Polycomb Group Proteins and Homeotic Gene

Polycomb Group Proteins and Homeotic Gene Silencing
in Drosophila
Dissertation
der Fakultät für Biologie
der Eberhard Karls Universität Tübingen
zur Erlangung des Grades eines Doktors
der Naturwissenschaften
vorgelegt
von
Aditya K. Sengupta
aus Kalkutta, Indien
2002
Sengupta, Aditya Kumar:
Polycomb Group Proteins and Homeotic Gene Silencing in Drosophila /
Aditya Kumar Sengupta. –
Als Ms. gedr.. – Berlin : dissertation.de – Verlag im Internet GmbH, 2003
Zugl.: Tübingen, Univ., Diss., 2002
ISBN 3-89825-573-5
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Parts of the work described in this thesis has been published as the
following:
Birve, A., Sengupta, A. K., Beuchle, D., Larsson, J., Kennison, J. A., RasmusonLestander, A. and Müller, J. (2001). Su(z)12, a novel Drosophila Polycomb group gene
that is conserved in vertebrates and plants. Development 128, 3371-9.
Müller, J., Hart, C.M., Francis, N.J., Vargas, M.L., Sengupta, A., Wild, B., Miller, E.L.,
O'Connor, M.B., Kingston, R.E., and Simon, J.A. (2002) Histone methyltransferase
activity of a Drosophila Polycomb group repressor complex. Cell Published online
September 13, 2002. 10.1016/S0092867402009765.
Tag der mündlichen Prüfung:
06. December 2002
Dekan:
Prof. Dr. H.U. Schnitzler
1. Berichterstatter:
Prof. Dr. C. Nüsslein-Volhard
2. Berichterstatter:
Prof. Dr. R. Reuter
CONTENTS
Acknowledgements
v
Zusammenfassung
1
Abstract
2
CHAPTER ONE: Introduction
3
§1.1. Homeotic Genes and Cell Determination
3
§1.2. Maintenance of Transcriptional States: the Trithorax and Polycomb Groups of
Proteins
5
§1.3. The Polycomb Group
5
§1.4. PcG Protein Complexes
7
§1.5. Polycomb Response Elements
9
§1.6. PcG Proteins and DNA Binding
9
§1.7. Targeting PcG Complexes: PRC1
11
§1.8. Targeting PcG Complexes: Esc-E(z) Complex
12
§1.9. PcG and the Mechanism of Repression
12
CHAPTER TWO: Requirement of the PRE Throughout Development
15
§3.1. Introduction
15
§3.2. Results
16
§2.2.1 PREs and Heterologous Enhancers
16
§2.2.2 Requirement for PRE During Development
23
§2.2.3. Mechanism of PRE-Mediated Silencing
27
§3.3 Discussion
29
CHAPTER THREE: Yeast 2-Hybrid Screen to Identify Interactors of Psc
33
§3.1. Introduction
33
§3.2. Results
35
§ 3.2.1 cDNA Library
35
§ 3.2.2 Yeast 2-Hybrid Screen with Psc
36
§3.3. Discussion
45
§3.4. Sequences
50
iii
CHAPTER FOUR: Su(z)12: A Novel Member of the Polycomb Group
55
§4.1. Introduction
55
§4.2. Results
57
§4.2.1 Su(z)12 is a Member of the Polycomb Group
57
§4.2.2 Silencing Through PRE is Dependant on Su(z)12
58
§4.2.3 The Su(z)12 Protein is Conserved in Vertebrates and Plants
59
§4.2.4 The Su(z)12 Zinc-finger Does Not Bind DNA
62
§4.2.5 Su(z)12 Binds to Chromatin
64
§4.2.6 Su(z)12 is a Member of the Esc-E(z) Complex
66
§4.3. Discussion
68
CHAPTER FIVE: Materials and Methods
71
§5.1. Buffers and Reagents
71
§5.2. General Protocols
73
§5.3. Transgenic Lines
80
§5.4. PRE Excision Experiments
81
§5.5. cDNA Library
81
§5.6. Yeast 2-Hybrid Screen with Psc
83
§5.7. Sequencing of Su(z)12 Alleles
82
§5.8. DNA Mobility Shift Assay
84
§5.9. Antibody Generation
85
§5.10. Staining of Polytene Chromosomes with Antibodies
86
References
87
Lebenslauf
97
iv
Acknowledgements
I would like to express my gratitude to Dr. Jürg Müller, who supervised this work;
his stimulating ideas, helpful discussions and infinite patience contributed immeasurably
to this dissertation work, and to my personal intellectual development.
I am very grateful to Prof. Christiane Nüsslein-Volhard, in whose department at
the MPI für Entwicklungsbiologie, Tübingen, a major part of this work was carried out. I
thank her and Prof. Rolf Reuter for agreeing to examine my thesis.
I would like to thank the members of the Müller lab, especially Dirk and Cornelia
who, apart from providing a friendly atmosphere, also helped me in many ways at various
stages of my work. Dirk did the initial genetic characterization of Su(z)12 and Cornelia
gladly shared her flies, yeast plates and reagents. I would also like to thank members of
the Tübingen Fly group for discussions and general friendliness.
I would like to thank A. Birve, Dr. A. Rasmusson-Lestander and Dr. J. Kennison
for collaborating on the initial part of the work on Su(z)12. I would like to thank Dr. G.
Struhl for the FRT-PRE-IDE stocks and Drs. K. Basler, H. Brock, S Carroll and J. Simon
for plasmid constructs.
I would like to thank my friends in Tübingen and elsewhere for support at
difficult moments.
Last, but not the least, I would like to thank my parents and brother for their love
and faith in me.
v
Zusammenfassung
In Fliegen und Wirbeltieren erhalten Polycomb Gruppen (PcG) Proteine den
reprimierten Zustand homeotischer Gene außerhalb ihrer Expressionsdomänen aufrecht.
PcG Proteine binden an das Chromatin von cis-regulatorischen Silencer Elementen, die
als PREs bezeichnet werden. In dieser Arbeit wird gezeigt, dass PREs über die gesamte
Entwicklung hinweg gebraucht werden, um den reprimierten Zustand der von ihnen
regulierten Gene aufrecht zu erhalten, und dass PREs auch Enhancer-Elemente regulieren
können, die normalerweise nicht von PcG Proteinen reprimiert werden. Die durch diese
Experimente gewonnenen mechanistischen Einblicke werden ebenfalls diskutiert.
Zwei PcG Komplexe wurden bislang aufgereinigt, der PRC1 Komplex und der Esc-E(z)
Komplex. Dennoch ist nicht klar, wodurch diese Komplexe an das Chromatin der PREs
rekrutiert werden. Um diese Frage zu untersuchen, wurde eine Interaktorsuche in Hefe
(2-Hybrid-Screen) mit dem Posterior sex combs (Psc) Protein, einem wichtigen
Bestandteil des PRC1 Kernkomplexes, durchgeführt. Hierbei wurde nach möglichen
DNA-bindenden Psc-Interaktoren gesucht. Die Ergebnisse dieser Suche sowie die hierbei
aufgetretenen Probleme werden diskutiert.
Wir haben ein neues Mitglied des Esc-E(z) Komplexes identifiziert. Die
Klonierung und Charakterisierung dieses Gens, Supressor of zeste (Su(z)12), wird hier
beschrieben. Su(z)12 enthält einen einzelnen klassischen Zinkfinger, scheint jedoch nicht
in der Lage zu sein, an DNA zu binden. Es interagiert mit dem PcG Protein Enhancer of
zeste (E(z)) und bindet an Chromatin. Es ist in Wirbeltieren und interessanterweise auch
in Pflanzen konserviert.
1
Abstract
Polycomb Group (PcG) proteins maintain the repressed state of homeotic genes
outside their domains of expression in flies and vertebrates. PcG proteins bind to
chromatin of specific cis-acting silencer elements, called Polycomb Response Elements
(PREs). In this work, evidence is presented that PREs are required continuously during
development for maintenance of the silenced state of PcG target genes and that PREs can
act on enhancers from genes that are normally not under PcG regulation. Mechanistic
insights obtained from these experiments are discussed.
Two PcG complexes have been purified, the PRC1 complex and the Esc-E(z)
complex. However the DNA-binding proteins that target these complexes to PREs is not
known. To identify such proteins, a yeast 2-hybrid screen was performed with Posterior
sex combs (Psc), an important PcG member that is a component of the core complex of
PRC1. The idea was to hunt for possible DNA-binding interactors of Psc. The results of
this screen and the problems encountered during its course are discussed.
We have identified a new PcG member of the Esc-E(z) complex. The cloning and
characterization of this gene, Suppressor of zeste 12 (Su(z)12) is described. Su(z)12
contains a single classical zinc finger, but does not appear to bind DNA. It interacts with
the PcG protein Enhancer of zeste (E(z)) and binds to chromatin. It is conserved in
vertebrates and, interestingly, has relatives in plants.
2
CHAPTER 1
Introduction
Cell types differ from each other in the combination of genes that they express—
and this is the basis of cellular differentiation. The single celled zygote achieves this
differentiated multicellular state through the complex process of embryonic development,
whereby cells multiply, move and exchange signals. In Drosophila, for example,
maternal factors provide an initial asymmetry to the fertilized egg. In the developing
embryo this pre-pattern is read by complex cascades of regulatory signals of
segmentation genes into different positional identities along the embryonic axes,
eventually giving rise to appropriate structures in appropriate body positions (reviewed in
St Johnston and Nüsslein-Volhard, 1992; Pankratz and Jäckle 1993). During the course of
these processes, cascades of various gene products signal cells to switch on or off
particular genes thereby committing them to a particular fate. Once such a determined
state has been achieved, it has to be stably maintained by these cell types through cell
divisions throughout development. The Polycomb and trithorax groups of proteins are
among the factors that provide stability to determined states of cells (reviewed in
Kennison, 1995; Orlando and Paro, 1995; Simon, 1995; Pirrotta, 1997a; Pirrotta, 1997b;
Francis and Kingston, 2001).
§1.1. HOMEOTIC GENES AND CELL DETERMINATION
In metamerically segmented animals, segment identities are determined by the
combinatorial expression of master “selector” genes, called homeotic genes (reviewed in
3
McGinnis and Krumlauf, 1992; Mann and Morata, 2000). These genes encode
homeobox-containing transcription factors and are conserved throughout the animal
kingdom. In vertebrates, for example, homeotic gene expression provides segmental
identity to the segmented regions of the body like the branchial region of the head and the
paraxial mesoderm (McGinnis and Krumlauf, 1992). In Drosophila, loss of function of a
homeotic gene in cells that express it causes them to form structures characteristic of a
different segment (Lewis, 1978; Struhl, 1982). Segment-specific patterns of homeotic
gene expression are generated by localized segmentation gene products during early
development. For example, expression of homeotic gene Ultrabithorax (Ubx) is restricted
within a broad band near the middle of the embryo by the gap protein Hunchback (Hb)
(White and Lehmann, 1986; Qian et al., 1991; Zhang et al., 1991) and is activated by the
products of the pair-rule genes fushi tarazu (ftz) and even skipped (eve) (Ingham and
Martinez-Arias, 1986; Müller and Bienz, 1992; Qian et al., 1993). Hb and Ftz proteins
bind directly to specific cis-acting elements in the Ubx regulatory region to repress and
activate transcription, respectively (Qian et al., 1991, 1993; Zhang et al., 1991; Müller
and Bienz, 1992).
Expression of these gap and pair-rule gene signals that generate the homeotic
gene expression patterns are very transient. However it is crucial that the expression
patterns of homeotic genes are heritably maintained throughout development. This is
achieved by the protein products of the trithorax group (trxG) and the Polycomb group
(PcG) genes. The trxG proteins maintain the active state of homeotic genes while the PcG
proteins maintain their repressed state (reviewed in Kennison, 1995; Orlando and Paro,
1995; Simon, 1995; Pirrotta, 1997a; Pirrotta, 1997b;Francis and Kingston, 2001).
4
Like homeotic genes, many trxG and PcG genes have been found to have
vertebrate homologues that perform similar functions in restricting homeotic gene
expression within their domains of expression (reviewed in Gould, 1997; van Lohuizen,
1998).
§1.2. MAINTENANCE OF TRANSCRIPTIONAL STATES: THE TRITHORAX
AND POLYCOMB GROUPS OF PROTEINS
Members of the trxG and PcG do not appear to be required for positioning of the
boundaries of homeotic gene expression, but are required for maintaining expression of
these boundaries once they are set up. This maintenance is heritable, i.e., the pattern of
homeotic gene expression is stable through mitosis. Though they maintain different
expression patterns of homeotic genes in different cells, trx and PcG proteins are
expressed in all cells. Genetic and biochemical studies indicate that both groups of
proteins act as hetero-multimeric complexes and bind to chromatin (Franke et al., 1992;
Shao et al., 1999; Ng et al., 2000).
§1.3. THE POLYCOMB GROUP
The first Polycomb group mutants in Drosophila, extra sex combs (esc) and
Polycomb (Pc), were discovered in the 1940s due to an extra sex combs phenotype. Since
then, 12 other members of the group have been discovered (Table 1.1). Chapter 3
describes the genetic and biochemical characterization of a new PcG member, Suppressor
of zeste 12 (Su(z)12). Mutations in these genes cause transformations in flies similar to
gain of function mutations of the homeotic genes of the bithorax and Antennapedia
5
complexes (BX-C and ANT-C). It has been shown that null mutations (lacking both the
maternal and zygotic products) in these genes are lethal and result in severe
misexpression of homeotic genes in embryos and larvae (Beachy et al., 1985; Beuchle et
al., 2001; Cabrera et al., 1985; Fritsch et al., 1999; Ingham, 1985; McKeon and Brock,
1991; Simon et al., 1992; Struhl and Akam, 1985; White and Wilcox, 1985). PcG genes
are expressed in the female germline and maternally deposited PcG proteins often rescue
mutant embryos to a considerable extent (Struhl, 1981; Breen and Duncan, 1986; Soto et
al., 1995). Embryos that are homozygous for two different PcG mutations often exhibit a
much more severe phenotype than single mutants (Jürgens, 1985). This property of PcG
mutations to enhance each other’s phenotypes indicated that they might act together at the
molecular level. Indeed, many of these gene products have been found to physically
interact with each other and to form complexes in vivo (Kyba and Brock, 1998; Shao et
al., 1999;Ng et al., 2000; Saurin et al., 2001). Staining of polytene chromosomes with
antibodies for PcG proteins revealed that these proteins bind chromatin at about 100
distinct bands, with strongest signals at the BX-C and ANT-C loci (e.g. Franke et al.,
1992; Rastelli et al., 1993). As might be expected, many of the sites labeled by distinct
PcG members are identical.
6