching experiments indicate that both hypertonic and hypotonic conditions reduce the mobility of GFP-WNK1 in cells. The four WNK family members can phosphorylate the activation loop of OSR1 to increase its activity with similar kinetic constants. C-terminal fragments of WNK1 that contain three RFXV interaction motifs can bind OSR1, block activation of OSR1 by sorbitol, and prevent the OSR1-induced enhancement of ion co-transporter activity in cells, further supporting the conclusion that association with WNK1 is required for OSR1 activation and function at least in some contexts. C-terminal WNK1 fragments can be phosphorylated by OSR1, suggesting that OSR1 catalyzes feedback phosphorylation of WNK1.
Subject(s)
Intracellular Signaling Peptides and Proteins/metabolism , Protein Serine-Threonine Kinases/metabolism , Sodium-Potassium-Chloride Symporters/metabolism , Amino Acid Motifs/genetics , Amino Acid Sequence , Animals , Cytoplasm/metabolism , Fluorescence Recovery After Photobleaching , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , HeLa Cells , Humans , Hypertonic Solutions/pharmacology , Hypotonic Solutions/pharmacology , Immunoblotting , Immunoprecipitation , Intracellular Signaling Peptides and Proteins/genetics , Isoenzymes/genetics , Isoenzymes/metabolism , Microscopy, Fluorescence , Minor Histocompatibility Antigens , Protein Binding , Protein Serine-Threonine Kinases/genetics , Protein Transport/drug effects , RNA Interference , Rats , Sodium-Potassium-Chloride Symporters/genetics , Solute Carrier Family 12, Member 1 , Solute Carrier Family 12, Member 2 , WNK Lysine-Deficient Protein Kinase 1ABSTRACT
Prostate cancer is initially responsive to androgen ablation, but prostate cancer tumors invariably progress to an androgen-independent state that is ultimately lethal. The onset of the androgen-independent prostate cancer is often associated with up-regulation of the androgen receptor that can cause antagonists to exhibit agonistic activity, which could lead to the failure of androgen ablation therapy. We describe a unique protein-DOC-2/DAB2 (differentially expressed in ovarian cancer-2/disabled 2)-that antagonizes androgen receptor-mediated cell growth in prostate cancer cells via interaction with c-Src protein. This interaction causes inactivation of Erk and Akt proteins critical for proliferation and survival of prostate cancer cells. However, DOC-2/DAB2 does not change the capacity of androgen receptor to regulate the transcription of androgen-responsive reporter genes, indicating that DOC-2/DAB2 selectively inhibits androgen receptor-mediated cell growth in androgen-independent prostate cancer by disrupting the androgen receptor/c-Src complex. In normal prostatic epithelia, DOC-2/DAB2 protein levels are more abundant than androgen receptor protein levels and reduced endogenous DOC-2/DAB2 protein levels in these cells by DOC-2/DAB2 RNA interference result in enhancing androgen receptor-mediated cell growth. We conclude that DOC-2/DAB2 can modulate androgen receptor-mediated cell growth in both normal and malignant prostatic epithelial cells and the outcome of this study could evolve into a new therapeutic strategy of prostate cancer.
Subject(s)
Adaptor Proteins, Vesicular Transport/physiology , Genes, Tumor Suppressor/physiology , Phosphotransferases/metabolism , Prostatic Neoplasms/pathology , Proto-Oncogene Proteins/metabolism , Receptors, Androgen/physiology , Adaptor Proteins, Signal Transducing , Adaptor Proteins, Vesicular Transport/genetics , Adaptor Proteins, Vesicular Transport/metabolism , Androgen Receptor Antagonists , Apoptosis Regulatory Proteins , Binding, Competitive , CSK Tyrosine-Protein Kinase , Cell Growth Processes/drug effects , Cell Growth Processes/physiology , Dihydrotestosterone/pharmacology , Epithelium/enzymology , Epithelium/metabolism , Humans , Male , Prostate/cytology , Prostate/enzymology , Prostate/metabolism , Prostatic Neoplasms/enzymology , Prostatic Neoplasms/genetics , Prostatic Neoplasms/metabolism , Protein-Tyrosine Kinases , RNA Interference , Receptors, Androgen/metabolism , Signal Transduction , Transfection , Tumor Suppressor Proteins , src-Family KinasesABSTRACT
The down-regulation of DOC-2/DAB2 gene, which encodes a unique phosphoprotein modulating signal pathways elicited by exogenous stimuli, is often associated with several cancer types; however, the underlying mechanism is still unknown. Dramatically different expression levels of DOC-2/DAB2 mRNA and protein are observed among several human transitional cell carcinoma (TCC) cell lines, suggesting that transcriptional regulation may play a role in these cells. In this study, we have shown that the histone acetylation status associated with the 5' upstream regulatory sequence of DOC-2/DAB2 gene is one of the key determinants for its gene expression. In addition, GATA6 but not other GATA family members, such as GATA2 and GATA4, can specifically induce DOC-2/DAB2 promoter activity, although GATA transcription factors share a very similar DNA-binding sequence. We also show that increased histone acetylation and the presence of GATA6 have a synergistic effect on DOC-2/DAB2 promoter activity, which results in the elevation of DOC-2/DAB2 protein expression. Thus, we conclude that transcriptional regulation of DOC-2/DAB2 gene in human TCC is determined by histone acetylation and a specific transcription factor (i.e., GATA6), which underlie the reduced DOC-2/DAB2 protein expression in TCC cells.
Subject(s)
Adaptor Proteins, Vesicular Transport/genetics , Carcinoma, Transitional Cell/genetics , DNA-Binding Proteins/genetics , Gene Expression Regulation, Neoplastic , Histone Deacetylase Inhibitors , Transcription Factors/genetics , Acetylation , Adaptor Proteins, Signal Transducing , Adaptor Proteins, Vesicular Transport/biosynthesis , Apoptosis Regulatory Proteins , Base Sequence , Carcinoma, Transitional Cell/enzymology , Carcinoma, Transitional Cell/metabolism , Cell Line, Tumor , Chromatin/genetics , Chromatin/metabolism , DNA-Binding Proteins/biosynthesis , DNA-Binding Proteins/metabolism , Depsipeptides/pharmacology , Down-Regulation , GATA6 Transcription Factor , Genes, Tumor Suppressor , Genetic Vectors/genetics , Histone Deacetylases/metabolism , Histones/metabolism , Humans , Molecular Sequence Data , Promoter Regions, Genetic , Transcription Factors/biosynthesis , Transcription Factors/metabolism , Transfection , Tumor Suppressor ProteinsABSTRACT
Human DAB2IP (hDAB2IP), a novel GTPase-activating protein modulating the Ras-mediated signaling and tumor necrosis factor-mediated apoptosis, is a potent growth inhibitor in human prostate cancer (PCa). Loss of hDAB2IP expression in PCa is due to altered epigenetic regulation (i.e. DNA methylation and histone modification) of its promoter region. The elevated polycomb Ezh2, a histone methyltransferase, has been associated with PCa progression. In this study, we have demonstrated that an increased Ezh2 expression in normal prostatic epithelial cells can suppress hDAB2IP gene expression. In contrast, knocking down the endogenous Ezh2 levels in PCa by a specific small interfering RNA can increase hDAB2IP expression. The association of Ezh2 complex (including Eed and Suz12) with hDAB2IP gene promoter is also detected in PCa cells but not in normal prostatic epithelial cells. Increased Ezh2 expression in normal prostatic epithelial cells by cDNA transfection facilitates the recruitment of other components of Ezh2 complex to the hDAB2IP promoter region accompanied with the increased levels of methyl histone H3 (H3) and histone deacetylase (HDAC1). Consistently, data from PCa cells transfected with Ezh2 small interfering RNA demonstrated that reduced Ezh2 levels resulted in the dissociation of Ezh2 complex accompanied with decreased levels of both methyl H3 and HDAC1 from hDAB2IP gene promoter. We further unveiled that the methylation status of Lys-27 but not Lys-9 of H3 in hDAB2IP promoter region is consistent with the hDAB2IP levels in both normal prostatic epithelial cells and PCa cells. Together, we conclude that hDAB2IP gene is a target gene of Ezh2 in prostatic epithelium, which provides an underlying mechanism of the down-regulation of hDAB2IP gene in PCa.