The Polycomb Group Protein Suz12 Is Required for Embryonic Stem Cell Differentiation▿ †

Polycomb group (PcG) proteins form multiprotein complexes, called Polycomb repressive complexes (PRCs). PRC2 contains the PcG proteins EZH2, SUZ12, and EED and represses transcription through methylation of lysine (K) 27 of histone H3 (H3). Suz12 is essential for PRC2 activity and its inactivation results in early lethality of mouse embryos. Here, we demonstrate that Suz12−/− mouse embryonic stem (ES) cells can be established and expanded in tissue culture. The Suz12−/− ES cells are characterized by global loss of H3K27 trimethylation (H3K27me3) and higher expression levels of differentiation-specific genes. Moreover, Suz12−/− ES cells are impaired in proper differentiation, resulting in a lack of repression of ES cell markers as well as activation of differentiation-specific genes. Finally, we demonstrate that the PcGs are actively recruited to several genes during ES cell differentiation, which despite an increase in H3K27me3 levels is not always sufficient to prevent transcriptional activation. In summary, we demonstrate that Suz12 is required for the establishment of specific expression programs required for ES cell differentiation. Furthermore, we provide evidence that PcGs have different mechanisms to regulate transcription during cellular differentiation

Lean thinking to support RFQ process modelling: The Hutchinson case

Lean thinking promotes a new vision of habitual processes in order to highlight the hidden obstacles that preclude the achievement of the best performance. Typically, most of the enterprise's processes are currently revised according to the lean thinking perspective and, because of its holistic approach, the analysis also includes the relationships of processes operating along the whole product lifecycle. In this paper one of these processes - the Request For Quotation (RFQ) - and its impact on the enterprise's overall performance, is analysed. The Request For Quotation is understood as a sequence of activities that companies perform in the initial phase of each project where customer needs are analysed in order to make a proposal that contains both economic and schedule items. The quotation has to be conform with the product specifications and requirements for which the company has been consulted; additionally, it has to be competitive and made in a short time. This paper presents a model for RFQ developed for Hutchinson Company using mapping of the information flow to support Lean Organization in order to reduce information wastes and allow continuous process improvement activities. The model focuses on efforts and time reduction and evaluates its performanc

Mdm4 (Mdmx) Regulates p53-Induced Growth Arrest and Neuronal Cell Death during Early Embryonic Mouse Development

We report here the characterization of a mutant mouse line with a specific gene trap event in the Mdm4 locus. Absence of Mdm4 expression results in embryonic lethality (10.5 days postcoitum [dpc]), which was rescued by transferring the Mdm4 mutation into a Trp53-null background. Mutant embryos were characterized by overall growth deficiency, anemia, improper neural tube closure, and dilation of lateral ventricles. In situ analysis demonstrated increased levels of p21(CIP1/Waf1) and lower levels of Cyclin E and proliferating cell nuclear antigen expression. Consistent with lack of 5-bromo-2′-deoxyuridine incorporation, these data suggest a block of mutant embryo cells in the G(1) phase of the cell cycle. Accordingly, Mdm4-deficient mouse embryonic fibroblasts manifested a greatly reduced proliferative capacity in culture. Moreover, extensive p53-dependent cell death was specifically detected in the developing central nervous system of the Mdm4 mutant embryos. These findings unambiguously assign a critical role for Mdm4 as a negative regulator of p53 and suggest that Mdm4 could contribute to neoplasias retaining wild-type Trp53. Finally, we provide evidence indicating that Mdm4 plays no role on cell proliferation or cell cycle control that is distinct from its ability to modulate p53 function

PML-RAR induces promyelocytic leukemias with high efficiency following retroviral gene transfer into purified murine hematopoietic progenitors

Acute promyelocytic leukemia (APL) is associated with chromosomal translocations resulting in fusion proteins of the retinoic acid receptor (RAR). Here, we report a novel murine model system for APL, based on the transduction of purified murine hematopoietic progenitors (lin−) using high-titer retroviral vectors encoding promyelocytic leukemia–RAR (PML-RAR), and the green fluorescent protein (GFP) as a marker. PML-RAR–expressing lin− cells were impaired in their ability to undergo terminal myeloid differentiation and showed increased proliferative potential in vitro. Inoculation of transduced lin− cells into syngeneic, irradiated mice resulted in the development of retinoic acid-sensitive promyelocytic leukemias at high frequency (> 80%) and short latency (approximately 4 months). Morphologic and immunophenotypic analysis revealed no gross abnormalities of the preleukemic bone marrows. However, hematopoietic progenitors from PML-RAR preleukemic mice showed a severe impairment in their ability to undergo myeloid differentiation in vitro. This result, together with the monoclonality or oligoclonality of the leukemic blasts, supports a “multiple-hit” model, where the fusion protein causes a “preleukemic” phase, and leukemia occurs after additional genetic lesions. This model system faithfully reproduces the main characteristics of human APL and represents a versatile tool for the in vitro and in vivo study of mechanisms of leukemogenesis and the design of protocols for differentiation treatment