Characterisation of HPC3, A NEW HUMAN POLYCOMB GROUP PROTEIN Julia I. Bardos Thesis presented for the degree of Doctor of Philosophy from the University of London August 2001 Molecular Structure and Function Laboratory Imperial Cancer Research Fund 44 Lincoln’s Inn Fields London WC2A 3PX ProQuest Number: U643553 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. uest. ProQuest U643553 Published by ProQuest LLC(2016). Copyright of the Dissertation is held by the Author. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code. Microform Edition © ProQuest LLC. ProQuest LLC 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106-1346 A b s t r a c t A b str a c t Polycomb Group (PcG) proteins are a conserved group of transcriptional repressors, mainly known for their role in stably maintaining the repressed state of homeotic and Hox genes, after their expression patterns have been established early in embryonic development. Thus, the Polycomb Group constitutes an important part of a cellular transcriptional memory system. Loss of PcG function leads to homeotic transformations in Drosophila and corresponding shifts in Hox gene expression in vertebrates. PcG proteins form multiprotein complexes of varying composition that associate with chromatin, and it has been postulated that they achieve gene silencing by altering higher order chromatin structure. But the exact mechanism by which PcG proteins establish repression is still unclear. A yeast two-hybrid screen with the PcG protein RINGl as bait led to the identification of a new protein that shows strong sequence similarity with other Polycomb homologues and was therefore termed human Polycomb 3 (HPC3). The hpc3 gene and its murine homologue are ubiquitously expressed in adult tissues, but in contrast to other polycomb genes, mpc3 is highly expressed during embryogenesis up to 8.5 days post coitum and becomes drastically downregulated at later stages, indicating a possible unique role of pc3 early in development. Analysis of the protein-protein interactions showed that the HPC3 C-Box is necessary and sufficient for the interaction with RINGl, but that this interaction depends only partially on the RING finger domain of RINGl. HPC3 also interacts with the PcG protein BMIl and colocalises with BMIl and RINGl in distinct nuclear domains, called PcG bodies. Consistent with its role as a PcG member, HPC3 is able to act as a long-range transcriptional repressor when targeted to a reporter gene. Surprisingly, analysis of the different domains of HPC3 revealed that its conserved C-Box, which mediates the repression function of the other Pc homologues, is not the repression domain of HPC3. Instead, an internal part of the HPC3 protein mediates silencing of reporter gene activity. Transient transfection experiments show that the same domain is involved in mediating localisation of a HPC3-GFP fusion protein to distinct nuclear foci, indicating that it represents a functional domain of the HPC3 protein. These data suggest that HPC3 is a new member of the mammalian Polycomb Group in terms that it colocalises and interacts with other PcG proteins and is able to repress gene activity. At the same time, its expression profile suggests that it might have a unique role in embryogenesis. Another feature that distinguishes HPC3 from the other Pc homologues is that its conserved C-Box is not the domain responsible for the repression activity of HPC3, but that an internal part mediates transcriptional repression as well as localisation of the protein. Through interaction with other proteins, HPC3 potentially becomes involved in processes like cell proliferation, senescence, apoptosis and leukaemogenesis. T a b l e o f C o n teo ts T a b le o f C o n ten ts A b s t r a c t ................................................................................................................................................2 T a b l e o f C o n t e n t s ......................................................................................................................... 3 L ist o f F ig u r e s ...................................................................................................................................7 L ist o f T a b l e s .................................................................................................................................... 9 A bbreviations ..................................................................................................................................10 A cknowledgements .................................................................................................................... 12 Chapter 1: Introduction.............................................................................................................15 Regulators of homeotic gene expression ............................................................... 15 The Polycomb Group ..............................................................................................16 The Trithorax Group ...............................................................................................19 The Polycomb protein: Domains, repression and homologues ...........................22 The chromodomain ..............................................................................................22 The C-Box............................................................................................................24 Pc homologues.................................................................................................... 25 RinglA/RinglB/RINGl......................................................................................... 26 Psc/Bmil/BMIl and Su(z)2/Mell8........................................................................27 Other members of the PcG .....................................................................................29 Ph/MPh/HPH...................................................................................................... 29 E(z)/Ezh/EZH...................................................................................................... 30 Esc/Eed/EED ........................................................................................................31 RAo/yyy.......................................................................... 32 PcG complexes........................................................................................................ 33 Different PcG complexes have different compositions .....................................34 PcG complex assembly ....................................................................................... 38 Mechanisms of PcG-mediated repression.............................................................43 The heterochromatin connection ........................................................................43 Histone deacetylation vs. nucleosome remodelling ......................................... 44 Nuclear compartmentalisation ........................................................................... 46 Cellular memory ...................................................................................................... 50 Maintenance through cell division.....................................................................50 Long-term maintenance ......................................................................................51 Conclusions ..............................................................................................................54 This thesis................................................................................................................ 55 T a b l e o f C o n t e n t s C h a p t e r 2: HPC3, a new Polycomb homologue..................................................57 Comparison of HPC3 with other Polycomb proteins........................................... 57 Protein domains of HPC3....................................................................................... 58 A mouse homologue of HPC3 ............................................................................... 60 Expression of the human and mouse pc3 gene..................................................... 61 Northern Blot analysis ........................................................................................ 61 In situ hybridisation.............................................................................................63 Summary .................................................................................................................. 63 CHAPTERS: The HPC3 protein................................................................................77 Characterisation of polyclonal anti-HPC3 peptide antisera................................. 77 Characterisation by immunofluorescence.........................................................77 Characterisation by Western blot analysis ........................................................78 Analysis of the ASA 12 antiserum ..........................................................................79 Western blot analysis ...........................................................................................79 Immunoprécipitation analysis ...........................................................................