PMLs and SPPs David A. Stephens Introduction Biological Background Imaging Dynamic Imaging PMLs and SPPs Biological Hypothesis Data Analysis Statistical Analysis SPPs David A. Stephens Randomization Tests Likelihood Results Department of Mathematics Conclusions Imperial College London d.stephens@imperial.ac.uk http://stats.ma.ic.ac.uk/ ∼ das01/ 21st February 2006
PMLs and SPPs Biological Objective Introduction To understand organization in the mammalian cell Biological Background nucleus via analysis of its components. Imaging Dynamic Imaging Biological Hypothesis Data Analysis The mammalian cell nucleus is a membrane-bound Statistical Analysis organelle that contains the machinery essential for SPPs Randomization Tests gene expression. Although early studies suggested Likelihood that little organization exists within this compartment, Results more contemporary studies have identified an Conclusions increasing number of specialized domains or subnuclear organelles within the nucleus .... An extensive effort is currently underway by numerous laboratories to determine the biological function(s) associated with each domain. Spector (2001) J. Cell Sci.
PMLs and SPPs Nuclear Domains Introduction Biological Background Imaging Dynamic Imaging Biological Hypothesis Data Analysis Statistical Analysis SPPs Randomization Tests Likelihood Results Conclusions Spector (2001) J. Cell Sci.
PMLs and SPPs Nuclear Domains Introduction Biological Background Imaging • Cajal body • Nucleolar proteins Dynamic Imaging Biological Hypothesis Data Analysis • Nuclear Gems • Nuclear Lamina Statistical Analysis • Chromatin proteins • RNA pol II transcription SPPs Randomization Tests factors Likelihood • PML body Results • OPT domain • Splicing Factor Enriched Conclusions Speckles • Nuclear Pore • Sam68 body • Cleavage body • Nuclear Diffuse proteins • Heterochromatin proteins • Perinuclear Compartment • Paraspeckles
PMLs and SPPs Nuclear Domains Introduction Biological Background Imaging • Cajal body • Nucleolar proteins Dynamic Imaging Biological Hypothesis Data Analysis • Nuclear Gems • Nuclear Lamina Statistical Analysis • Chromatin proteins • RNA pol II transcription SPPs Randomization Tests factors Likelihood • PML body Results • OPT domain • Splicing Factor Enriched Conclusions Speckles • Nuclear Pore • Sam68 body • Cleavage body • Nuclear Diffuse proteins • Heterochromatin proteins • Perinuclear Compartment • Paraspeckles
PMLs and SPPs PML Bodies Introduction Biological Background The promyelocytic leukemia (PML) nuclear body Imaging Dynamic Imaging is a nuclear matrix-associated structure 250-500 Biological Hypothesis Data Analysis nm in diameter that is present in the nucleus of Statistical Analysis most cell lines ... this subnuclear domain has SPPs Randomization Tests been reported to be rich in RNA and a site of Likelihood nascent RNA synthesis, implicating its direct Results involvement in the regulation of gene expression Conclusions ... electron spectroscopic imaging (ESI) demonstrates that the core of the PML nuclear body is a dense, protein-based structure ... which does not contain detectable nucleic acid. Boisvert et al. (2000) J. Cell Sci.
PMLs and SPPs PML Bodies Introduction Biological Background Imaging Dynamic Imaging Biological Hypothesis Data Analysis Statistical Analysis SPPs Randomization Tests Likelihood Results Conclusions
PMLs and SPPs PML Bodies Introduction Biological Background Imaging • aggregation of PML and other proteins. Dynamic Imaging Biological Hypothesis • vary in size from 0.3 µ m to 1.0 µ m in diameter. Data Analysis • typical mammalian nucleus contains 10-30 of these Statistical Analysis SPPs structures. Randomization Tests Likelihood • also called ND10, PODs (PML oncogenic domains) Results and Kr bodies. Conclusions • several other proteins (Sp100, SUMO1, HAUSP and CBP) have been localized to this nuclear domain. • PML bodies have been suggested to play a role in aspects of transcriptional regulation and appear to be targets of viral infection.
PMLs and SPPs Protein composition of the PML body Introduction Biological Background Imaging Dynamic Imaging Biological Hypothesis Protein Function BLM DNA helicase; complexes with RP-A and RAD51 at PML bodies Data Analysis CBP Protein acetyl-transferase, co-activator Daxx Involved in Fas-mediated apoptosis, transcriptional Statistical Analysis repression, and chromatin remodeling SPPs Hipk2 Serine/threonine kinase; associates with p53 and Randomization Tests CBP and is recruited to PML bodies Likelihood Mdm2 Regulates p53 protein levels Results NBS1 Involved in DNA repair; complexes with Mre11 and Rad50 at PML bodies; Conclusions complexes with Mre11 and TRF1 at PML bodies in ALT cells p53 Tumour-suppressor, transcription factor PML Protein involved in several nuclear functions Sp100 Transciptional repressor SUMO-1 Small ubiquitin-like modifier (post-translational modification) TRF1 Telomere-binding protein; colocalizes with PML bodies in ALT cells TRF2 Telomere-binding protein; colocalizes with PML bodies in ALT cells Ching et al. (2005) J. Cell Sci.
PMLs and SPPs Formation The appearance of ND10 after mitosis must result Introduction from a nucleation event possibly through homo- or Biological Background Imaging heteromultimerization of PML. This event may take Dynamic Imaging Biological Hypothesis place at specific nuclear deposition sites... . Data Analysis Transcriptional activation, for instance by interferon, Statistical Analysis can upregulate PML expression ... nucleating SPPs additional aggregation sites. SUMO-1 Randomization Tests Likelihood modification-demodification of PML (third level) may Results lead to a reversible accumulation of Daxx to ND10 Conclusions (fourth level), increasing or decreasing the availability of this protein for alternative binding partners (DNA, CENP-C, Fas, Pax3, DNA methyltransferase), and thus regulate corresponding functions. The complexity and plasticity of such a supramolecular regulatory mechanism are evident and envisioned structurally as a network of interacting proteins with PML at its core. Ishov et. al. (1999), J. Cell. Biol.
PMLs and SPPs Formation Introduction Biological Background Imaging Dynamic Imaging Biological Hypothesis Data Analysis Statistical Analysis SPPs Randomization Tests Likelihood Results Conclusions
PMLs and SPPs Function Introduction Biological Background Imaging Dynamic Imaging PML bodies are implicated in many cellular processes Biological Hypothesis Data Analysis • tumour suppression Statistical Analysis • apoptosis, SPPs Randomization Tests Likelihood • DNA replication and repair, Results • proteolysis and response to viral infection Conclusions • nuclear protein storage, • gene regulation and transcription, PML protein is implicated in acute pro-myelocytic leukaemia (APL)
PMLs and SPPs Putative Functional Model Introduction Biological Background PML bodies consist of smaller PML-containing Imaging subunits that interact with high-order chromatin fibers Dynamic Imaging Biological Hypothesis possibly through matrix attachment regions (MARs). Data Analysis PML bodies function to control the concentration of Statistical Analysis transcriptional activators and repressors within the SPPs Randomization Tests local chromatin environment. Upon stimulation of Likelihood cellular pathways, these factors are released from Results PML bodies and bind enhancer/promoter elements in Conclusions the surrounding chromatin. Upon infection with double-stranded DNA viruses such as HCMV, viral genomes localize to the surface of PML bodies. This targeting is mediated through cellular factors such as Daxx, which are normally associated with PML bodies, and viral tegument proteins, such as pp71. Ching et. al. (2005), J. Cell. Sci.
PMLs and SPPs Schematic Schematic representation of PML bodies in the nucleoplasm. Introduction Biological Background Imaging Dynamic Imaging Biological Hypothesis Data Analysis Statistical Analysis SPPs Randomization Tests Likelihood Results Conclusions
PMLs and SPPs Imaging (Batty, 2006) Nuclear compartments imaged using Introduction immunofluorescence staining and confocal microscopy Biological Background Imaging Dynamic Imaging PML bodies Biological Hypothesis Data Analysis Statistical Analysis SPPs Randomization Tests Likelihood Results Conclusions
PMLs and SPPs Centromeres Introduction Biological Background Imaging Dynamic Imaging Biological Hypothesis Data Analysis Statistical Analysis SPPs Randomization Tests Likelihood Results Conclusions
PMLs and SPPs Nascent RNA Introduction Biological Background Imaging Dynamic Imaging Biological Hypothesis Data Analysis Statistical Analysis SPPs Randomization Tests Likelihood Results Conclusions
PMLs and SPPs RNA polymerase II Introduction Biological Background Imaging Dynamic Imaging Biological Hypothesis Data Analysis Statistical Analysis SPPs Randomization Tests Likelihood Results Conclusions
PMLs and SPPs Acetylated histone Introduction Biological Background Imaging Dynamic Imaging Biological Hypothesis Data Analysis Statistical Analysis SPPs Randomization Tests Likelihood Results Conclusions
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