Marleen Blom

Interactions among Polycomb Domains Are Guided by Chromosome Architecture

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.

Prolonged Ezh2 Depletion in Glioblastoma Causes a Robust Switch in Cell Fate Resulting in Tumor Progression

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.

Tight regulation of ubiquitin-mediated DNA damage response by USP3 preserves the functional integrity of hematopoietic stem cells.

In vivo deletion of USP3, a deubiquitinating enzyme involved in DNA damage repair, increases the incidence of spontaneous cancer and impairs the proliferation and repopulation ability of HSCs.

Tight regulation of ubiquitin-mediated DNA damage response by USP3 preserves the functional integrity of hematopoietic stem cells

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; Usp3{delta}/{delta}), 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 Usp3{delta}/{delta} HSCs. Beyond the hematopoietic system, Usp3{delta}/{delta} animals spontaneously developed tumors, and primary Usp3{delta}/{delta} 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.

Deletion of Polycomb Repressive Complex 2 From Mouse Intestine Causes Loss of Stem Cells

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.

GFAP-Cre-mediated transgenic activation of Bmi1 results in pituitary tumors.

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 p16INK4A/Rb rather than on regulation of p19ARF/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.

Chromosome conformation capture on chip in single Drosophila melanogaster tissues.

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.