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1.
Cells ; 9(5)2020 05 11.
Article in English | MEDLINE | ID: mdl-32403252

ABSTRACT

Chromatin remodeling, including histone modification, chromatin (un)folding, and nucleosome remodeling, is a significant transcriptional regulation mechanism. By these epigenetic modifications, transcription factors and their regulators are recruited to the promoters of target genes, and thus gene expression is controlled through either transcriptional activation or repression. The Mat1-mediated transcriptional repressor (MMTR)/DNA methyltransferase 1 (DNMT1)-associated protein (Dmap1) is a transcription corepressor involved in chromatin remodeling, cell cycle regulation, DNA double-strand break repair, and tumor suppression. The Tip60-p400 complex proteins, including MMTR/Dmap1, interact with the oncogene Myc in embryonic stem cells (ESCs). These proteins interplay with the stem cell-related proteome networks and regulate gene expressions. However, the detailed mechanisms of their functions are unknown. Here, we show that MMTR/Dmap1, along with other Tip60-p400 complex proteins, bind the promoters of differentiation commitment genes in mouse ESCs. Hence, MMTR/Dmap1 controls gene expression alterations during differentiation. Furthermore, we propose a novel mechanism of MMTR/Dmap1 function in early stage lineage commitment of mouse ESCs by crosstalk with the polycomb group (PcG) proteins. The complex controls histone mark bivalency and transcriptional poising of commitment genes. Taken together, our comprehensive findings will help better understand the MMTR/Dmap1-mediated transcriptional regulation in ESCs and other cell types.


Subject(s)
Cell Lineage , Mouse Embryonic Stem Cells/cytology , Mouse Embryonic Stem Cells/metabolism , Polycomb-Group Proteins/metabolism , Repressor Proteins/metabolism , Animals , Cell Differentiation , Chromatin Assembly and Disassembly , DNA Helicases/metabolism , DNA-Binding Proteins/metabolism , HEK293 Cells , Histones/metabolism , Humans , Lysine/metabolism , Lysine Acetyltransferase 5/metabolism , Methylation , Mice , Mice, SCID , Models, Biological , Polycomb Repressive Complex 2/metabolism , Promoter Regions, Genetic , Protein Binding , Repressor Proteins/chemistry , Trans-Activators/metabolism
2.
PLoS One ; 9(12): e113442, 2014.
Article in English | MEDLINE | ID: mdl-25438047

ABSTRACT

Cetuximab, a chimeric monoclonal antibody developed for targeting the Epidermal Growth Factor Receptor (EGFR), has been intensively used to treat cancer patients with metastatic colorectal cancer and head and neck cancer. Intact immunoglobulin G (IgG) antibody like cetuximab, however, has some limitations such as high production cost and low penetration rate from vasculature into solid tumor mass due to its large size. In attempt to overcome these limitations, we engineered cetuximab to create single chain variable fragments (scFv-CH3; Minibody) that were expressed in bacterial system. Among three engineered minibodies, we found that MI061 minibody, which is composed of the variable heavy (VH) and light (VL) region joined by an 18-residue peptide linker, displays higher solubility and better extraction properties from bacterial lysate. In addition, we validated that purified MI061 significantly interferes ligand binding to EGFR and blocks EGFR's phosphorylation. By using a protein microarray composed of 16,368 unique human proteins covering around 2,400 plasma membrane associated proteins such as receptors and channels, we also demonstrated that MI061 only recognizes the EGFR but not other proteins as compared with cetuximab. These results indicated that engineered MI061 retains both binding specificity and affinity of cetuximab for EGFR. Although it had relatively short half-life in serum, it was shown to be highly significant anti-tumor effect by inhibiting ERK pathway in A431 xenograft model. Taken together, our present study provides compelling evidence that engineered minibody is more effective and promising agent for in vivo targeting of solid tumors.


Subject(s)
Antibodies, Monoclonal, Humanized/genetics , ErbB Receptors/immunology , Head and Neck Neoplasms/drug therapy , Molecular Targeted Therapy , Protein Engineering , Single-Chain Antibodies/genetics , Single-Chain Antibodies/therapeutic use , Animals , Antibody Specificity , Base Sequence , Cell Line, Tumor , Cetuximab , Escherichia coli/genetics , Head and Neck Neoplasms/immunology , Head and Neck Neoplasms/pathology , Humans , Male , Mice , Signal Transduction/immunology , Single-Chain Antibodies/immunology , Xenograft Model Antitumor Assays
3.
Nucleic Acids Res ; 38(16): 5456-71, 2010 Sep.
Article in English | MEDLINE | ID: mdl-20421208

ABSTRACT

Data presented here extends our previous observations on α-globin transcriptional regulation by the CP2 and PIAS1 proteins. Using RNAi knockdown, we have now shown that CP2b, CP2c and PIAS1 are each necessary for synergistic activation of endogenous α-globin gene expression in differentiating MEL cells. In this system, truncated PIAS1 mutants lacking the ring finger domain recruited CP2c to the nucleus, as did wild-type PIAS1, demonstrating that this is a sumoylation-independent process. In vitro, recombinant CP2c, CP2b and PIAS1 bound DNA as a stable CBP (CP2c/CP2b/PIAS1) complex. Following PIAS1 knockdown in MEL cells, however, the association of endogenous CP2c and CP2b with the α-globin promoter simultaneously decreased. By mapping the CP2b- and CP2c-binding domains on PIAS1, and the PIAS1-binding domains on CP2b and CP2c, we found that two regions of PIAS1 that interact with CP2c/CP2b are required for its co-activator function. We propose that CP2c, CP2b, and PIAS1 form a hexametric complex with two units each of CP2c, CP2b, and PIAS1, in which PIAS1 serves as a clamp between two CP2 proteins, while CP2c binds directly to the target DNA and CP2b mediates strong transactivation.


Subject(s)
DNA-Binding Proteins/metabolism , Erythroid Cells/metabolism , Protein Inhibitors of Activated STAT/metabolism , Transcription Factors/metabolism , Transcriptional Activation , alpha-Globins/genetics , Animals , Cell Line , Cell Line, Tumor , Cell Nucleus/metabolism , DNA-Binding Proteins/antagonists & inhibitors , DNA-Binding Proteins/chemistry , Humans , Mice , Promoter Regions, Genetic , Protein Inhibitors of Activated STAT/antagonists & inhibitors , Protein Inhibitors of Activated STAT/chemistry , Protein Interaction Domains and Motifs , RNA Interference , Transcription Factors/antagonists & inhibitors , Transcription Factors/chemistry
4.
Biochem Biophys Res Commun ; 378(3): 629-33, 2009 Jan 16.
Article in English | MEDLINE | ID: mdl-19059211

ABSTRACT

We examined the role of zebrafish (Danio rerio) Jak2a, a homolog of mammalian Jak2, in the developing embryo by injecting in vitro synthesized Jak2a shRNA into zebrafish zygotes. Blood circulation was suppressed in Jak2a shRNA-injected embryos from 24hours post fertilization (hpf) and all embryos died with enlarged pericardium, shortened body lengths, and defects in some vasculature within 8 days post fertilization. O-dianisidine staining of red blood cells revealed normal blood island formation with no circulating red blood cells. As in Jak2(-/-) transgenic mice, expression of definitive Ba1 globin was significantly reduced in Jak2a knockdown embryos at 36hpf, whereas expression of other hematopoietic markers, primitive be1 globin, gata-1, and scl, were unaffected. More importantly, blood vessel formation was disturbed in Jak2a knockdown embryos as revealed by alkaline phosphatase staining at 72hpf. Thus, our data indicate that zebrafish Jak2a is important in both definitive hematopoiesis and blood vessel formation.


Subject(s)
Blood Vessels/embryology , Hematopoiesis , Neovascularization, Physiologic , Protein-Tyrosine Kinases/physiology , Zebrafish Proteins/physiology , Zebrafish/embryology , Animals , Blood Circulation/genetics , Embryo, Nonmammalian/enzymology , Gene Knockdown Techniques , Gene Silencing , Hematopoiesis/genetics , Mice , Mice, Transgenic , Neovascularization, Physiologic/genetics , Protein-Tyrosine Kinases/genetics , RNA, Small Interfering/genetics , Transgenes , Zebrafish/genetics , Zebrafish/metabolism , Zebrafish Proteins/genetics
5.
J Cardiovasc Pharmacol ; 49(5): 280-6, 2007 May.
Article in English | MEDLINE | ID: mdl-17513946

ABSTRACT

Hyperproliferation of platelet-derived growth factor (PDGF)-BB-induced vascular smooth muscle cell (VSMC) is a hallmark of atherosclerosis and related vascular disorders. In the previous study, we reported that KTJ740 [2-chloro-3-(4-(ethylcarboxy)-phenyl)-amino-1,4-naphthoquinone], a newly synthesized vitamin K derivative, has potent antithrombotic effects in mice and antiplatelet activity in vitro and ex vivo. In the present study, we have tested that KTJ740 could inhibit PDGF-BB-stimulated VSMC proliferation. We have examined the potential inhibitory effect of this compound on rat aortic smooth muscle cells (RASMCs). Our results show that this compound significantly inhibits PDGF-BB-stimulated RASMC number and DNA synthesis in a concentration-dependent manner. Furthermore, we have examined its effect on cell cycle progression by flow cytometry. KTJ740 treatment resulted in a significant arrest in cell cycle progression of RASMCs induced by PDGF-BB, and this effect was achieved by suppressing activation of PDGF-beta receptor (PDGF-Rbeta) tyrosine kinase pathway. These results suggest that a possibility of KTJ740 can be a potential agent to control vascular disorders and its antiproliferative mechanism may be mediated through PDGF-Rbeta tyrosine kinase-dependent signaling pathway.


Subject(s)
Angiogenesis Inducing Agents/pharmacology , Aorta/cytology , Cell Cycle/drug effects , Cell Proliferation/drug effects , Fibrinolytic Agents/pharmacology , Myocytes, Smooth Muscle/drug effects , Naphthoquinones/pharmacology , Platelet-Derived Growth Factor/metabolism , Analysis of Variance , Animals , Becaplermin , Cell Count , Cell Survival/drug effects , Dose-Response Relationship, Drug , Enzyme Activation/drug effects , Flow Cytometry , Mitogen-Activated Protein Kinase 3/drug effects , Muscle, Smooth, Vascular/cytology , Myocytes, Smooth Muscle/cytology , Myocytes, Smooth Muscle/metabolism , Phospholipase C gamma/drug effects , Proto-Oncogene Proteins c-akt/drug effects , Proto-Oncogene Proteins c-sis , Rats , Receptor, Platelet-Derived Growth Factor beta/drug effects , Receptors, Platelet-Derived Growth Factor/drug effects , Signal Transduction/drug effects
6.
Biochem Biophys Res Commun ; 351(3): 682-8, 2006 Dec 22.
Article in English | MEDLINE | ID: mdl-17078929

ABSTRACT

As the LIF-induced Jak1/STAT3 pathway has been reported to play a crucial role in self-renewal of mESCs, we sought to determine if Jak2, which is also expressed in mESCs, might also be involved in the pathway. By employing an RNAi strategy, we established both Jak2 and Jak2/Tyk2 knockdown mESC clones. Both Jak2 and Jak2/Tyk2 knockdown clones maintained the undifferentiated state as wild-type controls, even in a very low concentration of LIF. However, we observed not only faster onset of differentiation but also differential expression of tissue-specific lineage genes for ectodermal and mesodermal, but not endodermal origins from embryoid bodies generated from both types of knockdown clones compared to the wild-type. Furthermore, the reduced level of Jak2 caused differentiation of mESCs in the presence of LIF when the Wnt pathway was activated by LiCl treatment. Taken together, we demonstrated that Jak2 and Tyk2 are not involved in LIF-induced STAT3 pathway for self-renewal of mESCs, but play a role in early lineage decision of mESCs to various differentiated cell types.


Subject(s)
Cell Differentiation/physiology , Embryonic Stem Cells/cytology , Embryonic Stem Cells/metabolism , Janus Kinase 2/metabolism , STAT3 Transcription Factor/metabolism , Signal Transduction/physiology , TYK2 Kinase/metabolism , Animals , Cell Line , Cell Proliferation , Cell Survival , Mice
7.
Mol Cells ; 21(3): 343-55, 2006 Jun 30.
Article in English | MEDLINE | ID: mdl-16819296

ABSTRACT

Stem cells are unique cell populations with the ability to undergo both self-renewal and differentiation, although a wide variety of adult stem cells as well as embryonic stem cells have been identified and stem cell plasticity has recently been reported. To identify genes implicated in the control of the stem cell state as well as the characteristics of each stem cell line, we analyzed the expression profiles of genes in human embryonic, hematopoietic (CD34+ and CD133+), and mesenchymal stem cells using cDNA microarrays, and identified genes that were differentially expressed in specific stem cell populations. In particular we were able to identify potential hESC signature-like genes that encode transcription factors (TFAP2C and MYCN), an RNA binding protein (IMP-3), and a functionally uncharacterized protein (MAGEA4). The overlapping sets of 22 up-regulated and 141 down-regulated genes identified in this study of three human stem cell types may also provide insight into the developmental mechanisms common to all human stem cells. Furthermore, our comprehensive analyses of gene expression profiles in various adult stem cells may help to identify the genetic pathways involved in self-renewal as well as in multi-lineage specific differentiation.


Subject(s)
Embryo, Mammalian/metabolism , Gene Expression Profiling , Gene Expression Regulation , Hematopoietic Stem Cells/metabolism , Mesenchymal Stem Cells/metabolism , Oligonucleotide Array Sequence Analysis , Adult , Bone Marrow Cells/metabolism , Cell Differentiation , Embryo, Mammalian/cytology , Humans
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