CULTURED HUMAN CELLS
Dissertation
zur Erlangung des akademischen Grades eines Doktors der Naturwissenschaften
(Dr. rer. nat.)
an der Universität Konstanz
vorgelegt von Sandra Kreitz
Konstanz, Dezember 2002
Referent: Prof. Dr. W. Boos
Die vorliegende Arbeit wurde in der Zeit von Februar 1999 bis Dezember 2002 am Lehrstuhl für Molekulare Genetik unter der Leitung von Herrn Prof. Dr. Rolf Knippers an der Universität Konstanz angefertigt. Herrn Prof. Dr. Rolf Knippers danke ich für die Bereitstellung des Themas, sein stetes Interesse am Fortgang der Arbeit sowie seine Diskussionsbereitschaft.
Allen Mitgliedern der Arbeitsgruppe, natürlich auch den ehemaligen, die mich während meiner Promotion begleitet haben, danke ich für das gute Arbeitsklima und die Hilfsbereitschaft. Vor allem Martina Baack, Monika Kulartz, Ekkehard Hiller, Elena- Catalina Damoc, Esther Biermann, und Marion Ritzi möchte ich für die gute Zusammenarbeit danken! An dieser Stelle geht auch ein Dankeschön an Ferdinand Kappes für seine Hilfe bei der Durchführung der [32P]-Markierungsversuche. Ebenso möchte ich mich bei Dr. Frank O. Fackelmayer und seiner „Crew“ bedanken, die sehr viel Mühe und Zeit in den Versuch investiert haben, stabile GFP-Orc1p-exprimierende Zelllinien herzustellen.
Besonderer Dank gebührt Martina Baack. Sie hat mit vielen anregenden Diskussionen und Ideen sehr zum Gelingen dieser Arbeit beigetragen. Außerdem möchte ich mich bei ihr für ihre Freundschaft auch über die Grenzen des Labors hinaus bedanken.
Für die kritische Durchsicht des Manuskripts bedanke ich mich bei Martina Baack, Monika Kulartz, Daniel Schaarschmidt und vor allem bei Dr. Frank O. Fackelmayer und Dr. Eva-Maria Ladenburger.
Nicht zuletzt möchte ich mich bei meinen Eltern bedanken, die mir diese Ausbildung ermöglicht haben und auf die ich mich immer verlassen konnte. Ein besonderes Dankeschön geht auch an meine Schwester Claudia, die mir ihr Auto für längere Zeit zur Verfügung gestellt und mir damit so manch langwierige Zellsynchronisation wesentlich erleichtert hat.
PUBLICATIONS
THIS THESIS IS BASED ON THE FOLLOWING PUBLICATIONS:
Kreitz S, Fackelmayer FO, Gerdes J, Knippers R (2000) The proliferation-specific human Ki-67 protein is a constituent of compact chromatin. Exp Cell Res. 261(1): 284- 92.
Kreitz S, Ritzi M, Baack M, Knippers R (2001) The human origin recognition complex protein 1 dissociates from chromatin during S-phase in HeLa cells. J Biol Chem. 276(9):
6337-42.
Kreitz S, Baack M, Hiller E, Knippers R (2002) Changes in the Origin Recognition Complex in Proliferating Human Cells. (submitted)
ADDITIONAL PUBLICATIONS:
Kreitz S, Knippers R (2001) Regulation of genome replication and genome repair.
Futura 16(1): 5-12
Biermann E, Baack M, Kreitz S, Knippers R (2002) Synthesis and turnover of the replicative Cdc6 protein during the HeLa cell cycle. Eur J Biochem 269(3): 1040-6.
Kulartz M, Kreitz S, Hiller E, Damoc E-C, Przybylski M, Knippers R (2002) Expression and phosphorylation of the replication regulator protein Geminin.
(submitted)
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2
TABLE OF CONTENTS
Table of Figures VII
Abbreviations VIII
Summary X
Zusammenfassung XII
GENERAL INTRODUCTION:
NUCLEAR ARCHITECTURE AND CHROMATIN STRUCTURE
The Nucleus 1
The Structural Organization of DNA in Eukaryotes 2 The Functional Subcompartments of the Nucleus 3
The Nuclear Scaffold 4
PART I:
THE KI-67 PROTEIN
THE PROLIFERATION-SPECIFIC HUMAN KI-67 PROTEIN IS A CONSTITUENT OF COMPACT CHROMATIN
Summary 8
Introduction 9
Experimental Procedures 11
Cell Culture 11
Cell Fractionation 11
Chromatin Preparation 11
Chromatin Fractionation 12
Nuclear Matrix Preparation 12
Western Blotting 13
Results 14
Localization of Ki-67p in Nuclear Matrix Preparations 14
Chromatin Fractionation 15
A Nuclear Binding Site of Ki-67p is Chromatin 17
Discussion 23
Subsequent Investigations 25
4 3
PART II:
THE ORC1 PROTEIN
INITIATION OF GENOME REPLICATION:
AN OVERVIEW
The Eukaryotic Cell cycle 28
Origins of DNA Replication 30
Yeast Origins 31
Metazoan Origins 32
Establishment of the Pre-Replication Complex 33
The Origin Recognition Complex 35
The Loading Factors Cdc6p and Cdt1p 35
The MCM Proteins 36
Activation of Licensed Origins 38
Mechanisms to Prevent Re-Replication 39
THE HUMAN ORIGIN RECOGNITION COMPLEX PROTEIN 1 DISSOCIATES FROM CHROMATIN DURING S-PHASE IN HELA CELLS
Summary 41
Introduction 42
Experimental Procedures 44
Cell Culture 44
Cell Fractionation 44
Chromatin Fractionation 44
Chromatin Preparation 44
Antibodies 44
Results 46
Characterization of Antibodies 46
hOrc1p and hOrc2p on Chromatin 47
hOrc1p and hOrc2p in Synchronized Cells 50
Discussion 54
5
6
CHANGES IN THE ORIGIN RECOGNITION COMPLEX IN PROLIFERATING HUMAN CELLS
Summary 57
Introduction 58
Experimental Procedures 60
Cell Culture, Synchronization and FACS-Analysis 60 Cell Fractionation, Total Cell Extract and S20-Extract 60
Expression Plasmids 61
Proteasome Inhibition and
In Vivo Ubiquitination Assay 61
Transfection 62
In Vivo Labeling with [35S]-Methionine 62 In Vivo Labeling with [32P]-Ortho-Phosphate 62 In Vitro Transcription-Translation and
In Vitro Phosphorylation Assay 62
Antibodies and Immunoblotting 63
Results 64
ORC Proteins on Chromatin 64
Expression of hOrc1p 67
Conditions for hOrc1p Degradation 70
Discussion 76
DISCUSSION CONCERNING
THE HUMAN ORIGIN RECOGNITION COMPLEX
ORC is localized to two different Chromatin Compartments 80 The Subunit Composition of the hORC 81 Changes in the Composition of hORC during the Cell Cycle 83 The Release of hOrc1p Depends on Ongoing Replication 84 HOrc1p is Phosphorylated In Vitro and In Vivo 85 Degradation of hOrc1p During S-Phase 87 Possible Roles of hOrc1p in Re-Replication Control 89 Possible Roles of hOrc1p in the Selection of
Origin Binding Sites 90
REFERENCES
7
LIST OF FIGURES
FIG 1-1 A model of the condensed chromatin fiber 2 FIG 1-2 Functional subcompartments of the nucleus 4 FIG 1-3 A model for the structure of an interphase chromosome 5
FIG 2-1 Nuclear matrix preparations 15
FIG 2-2 Chromatin fractionation 16
FIG 2-3 Ki-67p on nuclei and chromatin prepared at low ionic strength 18 FIG 2-4 Sucrose gradient centrifugation 19 FIG 2-5 Micrococcal nuclease digestion of isolated chromatin 20 FIG 2-6 Sucrose gradient centrifugation of chromatin fragments 21 FIG 2-7 Precipitation of DNA fragments by magnesium salts 22 FIG 3-1 Overview of the cell cycle control in mammals 30 FIG 3-2 Origin structure in the yeast S. cerevisiae 31 FIG 3-3 A model for pre-replicative complex formation in yeast 34 FIG 4-1 Characterization of antibodies 46
FIG 4-2 Chromatin fractionation 48
FIG 4-3 Differential cell fractionation 49 FIG 4-4 Co-immunoprecipitation of hOrc1p and hOrc2p 50 FIG 4-5 Chromatin-bound hOrc1p and hOrc2p in synchronized
HeLa cells 51
FIG 4-6 hOrc1p/hOrc2p-complexes dissociate from their
chromatin sites in S-phase 53
FIG 5-1 Distribution of ORC proteins in cell extracts 65 FIG 5-2 Interactions between ORC proteins 66 FIG 5-3 hOrc1p is released from chromatin during S-phase followed
by degradation, and is resynthesized during G1-phase 69 FIG 5-4 Release of hOrc1p during S-phase is dependent on the
progression of the replication fork 70
FIG 5-5 hOrc1p is phosphorylated in vivo 71 FIG 5-6 hOrc1p is phosphorylated in vitro 73 FIG 5-7 hOrc1p is stabilized by proteasome inhibitors and is
polyubiquitinated in vivo 74
ABBREVIATIONS
Metric conversions:
k kilo- 103
m milli- 10-3
µ micro- 10-6
n nano- 10-9
A adenine
ALLN N-acetyl-leucyl-leucyl-norleucynal, Calpain Inhibitor I ATP adenosin triphosphate
bp base pair(s)
BrdU 5-bromo-2-deoxyuridine
C cytosine
Cg Cricetulus griseus, Chinese hamster Ci Curie, 3.7x1010 disintegrations/s Dm Drosophila melanogaster, fruit fly DNA deoxyribonucleic acid
DNase deoxyribonuclease
DTT dithiothreitol
e.g. exempli gratia, for instance EDTA ethylendiamin tetraacetate
EGTA ethylenglycol-bis-(2-aminoethyl)-tetraacetate
FIG figure
g gravitational accelaration
G guanine
GFP green fluorescent protein GST glutathione S-transferase
h human, hour(s)
HEPES N-2-hydroxyethylpiperazin-N’2-ethansulfonic acid
His histidine
i.e. id est, that is to say
kb kilobase; 1000 bases (or base pairs)
kD kilodalton
l liter(s)
M molarity; number of moles per liter of solution
m meter
Mg-132 carbobenzoxy-L-leucyl-L-leucyl-L-leucinal
min minutes
mRNA messenger ribonucleic acid
Mt Methanobacterium thermoautotrophicum Ni-NTA nickel-nitrilotriacetic acid
NTP ribonucleoside 5’-triphosphate
PIPES piperazine-N,N’-bis(2-ethanesulfonic acid) Rn Rattus norvegicus, Norway Rat
RNA ribonucleic acid
RNase ribonuclease
RPA replication protein A rpm revolutions per minute rRNA ribosomal ribonucleic acid
RT room temperature
S Svedberg
Sc Saccharomyces cerevisiae, budding yeast SDS sodium dodecyl sulfate
SDS-PAGE SDS (denaturing) polyacrylamid gel elecrophoresis
sec second(s)
Sp Schizosaccharomyces pombe, fission yeast
T thymine
TCA trichloracidic acid
Tris tris-(hydroxymethyl)-aminomethane TritonX-100 t-octylphenoxypolyethoxyethanol Xl Xenopus laevis, African Clawed Frog
SUMMARY
For an ordered expression and replication of the eukaryotic genome, structural as well as regulatory proteins are indispensable. This thesis deals with the characterization of an example of both categories: the Ki-67 protein involved in the organization of heterochromatin and Orc1, a protein essential for the initiation of DNA replication.
PART I: THE KI-67 PROTEIN
AN EXAMPLE FOR A STRUCTURAL NUCLEAR PROTEIN
The Ki-67 protein is a nuclear protein, which is tightly associated with cell proliferation and therefore widely used as a prognostic proliferation marker in histopathological research and practice. Its functional significance has long been elusive, and has still not been clarified completely. In the past few years, several studies concerning the localization of Ki-67p have lead to the picture that Ki-67p performs architectural functions by interacting with satellite DNA-containing heterochromatic regions of the genome. However, these results are based on immunofluorescence experiments, and a direct association of Ki-67p with DNA in vivo has not been presented yet. An aim of this study was therefore to perform a systematic biochemical analysis of the location of Ki-67p in the nucleus.
Application of cell fractionation and nuclease digestion revealed that Ki-67p resides at nuclear sites that are sensitive to moderately high salt concentrations, which could further be connected to chromatin regions hardly accessible to nucleases under isotonic conditions. However, chromatin prepared under very low ionic strength was more accessible and digestion with micrococcal nuclease released DNA fragments carrying Ki-67p. Results presented in this thesis demonstrate for the first time that Ki-67p is bound to DNA in vivo, and provide a useful basis for the future biochemical characterization of this protein.
PART II: THE ORC1 PROTEIN
AN EXAMPLE FOR A REPLICATION INITIATION PROTEIN
The precisely coordinated replication of the eukaryotic genome requires several key proteins involved in the initiation of replication. Although these replication factors are highly conserved among all investigated eukaryotes, differences in their behavior could be observed especially between yeast and higher eukaryotes. This thesis deals with the characterization of one of these factors, the human ORC complex. Consisting of six proteins, hOrc1p-hOrc6p, ORC recognizes replication origins on chromatin and recruits additional factors to eventually assemble the replication machinery at these sites.
Investigation of the composition of ORC in HeLa cells synchronized in G1-phase revealed the existence of two major forms of ORC, one comprised of hOrc1p-hOrc5p and one that lacks hOrc1p. Both forms differ in their binding properties to nuclear sites – while the tetrameric hOrc2p-hOrc5p complex is released at a salt concentration of 250 mM NaCl, the hOrc1p-containing form requires higher salt (320 mM) for dissociation and is also located to chromatin regions less accessible to micrococcal nuclease. HOrc6p, however, was not found to be part of these complexes and was predominantly localized to the cyto- and nucleoplasm.
Functional differences of both ORC forms become evident upon passage through S-phase when the hOrc1p-containing complexes are destabilized probably due to the dissociation of hOrc1p from chromatin. Evidence is provided that the release of hOrc1p is accompanied by its phosphorylation and depends on progression of replication forks.
Further experiments suggest that the released hOrc1p is ubiquitinated and subsequently degraded by the 26S-proteasome in a regulated manner. Thus, the cell cycle-regulated association of hOrc1p with DNA might represent a new mechanism to limit initiation at each origin to once per cell cycle, and therefore contributes to the maintenance of the genome by preventing re-replication.
ZUSAMMENFASSUNG
Für eine geordnete Expression und Replikation des eukaryotischen Genoms sind sowohl strukturelle als auch regulatorische Proteine von großer Bedeutung. In der vorliegenden Arbeit wurde jeweils ein Beispiel dieser beiden Kategorien näher charakterisiert: Ki-67, ein Protein, das an der Organisation von Heterochromatin beteiligt ist und das für die Initiation der Replikation essentielle Protein Orc1.
TEIL I: DAS KI-67 PROTEIN
EIN BEISPIEL FÜR EIN STRUKTURELLES KERNPROTEIN
B e i Ki-67p handelt es sich um ein Kernprotein, dessen Expression eng mit der Proliferation von Zellen verknüpft ist und daher als Proliferationsmarker sowohl in der histopathologischen Forschung als auch der Diagnostik vielfach Verwendung findet.
Die funktionelle Bedeutung von Ki-67p konnte bisher jedoch noch nicht geklärt werden. Lokalisationsstudien der letzten Jahre weisen jedoch darauf hin, dass Ki-67p eine Rolle beim Aufbau von Heterochromatin spielen könnte, da es mit hetero- chromatischen Bereichen des Genoms, die Satelliten-DNA enthalten, assoziiert vorliegt.
Da diese Ergebnisse jedoch mit Hilfe von Immunfluoreszenzstudien gewonnen wurden, liegen bisher noch keine überzeugende Daten für eine direkte Interaktion von Ki-67 mit DNA vor. Deshalb war es Ziel dieser Arbeit, die Lokalisation von Ki-67p in der Zelle mittels biochemischer Methoden systematisch zu untersuchen.
Fraktionierungen von HeLa Zellen und Nuklease-Verdaus von Kernen, die unter isotonen Bedingungen präpariert wurden, weisen darauf hin, dass Ki-67p bei mittleren Salzkonzentrationen von seinen Bindestellen dissoziiert und mit Bereichen des Chromatins assoziiert vorliegt, die für Nukleasen nur schwer zugänglich sind. Wurden die Kerne jedoch unter hypotonen Bedingungen präpariert, waren sie wesentlich zugänglicher für Mikrokokken Nuklease, so dass DNA-Fragmente freigesetzt wurden, die mit Ki-67p assoziiert vorlagen. Somit konnte zum ersten Mal in vivo eine direkte Bindung von Ki-67p an DNA nachgewiesen werden und die Basis für weitere biochemische Untersuchungen gelegt werden.
TEIL II: DAS ORC1 PROTEIN
EIN BEISPIEL FÜR EIN REPLIKATIONSPROTEIN
Für die koordinierte Replikation des eukaryotischen Genoms wird eine Reihe von Proteinen benötigt, die an der Initiation der Replikation beteiligt sind. Obwohl diese Faktoren in Eukaryoten hoch konserviert sind, konnten doch Unterschiede in ihrem Verhalten, insbesondere zwischen Hefen und höheren Eukaryoten, beobachtet werden.
In der vorliegenden Arbeit sollte einer dieser Faktoren, der humane „Origin Recognition Complex“ (ORC) näher charakterisiert werden. ORC besteht aus den sechs Unter- einheiten hOrc1p-hOrc6p und ist an der Erkennung der Replikations-Startstellen und der Rekrutierung weiterer Faktoren beteiligt, was letztendlich zur Einleitung der DNA- Replikation führt.
Bei den Untersuchungen zur Zusammensetzung von ORC in HeLa Zellen, die in der G1-Phase synchronisiert vorlagen, wurden zwei Hauptformen von ORC identifiziert — eine bestehend aus hOrc1p-hOrc5p und eine zweite, bei der hOrc1p nicht im Komplex vorhanden ist. Beide Formen zeigen ein unterschiedliches Bindeverhalten. Während hOrc1p-hOrc5p bei Salzkonzentrationen von 250 mM eluiert werden kann, benötigt man höhere Salzkonzentrationen (320 mM) für die Ablösung des hOrc1p-haltigen Komplexes, der außerdem in Nuclease-unzugänglichen Bereichen vorliegt. HOrc6p ist hingegen größtenteils im Cyto- und Nucleoplasma lokalisiert und kann in keiner der beiden ORC Fraktionen nachgewiesen werden.
Dass es zwischen den beiden Formen von ORC auch funktionelle Unterschiede gibt, konnte an S-Phase Zellen demonstriert werden. So konnte gezeigt werden, dass in Abhängigkeit des Fortschreitens der Replikationsgabel hOrc1p vom Chromatin dissoziiert und es dadurch zu einer Destabilisierung der verbleibenden Untereinheiten kommt. Es konnte außerdem gezeigt werden, dass hOrc1p parallel zu seiner Ablösung phosphoryliert wird. In weiteren Experimenten konnte belegt werden, dass hOrc1p vermutlich ubiquitin-abhängig über das 26S-Proteasom abgebaut wird. Diese zellzyklus-abhängige Regulation und Assoziation von hOrc1p an Chromatin könnte somit einen neuen Mechanismus darstellen, der gewährleistet, dass jeder Replikations- startpunkt nur einmal pro Zellzyklus aktiviert wird und somit eine Re-Replikation verhindert wird.
General Introduction:
Nuclear Architecture and Chromatin Structure
THE NUCLEUS
Eukaryotic cells, by definition, and in contrast to prokaryotic cells, have a nucleus to keep the genome separate from the rest of the contents of the cell, the cytoplasm, where most of the cell’s metabolic reactions occur.
The nuclear envelope is composed of two lipid bilayers of which the outer membrane is in structural continuity with the endoplasmatic reticulum. The inner membrane is attached to a fibrous meshwork of polypeptides, the nuclear lamina, which helps to determine nuclear shape and is involved in the dissolution and reformation of the nuclear envelope during cell division. At many sites the nuclear envelope is perforated by nuclear pores, which consist of multiprotein complexes to allow a regulated transport of macromolecules between the nuclear and cytoplasmatic compartments.
THE STRUCTURAL ORGANIZATION OF DNA IN EUKARYOTES
The nucleus has to maintain a highly organized structure to allow a stringent regulation of key genetic processes despite of the high concentrations of DNA, RNA and proteins.
This is accomplished by specialized proteins, which bind to and fold the DNA, generating a series of coils and loops in order to provide increasingly higher levels of organization. Although the DNA is very tightly folded, it is compacted in a way that allows it to become accessible to the many enzymes and factors involved in DNA metabolism.
The basic structure of chromatin is the organization of DNA into nucleosomes, where approximately 200 bp of DNA wind around a cylindrical protein core. The nucleosome core is composed of a histone octamer formed by each two H3-H4 and H2A-H2B dimers interacting with each other. However, this 10 nm-fiber structure, also known as
“beads on a string” is rarely adopted in a living cell. Instead, the nucleosomes are packed on top of one another, generating a compact fiber with a diameter of about 30 nm. Several models have been proposed to explain how nucleosomes are packed in the 30 nm-fiber and the one mostly propagated is known as the solenoid model. Crucial for compacting nucleosomal DNA is histone H1, although it is not understood in detail how H1 pulls nucleosomes together. A second mechanism for formation of the 30 nm-fiber probably involves the tails of the core histones, which may help to attach one nucleosome to another (FIG 1-1).
FIG 1-1 A model of the condensed chromatin fiber. The octameric core is shown as a disk. Each nucleosome associates with one molecule histone H1, and the fiber coils into a solenoid structure with a diameter of 30 nm. (adapted from M. Grunstein (1992))
The 30 nm-fiber is the basic constituent of both interphase chromatin and mitotic chromosomes. Several different remodeling complexes, that loosen DNA-histone contacts or change positions of nucleosomes along DNA, permit ready access to nucleosomal DNA by other proteins in the cell, particularly those involved in gene expression, DNA replication and repair (Wolffe and Guschin, 2000).
THE FUNCTIONAL SUBCOMPARTMENTS OF THE NUCLEUS
Besides the highly organized chromatin structure the nucleus is divided into functionally different compartments (FIG 1-2). The nucleolus is one of the most obvious structures seen in the nucleus of a eukaryotic cell when viewed in the light microscope, and is the site where ribosomal RNAs are transcribed, processed, and assembled into ribosomes together with the ribosomal proteins (Scheer and Weisenberger, 1994). Further compartments of the nucleus are formed by the occupation of discrete territories by interphase chromosomes (Lichter et al., 1988).
Interphase chromosomes are themselves dynamic and their positioning correlates with gene expression. For example, transcriptionally silent regions of the chromosomes are often associated with the nuclear envelope where the concentration of heterochromatin is believed to be especially high. When these same regions become transcriptionally active, they relocate towards the interior of the nucleus, which is richer in the components required for mRNA synthesis (Croft et al., 1999). The chromosome territories are separated from each other by a space called the “inter-chromatin- domain (ICD)-compartment” (Bridger et al., 1998a). The ICD-compartment surrounds the chromosome territories and may act as a channel system to direct newly synthesized RNA to the nuclear periphery. This is detectable by RNA-tracks leading from the place of synthesis to the periphery (Lawrence et al., 1989). Other subnuclear structures include interchromatin granule clusters (speckles), proposed to be storage site for fully mature snRNPs (small nuclear ribonucleoproteins), and nuclear bodies, whose precise function has not been elucidated yet (Matera, 1999). Like the nucleolus, these other nuclear structures lack membranes and are highly dynamic. Their appearance is probably the result of the tight association of protein and RNA components involved in the synthesis, assembly and storage of macromolecules involved in gene expression.
FIG 1-2 Functional subcompartments of the nucleus. The nucleus is an organelle with a tesselated structure composed of various functional compartments. The largest structures are the chromosome territories (represented by the colored “islands”) separated from each other by an interspace called the ICD-compartment. Embedded in the ICD-compartment are nuclear bodies, highly dynamic structures composed of protein and RNA components involved in the nucleic acid metabolism. (adapted from F. O. Fackelmayer (2000))
THE NUCLEAR SCAFFOLD
The spatial separation of these functional complexes may be accomplished by an intranuclear framework, on which chromosomes and other components of the nucleus are organized. The nuclear “scaffold” or “matrix” has been defined as the insoluble material left in the nucleus after a series of biochemical extraction steps. The nuclear scaffold contains the lamina with parts of the nuclear pore complexes, remaining structures of the nucleoli and a three-dimensional network of polymorphic fibers with a diameter of 20-50 nm (He et al., 1990). Attached to these fibers are protein complexes identified as transcription and replication factories (Hozak et al., 1994). After removal of these protein complexes the remaining fibers have a diameter of 2-3 nm. The main components of these fibers are RNA and hnRNP proteins (heterogeneous nuclear ribonucleoproteins), involved in the packing and processing of newly synthesized mRNA. The composition of additional proteins present in the nuclear matrix varies between different types of cells and tissues and involves transcription factors and steroid hormone receptors among others. Digestion with small amounts of RNase leads to a collapse of the fibers and a disintegration of the chromosome territories (Ma et al., 1999). Presumably, the RNA component plays an essential role in maintaining the structural integrity of the nuclear matrix, which is in turn important for the organization of the genome in chromosome territories.
The nuclear matrix has been proposed to play an important role in the organization of the genome in interphase cells. It has been postulated that the 30 nm-chromatin fiber is organized in loops of around 20,000-80,000 bp representing functional domains in vivo (FIG 1-3) (reviewed in Gasser et al., (1989)). Indeed, a rough correlation between active genes and the number of chromatin loops could be demonstrated with studies using Drosophila polytene chromosomes (Agard and Sedat, 1983). Using hybridization probes it could be demonstrated that certain loops always correspond to specific DNA sequences.
Specific DNA sequences called SARs or MARs (scaffold- or matrix-attachment regions) have been proposed to form the base of the chromosomal loops (Bonifer et al., 1991). SAR/MAR elements are stretches of about 200-3000 bp, characterized by an AT-rich sequence and they are usually localized in the non-coding region of the genome near actively transcribed genes in the neighborhood of regulatory elements such as enhancers and promoters.
FIG 1-3 A model for the structure of an interphase chromosome. A section of an interphase chromosome is shown folded into a series of looped domains containing double-helical DNA condensed into a 30 nm-fiber. The DNA loops are anchored to the nuclear matrix by certain DNA elements, called SARs (scaffold attachment regions) or MARs (matrix attachment regions), which are able to interact with proteins located in the nuclear matrix. Individual loops can decondense, when the cell requires direct access to the DNA packaged in these loops. (adapted from F. O. Fackelmayer (2000))
Different proteins that bind to SARs in a sequence-specific manner have been identified and further characterized in order to shed light on the in vivo function of SARs. Factors found so far include proteins with additional well known functions, as for example topoisomerase II, histone H1 and lamin B1 as well as scaffold attachment factors (SAF-A and SAF-B), both involved in nuclear architecture and mRNA processing.
SAF-A is one of the most abundant proteins found in nuclear matrix preparations and the only one among those, which bind specifically to SARs. It therefore seems to represent one of the more important SAR-binding proteins, at least in a quantitative manner.
Taken together, by means of such chromosomal attachment sites, the matrix might help to organize chromosomes, localize genes, and regulate gene expression and DNA replication.
The picture drawn so far gives a brief overview on the organization of the nucleus and chromatin. However, for an ordered expression and replication of the genome additional proteins are associated with chromatin. These include structural proteins, which are required for the organization of chromatin within the small space it confines and genetically more important proteins that play important roles in the regulation of these processes.
This thesis deals with an example from both categories of non-histone chromatin proteins. The Ki-67 protein is a structural protein involved in the organization of heterochromatin, and the Orc1 protein is involved in the initiation and regulation of genome replication.
The Ki-67 Protein:
An Example for a Structural Nuclear Protein
The Proliferation-Specific Human Ki-67 Protein is a Constituent of Compact Chromatin
SUMMARY
The human nuclear Ki-67 protein (Ki-67p) is expressed in proliferating but not in quiescent cells, and is therefore widely used as a proliferation marker in histopathological research and practice. However, information regarding its intranuclear location is scarce and controversial. Here we describe the results of cell fractionation and nuclease digestion experiments using nuclei isolated from human HeLa cells in interphase. Ki-67p dissociates from its nuclear binding sites at 0.3-0.4 M NaCl, and gradient centrifugations indicate that the released Ki-67p is most likely a single molecular entity and not complexed to other proteins. In nuclei prepared under physiological salt conditions, the binding sites are largely resistant against micrococcal nuclease. However, when prepared at very low ionic strength, chromatin regions with associated Ki-67p become accessible to micrococcal nuclease, producing chromatin fragments that carry bound Ki-67p. We conclude that Ki-67p is a chromatin protein and resides at densely packed regions, probably heterochromatin. Our data provide a profound basis for further biochemical research on this human nuclear protein.
INTRODUCTION
The nuclear Ki-67 protein (Ki-67p) is exclusively expressed in proliferating cells and can therefore serve as a marker to determine the proportion of proliferating cells in human cell populations (Brown et al., 1988; Gerdes et al., 1984a; Gerdes et al., 1984b;
Ralfkiaer et al., 1986). In fact, Ki-67p-specific monoclonal antibodies became popular reagents in histopathological research and practice, used widely to assess the actively dividing fraction of cells in immunostained tissue sections of neoplasms (Gerdes, 1990;
Scholzen and Gerdes, 2000). A recent Pubmed query reveals thousands of entries documenting the great interest in Ki-67 antibodies for diagnostic purposes.
The human Ki-67 gene transcript appears to be differentially spliced resulting in two prominent mRNAs of approximately 9.800 and 8.700 nucleotides, encoding proteins with calculated molecular weights of 360 kD and 320 kD (Gerlach et al., 1998; Schluter et al., 1993)). Both Ki-67p isoforms possess large central domains with 16 tandem repeats, each 322 amino acids in length, which are identical between 42 and 62% of their sequence. The repeats contain numerous motifs that could be targets for phosphorylating enzymes as well as PEST amino acid motifs as sites for proteolytic degradation (Schluter et al., 1993). Interestingly, a “fork head associated” (FHA) domain has been detected in the amino-terminal part of Ki-67p (Hofmann and Bucher, 1995). The FHA domain appears to be a phosphopeptide recognition motif (Durocher et al., 1999), but its function in Ki-67p is not yet known, and a detailed investigation will depend on the progress made in elucidating the role of Ki-67p for nuclear physiology.
Ki-67p seems somehow to be involved in the regulation of the cell cycle. This has been concluded from antisense oligonucleotide transfection experiments with cultured human myeloma cells, where the incorporation of [3H]-thymidine into replicating DNA was inhibited (Schluter et al., 1993). In addition, antibodies against the mouse Ki-67p homolog block cell cycle progression when microinjected into the nuclei of mouse 3T3 fibroblasts (Starborg et al., 1996).
The distribution of Ki-67p in the nuclei of proliferating cells has earlier been investigated using immunohistochemical techniques. During most of the interphase of proliferating cultured cells, Ki-67p is mainly found in nucleoli, in particular in the nucleolar periphery (Kill, 1996; Starborg et al., 1996; Takagi et al., 1999; Verheijen et al., 1989a). In mitosis, human Ki-67p is located as a proteinaceous network on the surface of the condensed chromosomes (Gerdes et al., 1984b; Verheijen et al., 1989b), a finding that was confirmed by investigations on the mouse and the rat kangaroo Ki-67p homologs (Starborg et al., 1996; Takagi et al., 1999). Soon after the exit from mitosis, Ki-67p is dispersed into numerous small nuclear foci, which later in the G1-phase condense at and around the reforming nucleoli and in heterochromatic regions of the nucleus.
The peculiar localization of Ki-67p on the surface of mitotic chromosomes and the cell cycle-dependent change of localization appear to be regulated by phosphorylation and dephosphorylation events (Endl and Gerdes, 2000a; Endl and Gerdes, 2000b;
MacCallum and Hall, 1999). It appears that Ki-67p is hyperphosphorylated in mitosis (Endl and Gerdes, 2000b; MacCallum and Hall, 1999), and that mitotic Ki-67p differs from interphase Ki-67p in its DNA-binding properties (MacCallum and Hall, 1999).
A convincing interpretation of the localization of Ki-67p to nucleoli was provided by a combination of immunofluorescence studies for the detection of Ki-67p with fluorescence in situ hybridization (FISH) to identify satellite DNA sequences (Bridger et al., 1998b).
In nuclei of synchronously proliferating primary human dermal fibroblasts the two signals colocalize, indicating that Ki-67p may associate with virtually all satellite sequences in the chromatin of early G1-phase cells, and later remain associated with the short arms of the five acrocentric human chromosomes with their rRNA gene batteries that are incorporated into reforming nucleoli (Bridger et al., 1998b). The short arms of the acrocentric chromosomes are rich in satellite DNA sequences in their telomers and centromers as well as interspersed between the rRNA genes (Frommer et al., 1988;
Yunis and Yasmineh, 1971).
Thus, a picture emerges where Ki-67p performs architectural functions in the nucleus by interacting with satellite DNA-containing heterochromatic sections of the genome.
However, convincing evidence for a binding of Ki-67p to DNA in living cells has not yet been presented. This prompted us to investigate the localization of Ki-67p in chromatin prepared from HeLa cells to demonstrate the in vivo significance of the DNA-binding properties of Ki-67p. Our data indicate that the majority of Ki-67p is bound to a densely packed fraction of chromatin, probably heterochromatin.
EXPERIMENTAL PROCEDURES
Cell Culture
HeLa S3 cells were cultivated on 145 mm plastic dishes in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 5% fetal calf serum at 37°C in a humidified atmosphere containing 5% CO2.
Cell Fractionation
Cells were washed with phosphate-buffered saline (130 mM NaCl, 2 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4: pH 7.4) and resuspended in hypotonic buffer A (20 mM NaCl, 20 mM HEPES, pH 7.8). After swelling on ice for 10 min, cells were broken by Dounce homogenization. Soluble proteins were separated from nuclei by centrifugation at 600 g for 5 min. Subsequently the nuclear pellets were resuspended in buffer A containing increasing concentrations of NaCl (see below). After incubation for 10 min on ice, the released proteins were separated from the nuclei by centrifugation at 12,000 g for 10 min.
Chromatin Preparation
Chromatin was prepared at 0-4°C under low ionic strength conditions according to Hancock (Hancock, 1974) with slight modifications. Cells on plates were quickly washed four times with 50 ml of 0.25 mM EDTA and subsequently incubated with 40 ml of 0.25 mM EDTA for 10 min. Nuclei released from osmotically disrupted cells were collected by centrifugation at 600 g for 10 min and resuspended in 4 ml 0.25 mM EDTA with 0.5% Nonidet P-40 (NP40). The nuclei were then centrifuged for 10 min at 2000 g through a 10 ml sucrose cushion (0.1 M sucrose in 0.5 mM Tris, pH 8.0). The pellet was resuspended in 2 ml of 0.25 mM EDTA and centrifuged through a second sucrose cushion. The final pellet containing spherical particles without the nuclear envelope (“chromatin bodies”) was resuspended in 0.25 mM EDTA at a final concentration of 150 ng DNA/µl. DNA concentrations were determined with Hoechst 33258 by fluorimetry (Hoefer Scientific Instruments, San Francisco).
Extraction of proteins from chromatin bodies was achieved by resuspension of the pelleted chromatin bodies in 0.25 mM EDTA containing increasing concentrations of NaCl. After incubation on ice for 10 min, released proteins were separated from the chromatin bodies by centrifugation at 12,000 g for 10 min. The supernatant was applied to a sucrose gradient (12 ml, 5-25% sucrose in 0.25 mM EDTA containing 0.4 M NaCl) and spun in a Beckman SW40 rotor at 4°C for 17 h at 40,000 rpm.
Chromatin bodies were treated with micrococcal nuclease (MBI Fermentas, 4 units/µg of DNA) in the presence of 0.25 mM EDTA and 2 mM CaCl2 at 14°C. Aliquots (15 µg of DNA) were taken at various times and the reaction was stopped by addition of 8 mM EDTA. Insoluble material was removed by centrifugation (12,000 g, 10 min) and the concentration of released DNA in the supernatants was determined with Hoechst 33258
by fluorimetry (Hoefer Scientific Instruments, San Francisco). In addition, the released DNA fragments were analyzed after deproteinization by agarose gel electrophoresis.
Chromatin Fractionation
A procedure for isolation of S1, S2 and P chromatin fractions was adapted from Rose and Garrard (1984) with slight modifications. 5x107 cells were washed with phosphate- buffered saline (130 mM NaCl, 2 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4: pH 7.4) and resuspended in 200 µl of isotonic buffer (100 mM NaCl, 250 mM sucrose, 5 mM MgCl2, 20 mM HEPES: pH 7.5). Then, 100 µl of isotonic buffer containing 1% NP40 was added dropwise. After incubation for 5 min on ice, the nuclei were pelleted for 5 min at 1000 g and resuspended in 600 µl of digestion buffer (20 mM HEPES, pH 7.5, 100 mM NaCl, 250 mM Sucrose, 0.5 mM MgCl2, 2 mM CaCl2). Micrococcal nuclease (900 units, MBI Fermentas) was added to the suspension at 14°C. After indicated times, aliquots of 200 µl were taken and centrifuged for 3 min at 12,000 g. The supernatant termed S1 was supplemented with EDTA and EGTA (5 mM each) to terminate digestion, while the nuclear pellet was suspended in 2 mM EDTA. After incubation for 10 min on ice and centrifugation, the resulting supernatant designated S2 was recovered and the pellet (P) was resuspended in 2 mM EDTA. An aliquot of the P fraction was adjusted to 600 mM NaCl and extracted for 5 min on ice followed by centrifugation (5 min, 1000 g). To prevent protein degradation both isotonic and digestion buffer were supplemented with protease inhibitor (Complete, Roche Molecular Diagnostics).
Nuclear Matrix Preparation
Nuclear matrix was isolated as described by de Graaf et al. (1992) with published modifications (Mattern et al., 1996). All incubations were carried out at 0-4°C at a cell density of 5x107 cells/ml unless stated otherwise. Cells were washed twice with phosphate-buffered saline, scraped off the plate and collected by centrifugation. Cells were then extracted for 5 min in CSK100 (10 mM PIPES, pH 6.8, 0.3 M sucrose, 100 mM NaCl, 3 mM MgCl2, 1 mM EGTA) containing 1% TritonX-100 and 20 units/ml RNase inhibitor (Roche Molecular Diagnostics). The nuclei were pelleted (2,000 g, 5 min) and resuspended in CSK100 containing 5 units/ml RNase inhibitor and 1 mM sodium tetrathionate in order to stabilize the nuclear matrix (Kaufmann and Shaper, 1984; Stuurman et al., 1992). After incubation of 30 min the nuclei were collected by centrifugation (2,000 g, 5 min) and washed twice with CSK50 (10 mM PIPES, pH 6.8, 0.3 M sucrose, 50 mM NaCl, 3 mM MgCl2, 1 mM EGTA, 5 units/ml RNase inhibitor). The nuclei were further extracted with CSK50 containing 250 mM ammonium sulfate for 10 min, followed by a centrifugation step (2,000 g, 5 min). The pellet was resuspended in CSK50 (+15 units/ml RNase inhibitor) and digested with RNase-free DNase I (Roche Molecular Diagnostics) for 30 min at room temperature.
The pelleted nuclear matrix was then extracted in CSK50 (+250 mM ammonium sulfate, 10 min), collected by centrifugation and finally extracted in 2 M NaCl in
CSK-buffer for 10 min. To prevent protein degradation, all buffers were supplemented with protease inhibitor (Complete, Roche Molecular Diagnostics).
Western Blotting
Proteins were concentrated using the method of Wessel and Flügge (1984) before they were analyzed by denaturing polyacrylamide gel electrophoresis (Laemmli, 1970) and immunoblotted according to Towbin et al. (1979) with slight modifications (Burkhart et al., 1995).
For immunostaining, we used monoclonal antibodies directed against Ki-67p (MIB-2) (Key et al., 1993) and Mcm3p (Gerdes, personal communications). Monospecific antibodies against the human Orc2p have been described by Ritzi et al. (1998), antibodies against SAF-A by Romig et al. (1992).
RESULTS
Localization of Ki-67p in Nuclear Matrix Preparations
The location of Ki-67p in the nucleus remains somewhat controversial as Verheijen et al. (1989a; 1989b) describe Ki-67p to be bound to the nuclear matrix, while MacCallum and Hall (1999) found only tiny amounts persisting in the nuclear matrix, and Bridger et al. (1998b) find an association of Ki-67p with satellite DNA sequences.
As a starting point for our investigations, we prepared the nuclear matrix from isolated HeLa S3 cell nuclei (FIG 2-1A). The procedure includes a treatment of isolated nuclei with the mild oxidizing agent sodium tetrathionate, which has been shown to stabilize the structure of the internal nuclear matrix and preserve the localization of matrix proteins (Neri et al., 1995). The stabilization step was followed by a treatment with 250 mM ammonium sulfate, releasing histone H1 and other nuclear proteins (lane S2, FIG 2-1B). The remaining nuclear structure was then treated with DNase I followed by 2 M NaCl extraction to eliminate loosely bound nuclear proteins (lanes S3 and NaCl, FIG 2-1B). The insoluble material is operationally defined as the nuclear matrix, which consists of a peripheral lamina-pore complex and an internal filamentous ribonucleo- protein network (Berezney et al., 1995). The most prominent proteins in this fraction are the lamins (lane P, FIG 2-1B). An RNase inhibitor was included in the extraction buffers to prevent degradation of nuclear RNA, which may have structural functions in the nuclear matrix (He et al., 1990; Ma et al., 1999). Aliquots of each step of the procedure were analyzed by denaturing polyacrylamide gel electrophoresis and immunoblotting using the monoclonal antibody MIB-2 directed against the central repeat region of Ki-67p (Key et al., 1993) (FIG 2-1C, left panel). Both isoforms of Ki-67p with apparent molecular masses of 315 kD and 350 kD were detected in the fraction containing the nuclear matrix, consistent with the results obtained by Verheijen et al. (1989a; 1989b).
It is known, however, that sodium tetrathionate stabilizes the nuclear matrix by oxidizing sulfhydryl groups to disulfides (Belgrader et al., 1991; Kaufmann and Shaper, 1984; Stuurman et al., 1992). Several experiments demonstrated that certain proteins can be crosslinked to the internal nuclear matrix via sodium tetrathionate (Desnoyers et al., 1996; Humphrey and Pigiet, 1987; Kaufmann and Shaper, 1984). To assess the possibility that the formation of disulfide bonds might be responsible for the linkage of Ki-67p to elements of the nuclear matrix, we also isolated nuclear matrix in the absence of sodium tetrathionate. In fact, a larger number of Coomassie-stained bands is detectable in the matrix prepared with sodium tetrathionate, than in the matrix prepared without sodium tetrathionate (compare: lanes P in FIG 2-1B). Without sodium tetrathionate treatment, most of Ki-67p was released during the early extraction with 250 mM ammonium sulfate (FIG 2-1C).
FIG 2-1 Nuclear matrix preparations. (A) Nuclear matrix was isolated from 5x107 HeLa S3 cell nuclei.
Where indicated, sodium tetrathionate (NaTT) was added after the TritonX-100 step. (B) Aliquots of each step were analyzed by denaturing polyacrylamide gel electrophoresis and Coomassie blue staining. (C) Equal aliquots were used for western blotting with the monoclonal antibody MIB-2 directed against Ki-67p. Note that only 10% of fraction S1 compared to the other fractions was analyzed.
Chromatin Fractionation
The experiment of FIG 2-1 demonstrates that Ki-67p resides at nuclear sites that are sensitive to moderately high salt concentrations. To investigate the plausible possibility that these sites could be certain chromatin regions, we used a chromatin fractionation procedure by treating isolated nuclei with micrococcal nuclease (Rose and Garrard, 1984). Briefly, HeLa cell nuclei, prepared under isotonic conditions, were incubated with micrococcal nuclease for 5 and for 10 min. Centrifugation yielded the supernatant fraction S1, which is depleted in histone H1 (FIG 2-2C, asterisk) and contains highly accessible chromatin enriched in HMG (high mobility group) proteins (Rose and Garrard, 1984). The pellet was extracted with EDTA yielding the supernatant fraction S2 representing non-transcribed, more densely packaged chromatin. The pellet fraction P contains nuclease resistant chromatin DNA bound to the nuclear matrix (Rose and Garrard, 1984). The DNA fragments in fractions S1, S2 and P differ (FIG 2-2A) as fraction S1 primarily contained DNA degraded to the size of mono- and dinucleosomes, while fraction S2 contained a more extended ladder of nucleosomal DNA fragments which were further degraded during longer digestion times. Fraction P contained DNA fragments of heterogeneous sizes, which remained largely unchanged upon longer incubation (FIG 2-2A). Aliquots of the S1, S2 and P fraction were analyzed by denaturing polyacrylamide gel electrophoresis and immunoblotting (FIG 2-2B). Ki-67p was predominantly localized in the P fraction and only about 5% of Ki-67p was released into the S2 fraction after 15 min of digestion. Further extraction of the
P fraction with 600 mM NaCl resulted in a release of most Ki-67p, but about 30%
remained insoluble under these conditions (FIG 2-2C). As controls, we investigated two other proteins, Mcm3p (minichromosome maintenance protein) (Schulte et al., 1995) and SAF-A (scaffold attachment factor A) (Fackelmayer et al., 1994), which are known to be bound to chromatin. The two control proteins distribute between all three chromatin fractions, and can be completely (Mcm3p) or to large degree (SAF-A) released from the fraction P in the presence of 600 mM NaCl.
FIG 2-2 Chromatin fractionation. Isolated nuclei of HeLa S3 cells were treated with micrococcal nuclease for 5 and 15 min as described in the text. Centrifugations yielded three fractions: Fraction S1, supernatant after nuclease digestion; fraction S2, supernatant of EDTA wash; fraction P, nuclease resistant chromatin and the nuclear matrix. Fraction P was further extracted with 600 mM NaCl. (A) DNA analysis of each fraction by agarose gel electrophoresis. (B) Immunoblotting using antibodies against Ki-67p (MIB-2), SAF-A and Mcm3p. (C) Coomassie blue staining of polyacrylamide gels.
(* histone H1)
It should be noted, though, that nuclei used in the experiment of FIG 2-2 were prepared under isotonic conditions and have a relatively dense structure. Ki-67p could be trapped in this structure due to its large size and extended conformation (see below). To exclude this possibility we prepared chromatin by procedures under low ionic strength conditions.
A Nuclear Binding Site of Ki-67p is Chromatin
In a first approach, HeLa cell nuclei were prepared using a procedure that involves swelling of cells in hypotonic buffer followed by the mechanical disruption of the cytoplasmic membrane. The nuclear pellets were washed several times and finally resuspended in buffers with increasing concentrations of salt. Both isoforms of Ki-67p remained in the nuclear fraction at low salt concentrations, but could be completely mobilized when nuclei were treated with 0.4 M NaCl (FIG 2-3A).
In a second procedure, we prepared chromatin from nuclei in buffers of extremely low ionic strength (0.25 mM EDTA) with 0.5% of the detergent Nonidet-P40 (NP40). As originally described by Hancock (1974), the structures obtained are spherical particles (“chromatin bodies”) without the nuclear membranes, but with an electron dense surface representing the lamin network (Richter et al., 1998). The spherical particles are larger and more transparent than conventionally prepared nuclei (FIG 2-3B, right panel) due to an electrostatic repulsion of linker DNA segments at low ionic strengths (Clark and Kimura, 1990). This appears not to disturb the linear arrangement of nucleosomes and the binding of major chromatin-associated proteins (Hancock, 1974; Richter et al., 1998). We determined by immunoblotting that Ki-67p remains on the chromatin structures prepared under these conditions, but can again be mobilized at 0.3-0.4 M NaCl (FIG 2-3B). As controls, we assayed for two additional chromatin proteins, namely the replication initiator proteins Mcm3p and Orc2p. Mcm3p can be found in both the soluble fraction and in chromatin bodies (FIG 2-3B), in line with earlier data that demonstrated a soluble and a bound form of this protein in asynchronously proliferating cells (Krude et al., 1996). In contrast, Orc2p remained bound to chromatin bodies under low ionic strength conditions, but could be released at 0.3-0.4 M NaCl just like Ki-67p (FIG 2-3B). Thus, results from experiments with nuclei and chromatin, both prepared under low ionic strength conditions, suggest that Ki-67p is bound to a nuclear structure in a manner that is sensitive to moderately high salt concentrations.
FIG 2-3 Ki-67p on nuclei and chromatin prepared at low ionic strength. (A) Nuclei were isolated in hypotonic buffer followed by mechanical disruption of the cytoplasmatic membrane. (B) Chromatin was prepared under the very low ionic strength conditions of Hancock (1974). The “chromatin bodies”
obtained are larger and more transparent than conventionally prepared nuclei (right panels). Nuclei (A) and chromatin bodies (B) were extracted with increasing salt concentrations as indicated. Equivalent aliquots from the fractionation steps and extracted proteins were analyzed by denaturing polyacrylamide gel electrophoresis and immunoblotting.
Even though both Ki-67p isoforms can be released under identical salt conditions, they could be components of different protein complexes. To investigate this possibility, we prepared nuclear extracts at 0.4 M NaCl (FIG 2-3) and analyzed the released proteins by sucrose gradient centrifugation (FIG 2-4A). We found a co-sedimentation of the two Ki-67p isoforms, perhaps with a small excess of the 315 kD form in slower sedimenting fractions of the gradient (FIG 2-4B). Thus, there is no evidence that the isolated Ki-67p forms are components of large protein complexes, at least not under the buffer conditions used. The sedimentation coefficient of Ki-67p was estimated to be between 5.5 and 7.5 S compared to sedimentation markers. These values are comparatively low for proteins with molecular masses of 315-350 kD and could mean that Ki-67p has an extended configuration.
FIG 2-4 Sucrose gradient centrifugation. Chromatin was prepared under low ionic strength conditions (Hancock, 1974) and extracted with 400 mM NaCl. The supernatant was centrifuged through a preformed sucrose gradient (Experimental Procedures). To determine the distribution of proteins the gradient was fractionated and individual fractions were analyzed by denaturing polyacrylamide gel electrophoresis and Coomassie blue staining (A) or by western blotting as above using monoclonal antibody MIB-2 (B).
Positions of sedimentation markers are indicated between panel A and B.
As Ki-67p can be released at 0.3-0.4 M NaCl from its nuclear binding sites like chromatin-associated control proteins, we considered the possibility that Ki-67p may also be bound to chromatin, but may not be released under isotonic conditions because it is trapped in the densely packed nuclear remnants (FIG 2-2). We therefore used
“chromatin bodies” with their more accessible conformation to further investigate whether Ki-67p may be associated with chromatin.
Treatment with micrococcal nuclease for up to 30 min digested up to 15% of total nuclear DNA (FIG 2-5A) that elute as mono- and oligonucleosomal fragments (FIG 2-5B). Ki-67p was released in parallel to fragmented DNA (FIG 2-5C). We estimate, however, that only 5% of total nuclear Ki-67p was mobilized during the 30 min digestion time in contrast to the control proteins, Mcm3p and SAF-A, of which about 15% could be mobilized. Thus, the sites where Ki-67p is located in nuclei are less accessible to micrococcal nuclease than the Mcm3p- and SAF-A-binding sites, even under the low ionic strength conditions chosen.
FIG 2-5 Micrococcal nuclease digestion of isolated chromatin. Isolated “chromatin bodies” (see FIG 2-3) were incubated with micrococcal nuclease (4 units/µg DNA). Aliquots corresponding to 15 µg DNA were taken at the indicated times. The reaction was stopped by the addition of EDTA and insoluble material was removed by centrifugation. The concentration of supernatant DNA was determined by fluorimetry (A) and by agarose gel electrophoresis (B). Mobilized Ki-67p and the control proteins Mcm3p and SAF-A were determined by western blotting as above (C). To estimate the percentage of released protein, total extract from 2.5% of the input chromatin bodies were analyzed as above (C, left panel).
To address the question whether the released Ki-67p remained bound to fragmented chromatin, we analyzed the supernatant obtained after 15 min of digestion by sucrose gradient centrifugation. We found that Ki-67p does not sediment as free protein as in FIG 2-4, but in association with chromatin fragments (FIG 2-6, upper panel). The chromatin-bound Ki-67p could be converted to free protein by 400 mM NaCl and partially by DNase I, but not by RNase (FIG 2-6). This indicates that Ki-67p is either directly or indirectly bound to DNA in fragmented chromatin.
FIG 2-6 Sucrose gradient centrifugation of chromatin fragments. Isolated chromatin (500 µg of DNA) was digested for 15 min (see FIG 2-5) with micrococcal nuclease. Chromatin fragments in the supernatant were digested with RNase or DNase I or were extracted with 400 mM NaCl and applied to a sucrose gradient (12 ml, 5-25% sucrose in 5 mM EDTA, supplemented with protease inhibitor).
Centrifugation was performed in a Beckman SW40 rotor at 4°C for 5 h at 40,000 rpm (upper panel). The sucrose gradients were made up in EDTA buffer with 400 mM NaCl (B), or with 200 mM NaCl plus 0.1% NP40 (C and D). Fractions were immunoblotted and stained with MIB-2 to visualize Ki-67p. In a parallel gradient, we have determined the positions of mono-, di-, tri- etc. nucleosomes as indicated at the upper margin. Protein sedimentation markers are shown below.
To support this conclusion, we added 10 mM MgCl2 to the supernatant obtained by micrococcal nuclease to precipitate chromatin (Thoma et al., 1979). We found that Ki-67p precipitated with chromatin under these conditions (FIG 2-7). This clearly indicates that Ki-67p is a chromatin-binding protein.
FIG 2-7 Precipitation of DNA fragments by magnesium salts. Supernatant obtained by micrococcal nuclease digestion was adjusted to 10 mM MgCl2, incubated on ice for 5 min and centrifuged (10 min, 12,000 g) to separate the precipitated chromatin (P) from soluble proteins (S). (A) Agarose gel electrophoresis of deproteinized DNA fragments. (B) Immunoblot of Ki-67p with MIB-2 antibodies.
(C) Denatured polyacrylamide gel stained with Coomassie blue.
DISCUSSION
Numerous studies have shown that the presence of Ki-67p in nuclei strictly correlates with cell proliferation, and made Ki-67p a target for routine diagnostics of malignant cells and tissues. However, it is not known why cells in quiescence cease to express Ki-67p, and why it is expressed again when cells reenter the cell cycle.
As a first step to investigate the biochemical properties of Ki-67p we performed cell fractionation experiments. Earlier work had suggested that Ki-67p might be bound to the nuclear matrix (see Introduction), defined as an insoluble structure that remains when nuclei are treated with nucleases under high salt conditions. We also recovered Ki-67p in the matrix, but only when buffers contained sodium tetrathionate, a compound that has been recommended to stabilize the matrix during cell fractionation (Belgrader et al., 1991; Kaufmann and Shaper, 1984; Stuurman et al., 1992). However, sodium tetrathionate is known to induce covalent cross-links between proteins (Desnoyers et al., 1996; Humphrey and Pigiet, 1987; Kaufmann and Shaper, 1984), which might tether proteins to the nuclear matrix artifactually. In fact, in the absence of sodium tetrathionate, Ki-67p almost completely dissociates from its nuclear binding sites at salt concentrations of 0.3-0.4 M NaCl, in agreement with MacCallum and Hall (1999).
Due to differential splicing, Ki-67p is expressed in proliferating human cells as two isoforms with 350 kD and 315 kD in size (Schluter et al., 1993). The extracted Ki-67p isoforms sediment together in a single peak with an estimated sedimentation coefficient of 5-7 S. This value is rather low for proteins of 315-350 kD and suggests that Ki-67p may have an extended configuration. The low sedimentation coefficient may also indicate that both isoforms of Ki-67p sediment as individual molecular units and are not complexed to other proteins in the nuclear protein extract.
The finding that Ki-67p can be extracted from human nuclei with 0.3-0.4 M NaCl provides a convenient basis for its isolation in a biochemically useful form (MacCallum and Hall, 1999). We noted, though, that extracted Ki-67p is very sensitive to proteolysis in unfractionated nuclear extracts and special care is needed to preserve its integrity after release from its nuclear binding sites.
To determine whether Ki-67p is bound to chromatin, we treated HeLa cell nuclei with micrococcal nuclease under the conditions described by Rose and Garrard (1984) in a study on the nature of chromatin alterations in immunoglobulin genes during lymphocyte differentiation. The chromatin fragments mobilized by short treatments with micrococcal nuclease have been shown to originate from extended and genetically active chromatin regions. More compact chromatin fragments are released by an EDTA wash of nuclease-treated nuclei. We detected no or only very small amounts of Ki-67p in these two fractions of solubilized chromatin fractions but recovered most Ki-67p in the pellet fraction. This fraction includes the nuclear matrix in addition to the 20-30% of
chromatin that is not accessible to nuclease digestion under standard assay conditions (Clark and Felsenfeld, 1971; Klempnauer et al., 1980). As Rose and Garrard (1984) have noted, the nuclease-resistant chromatin fraction is enriched in actively transcribed gene sequences, and its insolubility at physiological salt may at least partially be due to associated large protein complexes such as the RNA polymerase holoenzyme. Indeed, the nuclease-resistant chromatin fraction contains other multiprotein complexes like the human SWI/SNF chromatin-remodeling complex (Reyes et al., 1997) and possibly the human origin recognition complex (Ritzi et al., 2000). However, it is not likely that Ki-67p is a component of a similar multiprotein complex because it appears to be released at 0.4 M NaCl as a single molecular entity, and not as a large complex.
Therefore, it may be more likely that Ki-67p is linked to a form of chromatin that cannot easily be attacked by nucleases due to a highly compact conformation. This possibility is consistent with the results of Bridger et al. (1998b) who determined by immunofluorescence combined with fluorescence in situ hybridization (FISH) that Ki-67p colocalizes with satellite DNA in heterochromatin. It is also consistent with the work of Tagaki et al. (1999) who showed that ectopically expressed regions of the Ki-67p-related protein Chmadrin induce aberrant chromatin compaction in PtK2 rat kangaroo cells.
Our experiments with chromatin at low ionic strengths support the idea that Ki-67p is a component of compact chromatin. Low ionic strength conditions cause an artificial extension of chromatin due to electrostatic repulsion. We have treated these preparations with micrococcal nuclease and could produce chromatin fragments that carry bound Ki-67p. The mode of interaction between Ki-67p and fragmented chromatin appears to be similar to that in intact nuclei because 0.4 M NaCl mobilizes Ki-67p in both cases. This is a strong argument for a chromatin-associated binding site of Ki-67p. However, we do not know yet whether Ki-67p is directly or indirectly bound to the DNA in chromatin, even though isolated Ki-67p can bind to protein-free DNA in vitro (MacCallum and Hall, 1999).
In summary, Ki-67p in interphase nuclei appears to be located at sites in highly compact heterochromatin. These sites are characterized by their sensitivity to moderately high salt concentrations and their resistance against nuclease digestion. It has yet to be shown whether Ki-67p remains on these sites in mitosis when it forms a proteinaceous layer on the surface of chromosomes.
SUBSEQUENT INVESTIGATIONS
Several studies published in the last two years indeed confirm the DNA-binding properties of Ki-67p as well as its localization in heterochromatin.
Additional data concerning the DNA association of Ki-67p was provided by MacCallum and Hall, who mapped a DNA binding domain in the C-terminal part of Ki-67p (MacCallum and Hall, 2000). Moreover, they demonstrated that Ki-67p has a preference for supercoiled and AT-rich DNA. Overexpression of the C-terminal 321 residues of Ki-67p in cells induced chromatin disruption and apoptosis, thus providing additional evidence that Ki-67p is able to influence chromatin structure.
A role for Ki-67p in regulating higher-order chromatin structure was further characterized by Scholzen and coworkers who demonstrated a partial colocalization of Ki-67p with heterochromatin protein 1 (HP1) (Scholzen et al., 2002), a member of a family of proteins containing a motif known as the chromodomain (Singh et al., 1991).
These proteins appear to play a central role in regulating diverse processes ranging from silencing to cell cycle-regulated chromosome dynamics (Jones et al., 2000). The authors further showed that a C-terminal domain of Ki-67p is able to bind to HP1 in vitro and in vivo, suggesting that Ki-67p might be involved in the establishment and/or maintenance of heterochromatin domains in interphase nuclei. This idea is supported by the observation that expression of a C-terminal Ki-67p fragment leads to the formation of a condensed heterochromatin domain (MacCallum and Hall, 2000; Takagi et al., 1999). Further evidence for the relevance of the interaction between Ki-67p and HP1 was provided by ectopic expression of GFP-tagged HP1 in HeLa cells, which leads to a dramatic relocalization of endogenous Ki-67p from the nucleolar fraction to sites of high HP1 concentration (Scholzen et al., 2002).
Considering that this relocalization of Ki-67p does not seem to have an effect on the nucleolar structure, it has been suggested that targeting of Ki-67p to the nucleoli in interphase is used as a mechanism to sequester the protein until it is needed, as already described for other proteins (Endl and Gerdes, 2000a; Visintin et al., 1999; Zhang and Xiong, 1999). In fact, disruption of the nucleolar structure leads to relocalization of nucleolar Ki-67p to centromeric heterochromatin (Kill, 1996). An apparent change in the distribution of Ki-67p occurs during prophase of mitosis, when Ki-67p disperses from the dissolving nucleoli and relocates to condensing chromosomes. In metaphase, Ki-67p covers the chromosomes and remains associated with the condensed chromatin throughout anaphase (Starborg et al., 1996; Verheijen et al., 1989b). It has been suggested that the Ki-67p/HP1 interaction may be physiologically relevant during mitosis because HP1 begins to assemble at the polar surfaces chromosomes in anaphase (Endl and Gerdes, 2000a). Given the known localization of Ki-67p to the surfaces of the chromosomes, it is intriguing to speculate that Ki-67p might be involved in the recruitment of HP1 proteins to chromosomes to reestablish heterochromatin after mitosis.
A role for Ki-67p during mitosis is also suggested from the observation that the Ki-67p FHA domain binds the kinesin-like protein Hklp2 (Human kinesin-like protein 2) in vitro and in yeast two-hybrid assays (Sueishi et al., 2000). The Xenopus counterpart Xklp2 is a plus-end directed putative motor protein that has been reported to be required in centrosome separation (Boleti et al., 1996). Thus, the question arises whether Ki-67p plays a role in spindle function and whether the observed interaction between Ki-67p and Hklp2 is involved in transfer of Ki-67p to the spindle as it has been proposed for the inner centromer protein INCENP (Ainsztein et al., 1998). However, evidence for a colocalization of Hklp2 and Ki-67p is still lacking, thus the in vivo significance of this interaction remains unclear.
The ORC1 Protein:
An Example for a Replication Initiation Protein
Initiation of Genome Replication:
An Overview
THE EUKARYOTIC CELL CYCLE
The eukaryotic genome is organized into multiple chromosomes because of its size and complexity. To produce two daughter cells each receiving a copy of the entire genome, the DNA in each chromosome must first be duplicated accurately, before the two copies are distributed to the two daughter cells. These processes define the two major phases of the cell cycle. DNA replication occurs during S-phase (S for synthesis), which requires about half of the cell cycle time in a typical mammalian cell (~9 h). After S-phase, chromosome segregation and cell division occur in mitosis (or “M-phase”), which requires much less time (~1 h). At the same time, dividing cells must coordinate their growth (i.e. their increase in cell mass) to maintain their size. Time for cell growth is provided through gap-phases between M- and S-phase (G1) and between S-phase and mitosis (G2). Thus, the eukaryotic cell cycle is divided into four sequential phases: G1, S, G2 and mitosis. G1, S and G2 together are called interphase (see FIG 3-1). The two gap phases are not essential and can be skipped in rare cases when rapid cell division is essential, e.g. during embryogenesis. On the other hand, most cells in the body persist in G1 during most of their life time, or – once they exited the cell cycle upon differentiation, in a closely related phase called G0. The progression from one cell cycle phase to the subsequent one is tightly regulated, and cells sense the completion of critical processes (such as DNA replication, or repair after DNA damage) before the next phase is initiated. Information about the completion of cell cycle events, as well as signals from the environment, can cause a control system to arrest the cell cycle at specific “checkpoints”. The G1-phase is especially important in this respect. Its length can vary greatly depending on external conditions and extracellular signals from other cells. If signals to grow and divide are present, cells in early G1-phase progress through the commitment point near the end of G1, known as “Start” or restriction point, and enter a new cell cycle.
