ABSTRACT
BACKGROUND & AIMS: The polycomb repressive complex 2 (PRC2) regulates differentiation by contributing to repression of gene expression and thereby stabilizing the fate of stem cells and their progeny. PRC2 helps to maintain adult stem cell populations, but little is known about its functions in intestinal stem cells. We studied phenotypes of mice with intestine-specific deletion of the PRC2 proteins embryonic ectoderm development (EED) (a subunit required for PRC2 function) and enhancer of zeste homolog 2 (EZH2) (a histone methyltransferase). METHODS: We performed studies of AhCre;EedLoxP/LoxP (EED knockout) mice and AhCre;Ezh2LoxP/LoxP (EZH2 knockout) mice, which have intestine-specific disruption in EED and EZH2, respectively. Small intestinal crypts were isolated and subsequently cultured to grow organoids. Intestines and organoids were analyzed by immunohistochemical, in situ hybridization, RNA sequence, and chromatin immunoprecipitation methods. RESULTS: Intestines of EED knockout mice had massive crypt degeneration and lower numbers of proliferating cells compared with wild-type control mice. Cdkn2a became derepressed and we detected increased levels of P21. We did not observe any differences between EZH2 knockout and control mice. Intestinal crypts from EED knockout mice had signs of aberrant differentiation of uncommitted crypt cells-these differentiated toward the secretory cell lineage. Furthermore, crypts from EED-knockout mice had impaired Wnt signaling and concomitant loss of intestinal stem cells, this phenotype was not reversed upon ectopic stimulation of Wnt and Notch signaling in organoids. Analysis of gene expression patterns from intestinal tissues of EED knockout mice showed dysregulation of several genes involved in Wnt signaling. Wnt signaling was regulated directly by PRC2. CONCLUSIONS: In intestinal tissues of mice, PRC2 maintains small intestinal stem cells by promoting proliferation and preventing differentiation in the intestinal stem cell compartment. PRC2 controls gene expression in multiple signaling pathways that regulate intestinal homeostasis. Sequencing data are available in the genomics data repository GEO under reference series GSE81578; RNA sequencing data are available under subseries GSE81576; and ChIP sequencing data are available under subseries GSE81577.
Subject(s)
Adult Stem Cells/physiology , Intestines/cytology , Polycomb Repressive Complex 2/deficiency , Animals , Base Sequence , Cell Differentiation , Cell Lineage , Cell Proliferation , Chromatin Immunoprecipitation , Enhancer of Zeste Homolog 2 Protein/deficiency , Intestinal Mucosa/metabolism , Mice , Mice, Knockout , Polycomb Repressive Complex 2/genetics , Wnt Signaling PathwayABSTRACT
EZH2 is frequently overexpressed in glioblastoma (GBM), suggesting an oncogenic function that could be a target for therapeutic intervention. However, reduced EZH2 activity can also promote tumorigenesis, leading to concerns about the use of EZH2 inhibitors. Here, we provide further insight about the effects of prolonged Ezh2 inhibition in glioblastoma using preclinical mouse models and primary tumor-derived human GBM cell lines. Using doxycycline-inducible shRNAs that mimic the effects of a selective EZH2 inhibitor, we demonstrate that prolonged Ezh2 depletion causes a robust switch in cell fate, including significantly enhanced proliferation, DNA damage repair, and activation of part of the pluripotency network, resulting in altered tumor cell identity and tumor progression. Short-term Ezh2 depletion significantly improved survival without the tumor progression observed upon prolonged Ezh2 depletion, suggesting that precise dosing regiments are very important. These results could be of high clinical relevance with regard to how glioblastomas should be treated with epigenetic therapies.
ABSTRACT
Histone ubiquitination at DNA breaks is required for activation of the DNA damage response (DDR) and DNA repair. How the dynamic removal of this modification by deubiquitinating enzymes (DUBs) impacts genome maintenance in vivo is largely unknown. To address this question, we generated mice deficient for Ub-specific protease 3 (USP3; Usp3Δ/Δ), a histone H2A DUB which negatively regulates ubiquitin-dependent DDR signaling. Notably, USP3 deletion increased the levels of histone ubiquitination in adult tissues, reduced the hematopoietic stem cell (HSC) reserves over time, and shortened animal life span. Mechanistically, our data show that USP3 is important in HSC homeostasis, preserving HSC self-renewal, and repopulation potential in vivo and proliferation in vitro. A defective DDR and unresolved spontaneous DNA damage contribute to cell cycle restriction of Usp3Δ/Δ HSCs. Beyond the hematopoietic system, Usp3Δ/Δ animals spontaneously developed tumors, and primary Usp3Δ/Δ cells failed to preserve chromosomal integrity. These findings broadly support the regulation of chromatin ubiquitination as a key pathway in preserving tissue function through modulation of the response to genotoxic stress.
Subject(s)
DNA Damage/physiology , Hematopoietic Stem Cells/cytology , Hematopoietic Stem Cells/metabolism , Ubiquitin-Specific Proteases/metabolism , Animals , Carcinogenesis , Cell Proliferation , Cellular Senescence , DNA Breaks, Double-Stranded , DNA Repair/physiology , Female , Histones/metabolism , Homeostasis , Lymphopenia/etiology , Male , Mice , Mice, 129 Strain , Mice, Inbred C57BL , Mice, Knockout , Ubiquitin-Specific Proteases/deficiency , Ubiquitin-Specific Proteases/genetics , UbiquitinationABSTRACT
Bmi1 is a member of the polycomb repressive complex 1 and plays different roles during embryonic development, depending on the developmental context. Bmi1 over expression is observed in many types of cancer, including tumors of astroglial and neural origin. Although genetic depletion of Bmi1 has been described to result in tumor inhibitory effects partly through INK4A/Arf mediated senescence and apoptosis and also through INK4A/Arf independent effects, it has not been proven that Bmi1 can be causally involved in the formation of these tumors. To see whether this is the case, we developed two conditional Bmi1 transgenic models that were crossed with GFAP-Cre mice to activate transgenic expression in neural and glial lineages. We show here that these mice generate intermediate and anterior lobe pituitary tumors that are positive for ACTH and beta-endorphin. Combined transgenic expression of Bmi1 together with conditional loss of Rb resulted in pituitary tumors but was insufficient to induce medulloblastoma therefore indicating that the oncogenic function of Bmi1 depends on regulation of p16(INK4A)/Rb rather than on regulation of p19(ARF)/p53. Human pituitary adenomas show Bmi1 overexpression in over 50% of the cases, which indicates that Bmi1 could be causally involved in formation of these tumors similarly as in our mouse model.
Subject(s)
Glial Fibrillary Acidic Protein/metabolism , Integrases/metabolism , Nuclear Proteins/genetics , Pituitary Neoplasms/genetics , Proto-Oncogene Proteins/genetics , Repressor Proteins/genetics , Transgenes/genetics , Animals , Cyclin-Dependent Kinase Inhibitor p16/metabolism , Gene Expression , Humans , Medulloblastoma/genetics , Medulloblastoma/pathology , Mice , Mice, Transgenic , Pituitary Neoplasms/pathology , Polycomb Repressive Complex 1 , Retinoblastoma Protein/metabolism , Tumor Suppressor Protein p53/metabolism , beta-Endorphin/metabolismABSTRACT
Chromosomes are protein-DNA complexes that encode life. In a cell nucleus, chromosomes are folded in a highly specific manner, which connects strongly to some of their paramount functions, such as DNA replication and gene transcription. Chromosome conformation capture methodologies allow researchers to detect chromosome folding, by quantitatively measuring which genomic sequences are in close proximity in nuclear space. Here, we describe a modified chromosome conformation capture on chip (4C) protocol, which is specifically designed for detection of chromosome folding in a single Drosophila melanogaster tissue. Our protocol enables 4C analyses on a limited number of cells, which is crucial for fly tissues, because these contain relatively low numbers of cells. We used this protocol to demonstrate that target genes of Polycomb group proteins interact with each other in nuclear space of third instar larval brain cells. Major benefits of using D. melanogaster in 4C studies are: (1) powerful and tractable genetic approaches can be incorporated; (2) short generation time allows use of complex genotypes; and (3) compact and well annotated genome. We anticipate that our sensitized 4C method will be generally applicable to detect chromosome folding in other fly tissues.
Subject(s)
Chromosomes, Insect/genetics , Drosophila melanogaster/genetics , Oligonucleotide Array Sequence Analysis , Animals , Base Sequence , Chromosome Mapping , Data Interpretation, Statistical , Dissection , Epistasis, Genetic , Furans , Genes, Insect , Molecular Sequence Data , Nucleic Acid Conformation , Thiophenes , Tissue FixationABSTRACT
Despite intense investigation of intrinsic and extrinsic factors that regulate pluripotency, the process of initial fate commitment of embryonic stem (ES) cells is still poorly understood. We used a genome-wide short hairpin RNA screen in mouse ES cells to identify genes that are essential for initiation of differentiation. Knockdown of the scaffolding protein Mek binding protein 1 (Mp1, also known as Lamtor3 or Map2k1ip1) stimulated self-renewal of ES cells, blocked differentiation, and promoted proliferation. Fibroblast growth factor 4 (FGF4) signaling is required for initial fate commitment of ES cells. Knockdown of Mp1 inhibited FGF4-induced differentiation but did not alter FGF4-driven proliferation. This uncoupling of differentiation and proliferation was also observed when oncogenic Ras isoforms were overexpressed in ES cells. Knockdown of Mp1 redirected FGF4 signaling from differentiation toward pluripotency and up-regulated the pluripotency-related genes Esrrb, Rex1, Tcl1, and Sox2. We also found that human germ cell tumors (GCTs) express low amounts of Mp1 in the invasive embryonic carcinoma and seminoma histologies and higher amounts of Mp1 in the noninvasive carcinoma in situ precursor and differentiated components. Knockdown of Mp1 in invasive GCT cells resulted in resistance to differentiation, thereby showing a functional role for Mp1 both in normal differentiation of ES cells and in germ cell cancer.
Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Cell Differentiation/physiology , Embryonic Stem Cells/metabolism , Pluripotent Stem Cells/metabolism , RNA Interference , Adaptor Proteins, Signal Transducing/genetics , Animals , Carcinoma in Situ/genetics , Carcinoma in Situ/metabolism , Cell Line , Cell Proliferation , Embryonic Stem Cells/cytology , Fibroblast Growth Factor 4/genetics , Fibroblast Growth Factor 4/metabolism , Gene Expression Regulation/physiology , Genome-Wide Association Study/methods , Humans , Mice , Mice, Knockout , Neoplasms, Germ Cell and Embryonal/genetics , Neoplasms, Germ Cell and Embryonal/metabolism , Pluripotent Stem Cells/cytology , Signal Transduction/physiologyABSTRACT
Polycomb group (PcG) proteins bind and regulate hundreds of genes. Previous evidence has suggested that long-range chromatin interactions may contribute to the regulation of PcG target genes. Here, we adapted the Chromosome Conformation Capture on Chip (4C) assay to systematically map chromosomal interactions in Drosophila melanogaster larval brain tissue. Our results demonstrate that PcG target genes interact extensively with each other in nuclear space. These interactions are highly specific for PcG target genes, because non-target genes with either low or high expression show distinct interactions. Notably, interactions are mostly limited to genes on the same chromosome arm, and we demonstrate that a topological rather than a sequence-based mechanism is responsible for this constraint. Our results demonstrate that many interactions among PcG target genes exist and that these interactions are guided by overall chromosome architecture.
Subject(s)
Chromosomes/chemistry , Chromosomes/metabolism , Drosophila melanogaster/genetics , Repressor Proteins/metabolism , Animals , Brain/metabolism , Chromatin/metabolism , Chromosomes, Insect/chemistry , Chromosomes, Insect/genetics , Computational Biology , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Drosophila Proteins/genetics , Drosophila Proteins/metabolism , Drosophila melanogaster/metabolism , Gene Expression Profiling , Gene Expression Regulation , Genes, Homeobox/genetics , Histones/metabolism , Larva , Polycomb-Group Proteins , Protein Binding , Repressor Proteins/chemistryABSTRACT
Inappropriate Hedgehog (Hh) signaling underlies development of a subset of medulloblastomas, and tumors with elevated HH signaling activity express the stem cell self-renewal gene BMI1. To test whether Bmi1 is required for Hh-driven medulloblastoma development, we varied Bmi1 gene dosage in transgenic mice expressing an oncogenic Hh effector, SmoA1, driven by a glial fibrillary acidic protein (GFAP) promoter. Whereas 100% of SmoA1; Bmi1(+/+) or SmoA1;Bmi1(+/-) mice examined between postnatal (P) days 14 and 26 had typical medulloblastomas (N = 29), tumors were not detected in any of the SmoA1;Bmi1(-/-) animals examined (N = 6). Instead, small ectopic collections of cells were present in the region of greatest tumor load in SmoA1 animals, suggesting that medulloblastomas were initiated but failed to undergo expansion into frank tumors. Cells within these Bmi1(-/-) lesions expressed SmoA1 but were largely nonproliferative, in contrast to cells in Bmi1(+/+) tumors (6.2% vs 81.9% PCNA-positive, respectively). Ectopic cells were negative for the progenitor marker nestin, strongly GFAP-positive, and highly apoptotic, relative to Bmi1(+/+) tumor cells (29.6% vs 6.3% TUNEL-positive). The alterations in proliferation and apoptosis in SmoA1;Bmi1(-/-) ectopic cells are associated with reduced levels of Cyclin D1 and elevated expression of cyclin-dependent kinase inhibitor p19(Arf), two inversely regulated downstream targets of Bmi1. These data provide the first demonstration that Bmi1 is required for spontaneous de novo development of a solid tumor arising in the brain, suggest a crucial role for Bmi1-dependent, nestin-expressing progenitor cells in medulloblastoma expansion, and implicate Bmi1 as a key factor required for Hh pathway-driven tumorigenesis.
Subject(s)
Gene Expression Regulation, Neoplastic , Hedgehog Proteins/metabolism , Medulloblastoma/metabolism , Nuclear Proteins/physiology , Proto-Oncogene Proteins/physiology , Repressor Proteins/physiology , Animals , Brain Neoplasms/metabolism , Cell Proliferation , Cerebellum/metabolism , Cyclin D1/metabolism , Cyclin-Dependent Kinase Inhibitor p16/metabolism , Genotype , Glial Fibrillary Acidic Protein , Mice , Mice, Transgenic , Nerve Tissue Proteins/metabolism , Nuclear Proteins/metabolism , Polycomb Repressive Complex 1 , Promoter Regions, Genetic , Proto-Oncogene Proteins/metabolism , Repressor Proteins/metabolismABSTRACT
The Polycomb group and oncogene Bmi1 is required for the proliferation of various differentiated cells and for the self-renewal of stem cells and leukemic cancer stem cells. Repression of the Ink4a/Arf locus is a well described mechanism through which Bmi1 can exert its proliferative effects. However, we now demonstrate in an orthotopic transplantation model for glioma, a type of cancer harboring cancer stem cells, that Bmi1 is also required for tumor development in an Ink4a/Arf-independent manner. Tumors derived from Bmi1;Ink4a/Arf doubly deficient astrocytes or neural stem cells have a later time of onset and different histological grading. Moreover, in the absence of Ink4a/Arf, Bmi1-deficient cells and tumors display changes in differentiation capacity.