Expression and function of Suppressor of zeste 12

in Drosophila melanogaster

Sa Chen

Department of Molecular Biology Umeå University 901 87 Umeå Sweden 2009

Copyright ○C 2009 Sa Chen

ISBN: 978-91-7264-729-9

Print by Arkitektkopia, Umeå, 2009

In memory of my father

怀念我的父亲

He who knows does not speak

He who speaks does not know

Lao Zi

TABLE OF CONTENTS

ABSTRACT……………………………………………………………………………. 9 LIST OF PAPERS .……………………………………………………………………10 1. REVIEW OF LITERATURE ………………………………………………..11 1.1 Drosophila as a model system ……………………………………………..11 1.2 Concepts of genes, genetics, epigenetics and chromatin ………………..11 1.2.1 Genes …………………………………………………………...…..12 1.2.2 Genetics ………………………………………………………...…..12 1.2.3 Epigenetics …………………………………………………………13 1.2.4 Chromatin …………………………………………………………..15

1.3 Epigenetic phenomena ……………………………………...………….…15

1.4 The regulation of gene expression, an epigenetic perspective …………..17 1.4.1 General introduction ………………………………………………..17 1.4.2 Epigenetic mechanisms in Prokaryotes …………………………….17 1.4.3 Epigenetic mechanisms in Eukaryotes ……………………………..19 1.4.3.1 DNA methylation ………………………………………………19 1.4.3.2 Histone modifications ………………………………………….22

1.5 Regulation of gene silencing by PcG proteins …………………………...42 1.5.1 Introduction ………………………………………………………...42 1.5.2 Genetics and biochemical characterization of PcG proteins ……….43 1.5.3 Mechanisms of PcG regulation of gene silencing ………………….54 1.5.3.1 The Polycomb Response Element (PRE) ………………………54 1.5.3.2 Gene silencing at specific loci ………………………………….56 1.5.3.3 Silencing at the chromatin level ………………………………..58 1.5.3.4 Three-dimensional (3D) organization of the nucleus …………..61 1.5.4 Evolution of PcG genes …………………………………………….61 1.5.5 PcG genes and cell cycle ……………………………………………63

1.6 Epigenetic inheritance ……………………………………………………..65

1.7 Epigenetics and disease …………………………………………………...69

2. AIM ……………………………………………………………………………70 3. RESULTS AND DISCUSSION ………………………………………….….71 4. CONCLUSIONS ………………………………………………………….…..75 5. ACKNOWLEDGEMENTS ………………………………………………….76 6. REFERENCES ………………………………………………………………..79

PAPERS I-IV

ABSTRACT

The development of animals and plants needs a higher order of regulation of gene expression to maintain proper cell state. The mechanisms that control what, when and where a gene should (or should not) be expressed are essential for correct organism development. The Polycomb group (PcG) is a family of genes responsible for maintaining gene silencing and Suppressor of zeste 12 (Su(z)12) is one of the core components in the PcG. The gene is highly conserved in organisms ranging from plants to humans, however, the specific function is not well known. The main tasks of this thesis was to investigate the function of Su(z)12 and its expression at different stages of Drosophila development. In polytene chromosomes of larval salivary glands, Su(z)12 binds to about 90 specific euchromatic sites. The binding along the chromosome arms is mostly in interbands, which are the most DNA de-condensed regions. The binding sites of Su(z)12 in polytene chromosomes correlate precisely with those of the Enhancer-of-zeste (E(z)) protein, indicating that Su(z)12 mainly exists within the Polycomb Repressive Complex 2 (PRC2). However, the binding pattern does not overlap well with Histone 3 lysine 27 tri- methylations (H3K27me3), the specific chromatin mark created by PRC2. The Su(z)12 binding to chromatin is dynamically regulated during mitotic and meiotic cell division. The two different Su(z)12 isoforms: Su(z)12-A and Su(z)12-B (resulting from alternative RNA splicing), have very different expression patterns during development. Functional analyses indicate that they also have different functions he Su(z)12-B form is the main mediator of silencing. Furthermore, a neuron specific localization pattern in larval brain and a giant larval phenotype in transgenic lines reveal a potential function of Su(z)12-A in neuron development. In some aspects the isoforms seem to be able to substitute for each other. The histone methyltransferase activity of PRC2 is due to the E(z) protein. However, Su(z)12 is also necessary for H3K27me3 methylation in vivo, and it is thus a core component of PRC2. Clonal over-expression of Su(z)12 in imaginal wing discs results in an increased H3K27me3 activity, indicating that Su(z)12 is a limiting factor for silencing. When PcG function is lost, target genes normally become de-repressed. The segment polarity gene engrailed, encoding a transcription factor, is a target for PRC2 silencing. However, we found that it was not activated when PRC2 function was deleted. We show that the Ultrabithorax protein, encoded by another PcG target gene, also acts as an inhibitor of engrailed and that de-regulation of this gene causes a continued repression of engrailed. The conclusion is that a gene can have several negative regulators working in parallel and that secondary effects have to be taken into consideration, when analyzing effects of mutants. PcG silencing affects very many cellular processes and a large quantity of knowledge is gathered on the overall mechanisms of PcG regulation. However, little is known about how individual genes are silenced and how cells “remember” their fate through cell generations.

10 LIST OF PAPERS:

I. In vivo analysis of Drosophila Su(z)12 function.

Chen S, Birve A, Rasmuson-Lestander Å.

Mol Genet Genomics (2008) 279: 159-70. Epub 2007 Nov 22.

II. Regulation of the Drosophila engrailed gene by Polycomb repressor complex 2.

Chen S, Rasmuson-Lestander Å.

Mechanisms of Development. 2009. In press.

III. The role of Suppressor of zeste 12 in cell cycle regulation. Chen S, Rasmuson-Lestander Å. Manuscript

IV. In vivo analysis of Suppressor of zeste 12´s different isoforms.

Chen S, Larsson A.L, Tegeling E, Rasmuson-Lestander Å.

Manuscript

11 1. REVIEW OF LITERATURE

1.1 Drosophila as a model system

The fruit fly, Drosophila melanogaster, was first brought to laboratory by William E. Castle at Harvard University in 1901 (Kohler, 1994). It was his work that inspired Thomas Hunt Morgan to use Drosophila as a model (in 1908) and led to Morgan's Nobel Prize in 1933. He and his students established the linear organization of genes in chromosomes. Since then, Drosophila has remained at the forefront of genetics, developmental biology, evolution biology and genomics research.

Why? Because fruit flies are easy to keep and breed. During the past 100 years a whole range of mutants have been used to combine classical genetic techniques and modern molecular genetic tools, for example, UAS-GAL4 and FLP-FRT, Chip (-on- Figure 1. Thomas Hunt Morgen Chip), 3C (Chromatin, Conformation Capture), 4C (Chromosome Conformation Capture–on-Chip) and 6C (Combined 3C-ChIP-cloning) (Dekker, 2006; Tiwari et al., 2008a; Zhao et al., 2006) techniques have been fruitful. Drosophila is an advanced representative of insects, an order that has undergone rapid evolution in the course of the past 300 million years. To understand the tree of life, and humans as a part of it, we need to understand genome evolution and gene regulation. Drosophila is an indispensable model organism in these studies.

1.2 Concepts of genes, genetics, epigenetics and chromatin

Genetics has seen a spectacular progress as a science. The interest on inheritance goes back to a time long before recorded history. Philosophers and scientists who have contributed to the ideas on heredity and individual development include Hippocrates (about 500 BC) and Aristotle (384-322 BC), who speculated about preformation and epigenesis, respectively; Darwin, with his ideas of natural selection and pangenesis (ca 1859-1872); Mendel, with his theory of inheritance (1866); August Weissmann (1885) with his germ plasm theory; Watson and Crick (1953) who discovered the structure of

12 DNA. Accordingly, the meanings of “gene, genetics and epigenetics” have been subject to an evolution that parallels our dramatically increasing knowledge of the mechanisms underlying heredity and the regulation of gene expression.

1.2.1 Genes

The modern concept of the word “gene” originates from the work of Gregor Mendel (1822-1884), who first showed the existence of a particulate hereditary material. In 1889, Hugo de Vries invented the concept pangen which is derived from Darwin´s word pangenesis, where pan is a Greek prefix meaning “whole” and genesis means “birth” or “origin”. Two decades later, Wilhelm Johannsen abbreviated this term to "gene" ("gen" in Danish and German). People had, of course, used artificial selection ever since the domestication of animals and plants. Nevertheless, the mechanism of heredity has been subject to serious study only since the rediscovery of Mendel’s work in 1900. Since then, the definition of the gene has followed our increasing knowledge about the hereditary material. For example, the gene has been defined as a distinct locus (the 1910s), a blueprint for a protein (the 1940s), a physical molecule (the 1950s), a transcribed code (the 1960s) and an open reading frame (the 1970s-1980s). Recently, the Encyclopedia of DNA Elements (ENCODE) Project defined the gene as “a union of genomic sequences encoding a coherent set of potentially overlapping functional products” (Gerstein et al., 2007).

1.2.2 Genetics

Genetics (from Greek “genesis”, “original”) was first coined in 1831 by a historian, Thomas Carlyle (1795-1881) and the concept was extended to biology by Charles Darwin in 1859 so that he used it in 1872 in the meaning of “Law of origination”. Later on, William Bateson (1861-1926) put forward Genetics as the “study of heredity and variation” (1905) as understood through Mendel´s work.

Unlike the definition of the word “gene”, the definition of genetics has not been changed dramatically during the past 50 years. Some oversimplifications exist, such as stating that it means the study of genes, which is incorrect. Therefore, the definition of genetics as the study of heredity and variation is still widely accepted

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1.2.3 Epigenetics

The history of epigenetics is linked to the study of genetics, evolution and development. This concept has been changed dramatically. Epigenetics was first defined by Conrad H. Waddington in the 1940s. The word is a combination of genetics and epigenesis. The root term of epigenesis can be traced back to Aristotle who proposed it in opposition to preformation (which means that individual development is just an increase of already existing structures). Epigenesis, the concept that individual development involves a differentiation of the originally undifferentiated, was shown to be the correct explanation in the 18th century, along with the advent of achromatic microscope and the discovery of the germ layers in the chick embryo. In the 19th century, epigenetics was used to describe the working of epigenesis. However, from the middle of the 20th century, the definition of epigenetics acquired a new meaning. Waddington (1940) defined it as a branch of biology which deals with the causal analysis of development. He illustrated his theory with drawings (Fig. 2), where the developing system is depicted as a landscape, and the bifurcating and deepening valleys run down from a plateau.

Figure 2. (A) Waddington's epigenetic landscape. (B) The interactions underlying the epigenetic landscape. (Jablonka and Lamb, 2002).

In figure 2A, the plateau is the fertilized egg, and the path that the ball would take represents the development route from the fertilized egg to a certain tissue or organ. In

14

figure 2B, the path, slopes, cross-sections of the valley are decided by genes (peg) and their interactions (guy ropes). Canalization (Waddington´s term for development) and plasticity are two sides of the same coin. Even though they refer to the opposite view of phenotypic change ability, they have one aspect in common: phenotypic variation is not always coupled with genetic variation. This is the central point of Waddington´s epigenetics theory. So the difference between epigenetics and developmental genetics is their different foci: epigenetics focuses on the complexity of developmental networks with redundancy and compensatory mechanisms, while developmental genetics is more concerned about how a gene (or genes) affects the phenotype.

In the 1990s the meaning of “epigenetics” changed again as the molecular mechanisms controlling gene activity and the inheritance of cell phenotype began to be understood. Holliday´s work on cell memory, mainly his findings concerning DNA methylation, made the main contribution. He narrowed down the definition of epigenetics as “The study of the changes in gene expression, which occur in organisms with differentiated cells, and the mitotic inheritance of given patterns of gene expression” (Holliday, 2006). Today, the scope of this subject is much wider and I will regard this concept as modern epigenetics. It includes studies of cellular regulatory networks that confer phenotypic stability; changes of DNA regulated during development (such as those seen in the immune system); cell memory mechanisms; self-propagating properties; studies of the controlled responses of cells to genomic parasites and severe environmental effects, which involve DNA methylation, RNA mediated gene silencing and enzyme-mediated DNA rearrangements and repair. The position of modern epigenetics in biology is at the junction of genetics, developmental biology and ecology. These sciences contribute knowledge from different sides of life, and should be considered as being perfectly compatible and complementary. The mechanisms and phenomena that I discuss later will be based on this concept of epigenetics.

15

1.2.4 Chromatin

In eukaryotic cells, nuclear DNA is tightly packed with proteins into chromatin. The basic structure is the nucleosomes. Each nucleosome is composed of an octamer of the four core histones (H3, H4, H2A and H2B) where a stretch of 147 base pairs of DNA is wrapped around (Fig. 3). Histone 1(HI) is the linker protein between two nucleosomes. The core histones have a predominantly global structure, but their N-terminal tails are unstructured. Many of the residues in these N-terminal tails can be either covalently or non-covalently modified by several post-translational modifications (PTMs) and these modifications play causal roles in gene regulation.

Figure 3. Nucleosome structure. The core DNA (147 base pairs) is wrapped around the octamer of histones 1.8 turns. The DNA that connects adjoining nucleosomes is called linker DNA and is associated with the linker histone 1 (H1).

1.3 Epigenetic phenomena

From a traditional point of view, the best known epigenetic phenomena in eukaryotes are position effect variation (PEV), genomic imprinting (Fig. 4), transvection and the dosage compensation by X-chromosome inactivation. In addition, according to the modern definition of epigenetics, epigenetic phenomena should also include the lambda bacteriophage switch between lysis and lysogeny (Ptashne, 2005) and pili switching in

16 uropathogenic Escherichia coli (Hernday et al., 2002). Here, I shall only give a short overview on PEV in Drosophila and genomic imprinting in mammals, as examples.

PEV has been observed in flies, mammals and yeast. It was first discovered in flies with a variegated eye pigmentation phenotype by Muller in 1930 (Fig. 4A). An inversion on the X-chromosome (In(1)wm4) juxtaposed the white gene with centromeric heterochromatin, which then causes repression of white gene expression in some cells. Later studies showed that PEV can be detected in a wide range of genes, actually, all genes can show a PEV-variegated phenotype if they are in the right rearrangement (Girton and Johansen, 2008).

Figure 4. PEV (A) and imprinting (B). Modified from (Ferguson-Smith et al., 2006; Girton and Johansen, 2008).

Imprinting is a process that causes genes to be expressed or repressed depending on their parental origin (Fig. 4B). So far only a small number of imprinting genes has been discovered. These genes are marked in germ cells and can “remember” their parental origin. As a result of this their alleles are either activated or repressed. They play important roles in prenatal growth and organ development, but also after birth, in the regulation of metabolic pathways and in behaviors (Ferguson-Smith et al., 2006).

17 1.4. Regulation of gene expression, an epigenetic perspective

1.4.1 General principles

Some fundamental mechanisms of gene expression and regulation are conserved from one organism to another. The central dogma of molecular biology has changed its concept from an original simple, unidirectional pathway:

where black arrows mean the ability for DNA to replicate itself and the general procedure for a cell to transcribe DNA to mRNA, then to translate the mRNA to protein. The gray straight arrow indicates one special case: reverse transcriptases use a single-strand RNA as a template to generate a double-stranded DNA copy, for example in the case of retroviruses such as HIV, retrotransposons or telomere synthesis in eukaryotes. The gray curved arrow over RNA shows how RNA can be self replicated into double strand RNA via RNA-dependent RNA polymerases in the case of viruses, or in RNA silencing in eukaryotes. The white curved arrow over protein indicates the case of prions, diseases caused by protein conformation change.

Any particular cell type needs certain types or amounts of proteins. Most of the specialized cells have the possibility to adjust their gene expression according to extracellular cues. As shown in the modified central dogma above, there are many steps from DNA to protein. That is to say, in general, gene expression can be controlled at several levels: transcriptional control (initiation of transcription, transcription rate), RNA processing control, RNA turnover control and translational control. However, the transcription initiation controls appear to be the most important.

1.4.2 Epigenetic regulation in Prokaryotes

Phenotypic variation in bacteria, and in pathogenic bacteria in particular, has long been studied. The phenomenon that the individual bacterial cell in a clonal population can have a reversible switch of expression phase between “ON/OFF” is usually defined as phase

18 variation. Molecular mechanisms of phase variation include genetic and epigenetic regulation. The most extensively studied epigenetic regulations are the PAP and Ag43 phase variations (van der Woude and Baumler 2004). The PAP (pyelonephritis- associated pili) plays an important role in attachment to urinary epithelia of uropathogenic E.coli cells. The pap operon contains a 416bp regulatory region, as well as sites for the global regulator leucine-responsive regulator protein (Lrp), regulator proteins PapI and PapB, a global regulator catabolic activator protein (CAP) (Fig. 5).

Figure 5. DNA methylation-dependent phase variation of the pap operon in E.coli. Proteins that are essential are depicted. Modified from (van der Woude and Baumler, 2004).

It is a dual operon, both for transcription of pI promoter (which encodes PopI ) and pBA promoter (which is the main promoter for the pap operon). Expression of the pap operon phase variation is dependent on deoxyadenosine methyltransferase (Dam), which has two binding sites in the transcription region: GATCdist and GATCprox . In the ON state, Lrp- PapI binds to nonmethylated GATCdist sites, protecting GATCdist from Dam, but GATCprox is methylated. The expression of PapI needs PapB and CAP proteins. In the OFF state, Lrp, but not PapI protein, binds to non-methylated GATCprox sites leading the GATCdist to be methylated. Regulation of the switch from OFF to ON can be controlled

19 by environmental factors, for example, the binding of cAMP-CAP enhances papBA transcription, and the cAMP level is controlled by a carbon source in the environment. Lrp may bend pap DNA between the CAP and the pap BA promoter to facilitate the contact between cAMP-CAP (Goransson et al., 1989); Other environmental factors, such as H-NS can modulate Pap gene expression and Pap switch rates.

1.4.3 Epigenetic mechanisms in eukaryotic gene regulation

1.4.3.1 DNA methylation

DNA methylation has been found in mammals, plants, fungi and bacteria. There is, however, little evidence for any DNA methylation in Drosophila.

In vertebrates, the cytosine base in the dinucleotide sequence 5´CpG3´ is covalently modified by methylation through DNA methyltransferase enzymes (Dnmt). This CpG methylation is a common phenomenon in the vertebrates examined thus far. From 60 to 90% of the mammalian CpG sites are methylated; they include a wide range of DNA sequences such as satellite DNAs, repetitive elements and exons of genes. The non- methylated CpG regions usually cluster together and are called CpG islands. Most CpG islands mark the 5´ regulatory and promoter regions of genes and 60% of the human genes have CpG island promoters.

Molecular and genetic studies have showed that DNA methylation has a variety of functions in gene regulation; it is mainly associated with mutagenesis, gene silencing (genomic imprinting, X-chromosome inactivation). Two recent reports show that cycles of DNA methylation are also involved in transcription activity (Kangaspeska et al., 2008; Metivier et al., 2008). They found that the active X-chromosome has a higher DNA methylation level in the transcribed region (gene body) than the inactive X-chromosome.

Before going into the mechanism of CpG methylation in regulation gene expression, I would like to discuss first about how CpG is methylated. DNA methyltransferases (Dnmts) catalyze genome-wide DNA methylation (Table 1). There are two types of CpG methylation: initiation methylation and maintenance methylation (Fig. 6). Initiation

20 methylation is carried out by the Dnmt3a and Dnmt3b enzymes (de novo DNA methyltransferases). They are responsible for establishing new DNA methylation patterns. Mice lacking Dnmt3a die at about four weeks of age, while Dnmt3b null mice are not viable (Li, 2002). Maintenance methylation is catalyzed by Dnmt1, which only catalyzes the hemimethylated CpGs during DNA replication, so that it is regarded as cell memory holder. DNMT3L has a homology with Dnmt3a and Dnmt3b in the N-terminal regulatory

Table 1. The mammalian Dnmts family.

Dnmts Schematic structure Phenotype in Function Interacting knockout mice proteins -Embryo lethality -Maintence DMAP1/B Dnmt1 -Global DNMT; MI1, hypomethylation - NP95, -loss of Demethylation PCNA imprinting Dnmt2 -Viable, minor -Preference for defects centromeric structures Dnmt3a -Postnatal die -De novo EZH2, -loss of de novo methylation hSNF2H, methylation - HDAC, -spermatogenesis Demethylation LSH, defects SUMO1, UBC9 Dnmt3b -Embryonic -De novo EZH2, lethality methylation hSNF2H, -Loss of de novo - HDAC, methylation Demethylation LSH, -Demethylation of SUMO1, centromeric UBC9 repeat sequences Dnmt3L -Impaired -Has no spermatogenesis methyl- -Females have no transferase offspring activity -Loss of maternal -Enhancer of and paternal DNMT3A and imprints in DNMT3B gametes

21 region. It has no methyltransferase activity on its own, but it enhances Dnmt3a and Dnmt3b activity, and is required for establishing genomic imprints.

The DNA methylation patterns are established in the embryo and maintained through several cell divisions, and therefore constitute a cellular memory system (Riggs, 1975) (Fig 6).

Figure 6. Initiation methylation and maintenance methylation of DNA. Unmethylated DNA is shown as black vertical bars, the newly synthesized DNA strand is shown as the red bars, methylated CpG pairs are shown in red bars with pink dots. Unmethylated DNA is methylated de novo by Dnmt3a and Dnmt3b. During cell division, the newly synthesized DNA strand is unmethylated, forming a semimethylated double strand DNA. The methylation of semi-methylated sites to fully methylated ones is carried out by Dnmt1.

DNA methylation is associated with transcriptional silencing, either through interference with transcription factors binding (Fig. 7) or through recruitment of repressors (which include histone tail modification or chromatin remodeling factors). A number of transcription factors recognize and can bind to an unmethylated CpG sequence, but

22 cannot bind to a methylated CpG sequence. Examples include the CTCF (CCCTC binding factor) protein in imprinting at the H19/Ifg2 locus in mice (Bell and Felsenfeld, 2000) (Fig. 7).

Figure 7. CTCF mediated maternal imprinting regulation of Igf2 through an interaction with SUZ12. CTCF binds to the unmethylated ICR and the Igf2 promoter on the maternal allele and forms an intrachromosomal loop through CTCF dimerization. CTCF recruits the PRC2 complex via SUZ12, which results in histone methylation and formation of an inactive chromatin configuration around the Igf2 promoter. On the paternal allele, the CTCF cannot bind to methylated ICR, so, it cannot form a CTCF- CTCF dimer, resulting in an active Igf2 promoter. Modified from (Li et al., 2008)

1.4.3.2 Histone modifications

Regulation of gene expression occurs at the level of individual genes (DNA sequence) level, chromosome regions and even on entire chromosomes. The chromatin structure is dynamic and affects gene activity, depending on the cellular signals and/or environmental cues. ATP-dependent chromatin remodeling complexes, reversible histone modifications and histone variants are the main causal reasons for the changes of chromatin structure. Here I shall focus on the function of histone modifications and histone variants in gene regulation.

23 Eight types of post-translational modification (PTM) have been found on histones (Fig. 8): acetylation/deacetylation, methylation/demethylation and phosphorylation are those that have small chemical group modifications (covalent modifications), while ubiquitylation, sumoylation, ADP ribosylation, deimination and proline isomerization modifications include larger chemical modifications.

Figure 8. Some of the histone post-translational modifications (PTMs). They include methylation/demethylation, acetylation/deacetylation, phosphorylation, ubiquitination.

Acetylation The N-terminal tails of the core histones are rich in lysines, and thus are, accordingly, positively charged. This allows an intimate interaction with either the negatively charged backbone of the DNA and/or with adjacent nucleosomes, which then lead to a “tight” chromatin formation. Acetylation neutralizes this charge and weakens the interactions, resulting in a looser chromatin structure which facilitates the binding of transcription factor. In general, actively transcribed chromatin regions are associated with hyperacetylation and histone acetyl-transferases (HATs) recruitment, while histone deacetylases (HDACs) are thought to promote the return to a repressive, higher-order

24 chromatin structure. All core histones can be acetylated, but a considerable amount of evidence suggests that the acetylation of histones H3 and H4 has distinct functions and

Table 2. The function of histone acetylation and deacetylation in different species.

Chromatin Residues Enzymes that modify Histones Functions modifications Mammals Drosophila yeast Acetylation H2A K5 CBP/P300 Enok Hzt1K14 NuA4, SAGA

H2B K12 K15 H3 K3 Gcn5 K9 PCAF dAda2, dAda3 K14 Tip60, dAda2, Gcn5 PCAF dAda3 K18 PCAF K23 K36 Gcn5 K56 Rtt109 DNA repair, SPT10 Chromatin (H3/H4) disassembly H4 K5 Tip60, dAda2, Sas2p Initiation of DNA HAT1, Chameau replication; silencing Hbo1 K8 Tip60, Gcn5, Sas2p PCAF, Hbo1 K12 Tip60, dAda2, Hat1 DNA repair HAT1, dAda3 Hbo1 K16 Tip60, dMOF NuA4, Chromatin decondensation hMOF ScSAS2

Deacetylation H1K26Ac SirT1

H3K9Ac SirT1, SirT3

H4K16Ac SirT1, Sir2 Chromatin condensation SirT2, SirT3

25 temporal patterns: histone H3 acetylation seem to be involved in gene expression, while histone H4 acetylation seems to be more important in histone deposition to newly synthesized DNA in the S phase of cell division and chromatin structure (Vaquero et al., 2007). The acetylation/deacetylation of histones is catalyzed by HATs or HDACs. The balance between this dynamic equilibrium is a crucial component in generating a proper chromatin state as part of the response to environmental changes (Vaquero et al., 2007).

HAT proteins can acetylate the lysines on all four core histones, but different enzymes have their specific substrates (Table 2). There are three HAT families: GNATs (Gcn5 related N-acetyltransferase) family, which mainly targets H3 as its substrate; MYST proteins, which mainly target H4 and CBP/p300 (CREB-binding protein/E1A-associated protein of 300kD), which targets both H3 and H4 (Allis, C.D, epigenetics, 2006).

The GNATs are an enormous super family (about 10,000 members) of enzymes that are universally distributed in nature and that use acyl-CoA to acylate their cognate substrates (Vetting et al., 2005). Histone acetyltransferases are part of this family. The first HAT shown to be involved in transcription regulation was Hat A from Tetrahymena (Brownell et al., 1996), a homologue of yeast Gcn5. Humans express two homologs: Gcn5 and PCAF (p300/CBP associated factor). Gcn5/PCAF can acetylate H3K9, K14, and K18. Gcn5 and CBP/p300 have a bromodomains, which can specifically bind to acetylated lysines. CBP/p300 proteins are unique to metazoans.

The MYST (Moz, YBF2, Sas2p, Tip) protein family is defined by a highly conserved histone acetyl-transferase domain, the MYST domain. Five proteins have been identified in mammals: Querkipf (Qkf), Monocytic leukemia zinc finger protein (Moz), males absent on the first (Mof), HIV tat-interacting protein 60 (Tip60) and histone acetyl- transferase bound to ORC (Hbo1) (Thomas and Voss, 2007).

Qkf is required in neural stem cells, while Moz is essential for hematopoietic stem cells. They are closely related and belong to the same group in the MYST family. Moz and Qkf have several highly conserved domains: the PHD-type zinc fingers domain (which can bind to H3K4me3), the MYST domain (which has the acetyl-transferase activity), an acidic region in the central portion of the proteins, and serine-rich and methionine-rich

26 domains. The Drosophila protein Enok (enok is a mushroom) displays similarities in the MYST domain and N-terminal compared to Moz. Enok is important in the Drosophila mushroom body development (Scott et al., 2001)

Mof is closely related to Tip60. They share a conserved chromodomain and a MYST domain. The chromodomain in Tip60 is found to bind to methylated histone tails, while the Drosophila Mof chromodomain was found to mediate binding to a noncoding RNA (Akhtar et al., 2000). In Drosophila, Mof is one of the catalytic subunits (histone acetylation (H4K16)) of the male sex lethal (MSL) complex, which regulates the X- chromosome gene expression. Tip60 has a broad range of functions: for example, it has been shown to be involved in cell cycle control (by interacting with c-Myc) (Frank et al., 2003; Patel et al., 2004), and in the regulation of apoptosis (by acting as coactivator of p53 and NF-B) (Gaughan et al., 2002; Legube et al., 2004). Hbo1 in mammalian has multiple functions; it is involved as an activator, in the initiation of DNA replication. On the other hand, it can also interact with the androgen receptors where it acts as a repressor of transcription. The Drosophila homologue of Hbo1 is Chameau. It has been shown to interact with the origin recognition complex (Orc1), which not only binds to DNA replication initiation sites but also interacts with the Heterochromatin protein 1 (Hp1) (Shareef et al., 2001). In addition, a full Chameau activity is needed to maintain Hox gene silencing mediated by Polycomb group proteins (PcG) (Grienenberger et al., 2002).

Deacetylation Histone deacetylases (HDACs) catalyze the removal of acetyl groups from the N-terminal tails of histone proteins. There are two families: Rpd3/Hda1 family and sirtuin family. In humans, the Rpd3/Hda1 family contains HDAC1, -2,-3, -8 (class I, similar to yeast Rpd3 ); HDAC4, -5, -6, -7, -9, -10 (class II, similar to yeast Hda1); and HDAC11 (class IV). The sirtuin family has seven members in humans: SIRT1-7 (class III, similar to yeast Sir2).

Methylation Histone methylation contributes to transcriptional regulation, the maintenance of genome integrity and epigenetic inheritance. It is also the most complex

27 covalent modification among modifications. Methylation can not only occur at different residues (lysines, arginines) in different histone tails, but can also give rise to a different status of methylation (mono-, di- or tri-methylation) occurs at the same residue, and these different modifications may contribute to differential gene activity depending on the affinity of binding proteins. (Table 3).

Table 3. The function of histone methylation and demethylation.

Residues Catalyzing enzymes Functions Methylation H3K4 SET1/Compass KMT2) Activation of transcription MLL1 (KMT2A) Activation of transcription MLL2(KMT2B) Activation of transcription MLL3 (KMT2C) Activation of transcription MLL4 (KMT2D) Activation of transcription hSet1A (KMT2E) Activation of transcription hSet2B (KMT2F) Activation of transcription ASH1 (KMT2G) N/D SET7/9 (KMT7) N/D H3K36 ySet2 (KMT3) Elongation form of Pol II SET2 (KMT3A) Elongation form of Pol II NSD1 (KMT3B) N/D SYMD2 (KMT3C) Transcription activation H3K79 yDot1 (KMT4) Activation of transcription DOT1L (KMT4A) Activation of transcription H3K9 Su(var)3-9(D)/ Clr4(fy) Heterochromatin formation silencing (KMT1) Heterochromatin formation silencing SUV39H1 (KMT1A) Heterochromatin formation silencing SUV39H2 (KMT1B) Euchromatic H3K9 methylation G9a (KMT1C) Heterochromatin formation silencing EuHMTase (KMT1D) Transcriptional repression SetDB1 (KMT1E) N/D CLL8 (KMT1F) Transcriptional repression RIZ1 (KMT8)

28 H3K27 EZH2 (KMT6) Polycomb silencing E(z)(D) H4K20 sp Set9 (KMT5) DNA repair Pr-SET7/8 (KMT5A) Transcriptional repression SUV4-20H1 (KMT5B) DNA repair SUV4-20H2 (KMT5BC) N/D H4R3 PRMT1 Activation of transcription PRMT7 Imprinting in male germ cell H3R2, R17, R26 PRMT4, CARM1 Activation of transcription H3R8, H4R3 PRMT5 Repression of transcription Demethylation H3K4me1/2 LSD1 Repression of transcription H3K4me2/3 Yjr119Cp(y), JARID1a-d(y) H3K9me1/2 LSD1-AP Activation of transcription H3K9me1/2 JHDM2A Activation of transcription H3K9me3 JHDM3A, JMJD2 Activation of transcription H3K36me1/2 JHDM1 H3K36me3 JHDM3A, JMJD2A H3K27me2/3 UTX, JMJD3 Activation of transcription

Histone lysine methylation In most eukaryotes, histone methylation patterns regulate chromatin architecture and function. H3K4 methylation demarcates euchromatin. All histone methyltransferases, except Dot1, contain a SET domain, which has catalytic activity. Six of the methylated substrates in histones have been well studied: H3 (K4, K9, K27, K36, K79) and H4(K20). Among them, H3K4, H3K36, H3K79 are in general related to the activation of transcription, while the others are related to repression.

H3K4 methylation was first discovered in trout testes chromatin (Honda et al., 1975a; Honda et al., 1975b). It is associated with gene activation. This residue can be mono-, di- or trimethylated. High levels of H3K4me3 are associated with the 5´regions of virtually all active genes and that there is a strong positive correlation between H3K4me3 and

29 transcription rates, RNA pol II, and histone acetylation (Ng et al., 2003; Santos-Rosa et al., 2002; Schubeler et al., 2004). H3K4me1 is most abundant in the 3´ends of genes (van Dijk et al., 2005). On the other hand, the distribution patterns of H3K4me2 are quite different from yeast to mammals: In Saccharomyces cerevisiae, H3K4me2 appears to spread out over (among?) the genes, and is associated with a transcriptionally active gene. In mammals, however, H3K4me2 mainly colocalizes with H3K4me3 in discrete zones about 5-20 nucleosomes in length (Bernstein et al., 2005; Schneider et al., 2004). Only a small subset of H3K4me2 does not overlap with H3K4me3 and these regions do not correlate to transcription start sites, but are highly dependent on the cell types, suggesting that they may have a role in determining lineage specific chromatin states.

In the yeast, only one enzyme so far has been found to methylate H3K4. But in mammals, there are at least ten H3K4 methyltransferases; those that are related to the yeast Set1 and Drosophila Trx proteins (the MLL family) or those unrelated (ASH1, SET7/9, SMYD3 and Meisetz) (Beisel, 2002, Wang 2001, Hamamoto, 2004, Hayashi, 2005). The MLL family contains MLL1 (Mixed Lineage Leukemia 1), MLL2, MLL3, MLL4, SET1A and SET1B. The H3K4 methylase activities in mammals are not redundant, as the deletion of either gene causes embryonic lethality. Like most histone modifying enzymes, the MLL family exists in multi-protein complexes. There are three common subunits (WDR5, RbPB5 and ASH2) which are required for H3K4 methylation by MLL1 in vitro and in vivo (Dou et al., 2006).

In S.cerevisiae, the recruitment of Set1 to H3K4 is accomplished first by the phosphorylated form of RNA polymerase II c-terminal domain (phosphorylated Pol II CTD). This phosphorylation recruits Set1 to the H3K4 residue close to the promoter. The RNApol II is released from the transcription initiation complex into the elongation complex (Promoter escape). The second factor that recruits H3K4me3 is the PAF elongation complex, which is involved in RNA metabolism and interacts with Set1 and H2B monoubiquitylation. This H2BK123ub1 is the third component involved in H3K4 methylation. This mechanism in yeast is regarded as a general H3K4 methylation regulation mechanism. The existence of a larger amount of H3K4 methyltransferase in mammals makes it reasonable to envision several possibilities: H3K4 metyltransferases

30 may be recruited 1) directly or indirectly by sequence specific DNA binding proteins; 2) by association with basal transcriptional factors; 3) through chromatin modification readers; or 4) by RNA from the specific RNApol II binding regions. The available evidence suggests the existence of a combination of gene specific and general mechanisms. Ruthenburg et al. (2007) suggest that the initial recruitment is gene specific, mediated by specific transcription factors and/or RNAs, and is sensitive to the signaling cues within the cell, whereas further stabilization of the complex on chromatin and stimulation of its catalytic activity occur through general mechanisms and stabilization of the methyltransferases on chromatin by the association with acetylated and H3K4 methylated histones .

H3K36 methylation is catalyzed by the Set2 histone methyltransferase and is necessary for the elongation phase of transcription. It also promotes deacetylation of transcribed chromatin. Methylated H3K36 is found within the coding regions of actively transcribed genes. Set2 can catalyze all three states of H3K36 and negatively influence gene expression (for example the repression of cryptic promoters within coding regions of the gene STE11). Different states of H3K36 (H3K36me2 and H3K36me3) have different functions: the N-terminal of Set2 (containing SET domain) is sufficient for H3K36me2, and is correlated to whether a gene is expressed or not, with histone deacetylation and repression of cryptic promoters at STE11(Youdell et al., 2008)), while H3K36me3 tends to correlate to highly expressed genes via the C-terminal domain of Set2 and requires Spt6, the H3P38 (histone proline 38), and the CTD of RNAPII (Youdell et al., 2008)).

H3K79 methylation, unlike the other histone tail modifications, is located within the globular domain, and is thus exposed on the nucleosome surface. The yeast Dot1 (disruptor of telomeric) and its homologues in other species are the only known H3K79 methyltransferases (Feng et al., 2002). Unlike the other methyltransferases, the Dot1 family members do not have a SET domain (Feng et al., 2002). Their catalytic domain contains conserved sequence motifs characteristic of class I methyltransferases such as DNMTs and arginine methyltransferases (PRMT1) (Cheng et al., 2005). In the yeast, H3K79me is localized to euchromatic regions and it is associated with the coding region of active genes.

31 H3K9 methylation is intimately connected with heterochromatin. It is, next to deacetylated histones, the evolutionary most stable heterochromatic marker (Krauss, 2008). However, it has also been found to be enriched in transcribed active genes (Vakoc et al., 2005). There are three locally and functionally distinct distributions of H3K9me: 1) heterochromatin regions, 2) some epigenetically modified silenced promoter regions of euchromatic genes (Tachibana et al., 2005), 3) within active transcription units where it participates in the repression of illegitimate initiations of transcription (Vakoc et al., 2006). As in other lysine methylation residues, H3K9 can be methylated in three levels: mono-, di-, trimethylation, and these different levels of methylation status have distinct locations: H3K9me1 (in mouse), H3K9me2 (in maize) or H3K9me3 (in Arabidopsis, maize and Chlamydomonas) are mainly found in euchromatin; while H3K9me3 seems common in the heterochromatin of animals, fungi, but not or rarely in plants. Recently Folco and colleagues found that, together with the RNAi pathway, H3K9me2 is needed to mark the neighboring heterochromatin region to recruit CENP-ACnp1 (Centromere- specific protein) in the central domain in fission yeast Schizosaccharomyces pombe (Folco et al., 2008). Drosophila seems to have a similar distribution pattern of H3K9me1, 2, 3, they are all localized to heterochromatic regions (Krauss, 2008).

H3K9 is methylated by SUV39 methyltransferases family. The first HKMT (histone lysine methyltransferase) was HEK9-SUV39H1 (Rea et al., 2000). Proteins in this family contain a N-terminal chromo domain and a histone methyltransferase (HMTase) catalytic domain consisting of a SET domain flanked at the N-terminus by a cysteine-rich pre-SET domain (required for specificity toward H3K9) and a C-terminus post-SET domain.

The formation of heterochromatin involves the cooperation of two proteins: SUV39H and its binding protein HP1. HP1 has three forms in mammals:  and. HP1 and HP1 interact with methylated H3K9 within the heterochromatin and the silenced regions of euchromatin. However, HP1 interactions with methylated H3K9 are found on the actively transcribed gene regions (Vakoc et al., 2005). The model for these two proteins interaction is that methylated H3K9, which is carried out by SUV39H, works as a binding platform for HP1 through its chromodomain. Once HP1 binds, it can spread into adjacent nucleosomes by its association with SUV39H, which then further methylates histones.

32 On the other hand, HP1 has the possibility to self-associate with its chromoshadow domain, which in turn facilitates the spread of heterochromatin. This is like the writer (SUV39H) and the reader (HP1) relationship. How the writer can find the specific place to write was revealed by the discovery of siRNAs (small interfering RNAs) (Volpe et al., 2002). These RNAs come from the bidirectional transcription of centromeric repeats via the Dicer enzyme, and these RNAs are packed into the RITS complex, which contains the H3K9 methylated binding protein Chp1.

H3K27 methylation is crucial in gene silencing during development (Cao and Zhang, 2004), X-inactivation (Plath et al., 2003), stem-cell pluripotency (Boyer et al., 2006), cancer (Sparmann and van Lohuizen, 2006) and inflammation (De Santa et al., 2007). The control of H3K27 methylation is dynamic. It is catalyzed by EZH2 ( E(z) in Drosophila) and can be methylated at three levels as well. EZH2 is a SET domain (Su(var)3-9, Enhancer-of-zeste, Trithorax) containing enzyme, and is a core component of PRC2 (Polycomb repressive complex 2). It has been shown that E(z) can methylate all three levels of H3K27 in Drosophila, while in mammals, EZH2 can only methylate di-, and tri- H3K27. In fact, the three levels of methylation have very different distributions: in both Drosophila and mammals, H3K27me2 is virtually ubiquitous in euchromatic regions (more than 50%); H3K27me3 is found essentially in PcG target sites (5-10%) (Schwartz and Pirrotta, 2008). A heterochromatic enrichment of H3K27me1, H3K27me2 and H4K20me1 has been specifically found in angiosperms (plants). In Drosophila, E(z) or (PRC2) is recruited to chromatin via the interaction of PRE (Polycomb response elements) binding proteins. But in mammals, the targeting of the EZH2 complex may be mediated by a variety of transcription factors, like YY1, GAGA factor and MYC.

H4K20 mono-methylation, H4K20me1, is associated with facultative heterochromatin (Fang et al., 2002; Martens et al., 2005; Sims et al., 2006), and the inactive X chromosome (Kohlmaier et al., 2004). H4K20 me3 is a marker of constitutive heterochromatin (Mikkelsen et al., 2007). Mono- methylation of H4K20 is carried out by

33 PR-Set7 HKMT. SUV4-20H1 and SUV4-20H2 methylates catalyse the formation of H4K20 me2 and H4K20me3. There is an exception, however; H4K20 has been linked to DNA repair via the binding of the DNA damage checkpoint protein CrB2.

Histone Arginine methylation In mammals, histone arginine methylation is found on the H3R2, H3R8, H3R17, H3R26 and H4R3 residues. It is catalyzed by the PRMTs (protein arginine methyltrasferases) class of histone methyltransferases and contributes to both active and repressive effects on gene expression (Wang et al., 2001). Histone arginine residues can be methylated in mono-methyl, symmetrical di-methyl or asymmetrical di-methyl states (Fig. 9). So far, eleven arginine methyltransferases (PRMT1-11) have been found in mammals and six of them have the ability to methylate histone arginine (PRMT1,4,5,7,8,9). Drosophila has 9 homologues (DART1-9), and they all have the histone arginine methyltransferase ability. In yeast the PRMT1 (the Drosophila homologue is DART1,2,3,6,8,9) is recruited to actively transcribed genes during elongation, and its activity is important for heterogenous nuclear ribonucleoprotein (hRNP)-mediated mRNA processing and export. In mammals, PRMT1 interacts with the p160 family of nuclear receptor coactivators and facilitates transcription driven by the androgen receptor through the methylation of histone H4R3 (Wang et al., 2001). PRMT4 can methylate both the N-terminal (R2, 17, 26) and C- terminal (R128, 129, 131, 134) arginine residues in histone H3 and enhance transcription activation by methylating both histone arginine and coactivators (Bauer et al., 2002). PRMT5 symmetrically methylates histones H3R8 and H4R3 along with other cellular proteins. This enzyme has been found in multiple complexes where it mediates diverse functions including RNA processing, transcriptional regulation, and muscle as well as germ line differentiation (Dacwag et al., 2007). It has been co-purified with chromatin

34

Figure 9. Histone arginine methylations and deimination by PRMTs enzymes.

remodeling complexes, hSWI/SNF and NURD, and has therefore been associated with transcriptional repression of cell cycle regulators (by methylating the promoters of CYCLIN E, P14ARF, and P16INK4a) and tumor suppressor genes (ST7 and NM23 (Bachman et al., 2003; Le Guezennec et al., 2006). PRMT5 has also been shown to interfere with gene expression by methylating and promoting the dissociation of the transcription elongation factor SPT5 (Kwon et al., 2003). On the other hand, PRMT5 was found as a component of the androgen receptor-driven transcription, which is independent of its catalytic activity (Chie et al., 2003). Accordingly, PRMT5 affects transcription in a methylase-dependent as well as a methylase-independent fashion. PRMT7 can methylate H2A and H4 (Lee et al., 2005). It mediates H4R3 methylation at the H19 and GTL2 loci

35 in male germ cells that contributes to the imprinting of H19 and GTL2 genes (Jelinic et al., 2006). PRMT8, 9, 10, 11 are recently found and their function is still unknown.

Demethylation The discovery of the first histone demethylase, lysine-specific- demethylase-1 (LSD1) (Metzger et al., 2005; Shi et al., 2004a), and the peptidyl arginine deiminase 4 (PAD14/PAD4) (Wang et al., 2004), open a whole new view on the dynamic regulation of chromatin.

LSD1 is an amine oxidase that catalyzes lysine demethylation in a FAD (flavine adenine dinucleotide) dependent manner and is involved in repression. Interestingly, the specificity of LSD1 can be changed if it binds to a partner, for example, the androgen receptor (AP). When LSD1 and AP form a complex, it will demethylate H3K9 instead of H3K4, acting as an activator instead of a repressor (Metzger et al., 2005). But, LSD1 can only demethylate mono-and dimethyl marks on H3K4me1/2 and H3K9me1/2. It can not demethylate tri-methylated lysines (example: H3K4me3 and/or H3K9) due to the absence of a protonated nitrogen required for oxidation (Metzger et al., 2005; Shi et al., 2004b). Demethylation of trimethyl marks on H3K4me3 in budding yeast is carried out by YJR119Cp (or JARID1) (Liang et al., 2007; Seward et al., 2007). Both groups show that H3K4 methylation and demethylation can be dynamically regulated, but Liang et al. (2007) also showed that YJR119Cp contributed to the regulation of telomeric silencing as well. The H3K4me3 demethylase is evolutionarily conserved. The Drosophila YJR119Cp homolog lid (little imaginal discs) is a member of trithorax group of genes (Klose et al., 2006). Lid can demethylate H3K4me2/3 in vitro, but not in vivo for unknown reasons.

The demethylation of H3K36 is carried out by JHDM1 for H3K36me1/2 and JHDM3A/JMJD2A for H3K36me3 (Klose et al., 2006). There are two things here which are interesting to note: that the different enzymes are carried out the removal of the various methyl group in the same lysine residue. JHDM1 is only capable of removing the H3K36me1/2 modification states (Tsukada et al., 2006; Whetstine et al., 2006), while H3K36me3 demethylation is catalyzed by JHDM3A/JMJD2A. The second interesting

36 issue is that the enzyme JHDM3A/JMJD2A can demethylate both the repressive mark H3K9me3 and the active mark H3K36 me3. These dual functions are probably due to interactions with different co-factors.

H3K79 methylation might be a more static mark since so far no demethylase for this modification has been found. In contrast to the rapid deacetylation of H3, levels of H3- K79me2 decrease gradually through the developmental time, which indicates that replication-mediated dilution can largely account for the loss of di-methylated H3-K79; while the more rapid removal of H3-K4 methylation requires a replication-independent component that is specific for the H3-K4 modification (Katan-Khaykovich and Struhl, 2005).

The demethylation of H3K27me3 and H3K27me2 to H3K27me1 is catalyzed by the JmjC-domain proteins UTX and JMJD3 (Agger et al., 2007; Lan et al., 2007; Lee et al., 2007; Xiang et al., 2007). So far, no enzyme has been found that catalyzes demethylation of H3K27me1, but a recent genomic study revealed that H3K27me1, me2 and me3 are enriched downstream from transcription start sites of active genes (Barski et al., 2007). In pluripotent cells, Hox genes are kept in a silent state by Polycomb group complex (PcG). The genes are marked by H3K27me3, a modification which is catalyzed by EZH2 in the PRC2 complex (PcG function will be discussed later). During differentiation, Hox genes are derepressed colinearly, UTX was found to be recruited to the promoters of active Hox gene while SUZ12 and EZH2 showed decreased occupancy. In differentiated cells, UTX controls a steady-state level of non-methylated H3K27 at transcription start sites, but not at coding and intergenic regions.

De Santa and co-workers (De Santa et al., 2007) showed that JMJD3 is rapidly induced in macrophages in response to an inflammatory stimulus. Recruitment of JMJD3 affected the expression of a subset of genes induced by inflammation, Bmp-2 being one of the genes. Upon treatment, H3K27me3 levels decrease at the Bmp-2 locus, depending on JMJD3.

Unlike lysine demethylation, there is no true histone arginine demethylase identified, so far, which can remove methyl groups from either symmetric or asymmetric dimethylation

37 marks. Instead, at least so far, PADI4/PAD4 is the only enzyme which has been found to antagonize histone arginine methylation by converting either arginines or monomethyl- arginine to citrulline in a Ca2+ and DTT dependent reaction.

Ubiquitination / Deubiquitination

Ubiquitin is an 8.5kKD (76-amino acid) protein (Goldknopf et al., 1975). Its C-terminal four amino acids are in a random coil, whereas the N-terminal 72 amino acids form a compact globular structure.

Ubiquitination is the process where ubiquitin covalently conjugates to substrate proteins. This involves three separate enzymatic activities (Fig. 10): Ubiquitin is activated by a covalent attachment to E1 enzyme in an ATP-dependent reaction and is subsequently transferred to an E2 (ubiquitin-conjugating) enzyme. Ubiquitin is transferred from E2 to target substrate with an E3 (ubiquitin ligase) enzyme. Substrates can be mono- or polyubiquitinated; whereas polyubiquitination (at lease four units of linked ubiquitin) of target proteins are recognized by proteasomes (Pickart, 2001), monoubiquitinated proteins are stable and are usually intracellular compartments (Hicke, 2001; Hicke and Dunn, 2003)

Figure 10: The process of ubiquitination. Modified from (Pickart, 2001)

38 The histone H2A was the first protein identified to be ubiquitinated (Goldknopf et al., 1975). The ubiquitination site for H2A is lysine 119 (Nickel and Davie, 1989) and ubiquitinated H2A (ubH2A) has been detected in many eukaryotic organisms except in S. cerevisiae. The majority of ubH2A is monoubiquitinated, however, polyubiquitinated H2A has also been detected (Nickel and Davie, 1989). In addition to H2A, H2B can also be ubiquitinated (West and Bonner, 1980). ubH2B is less abundant (1%-2%) than ubH2A (5%-15%) and it can be either mono- or polyubiquitinated (Geng and Tansey, 2008). Unlike ubH2A, however, the site for H2B ubiquitination (ubH2B) is different among organisms: for example, the ubiquitin site in S. cerevisiae is Lys-123, in S. pombe is Lys- 119, in Arabidopsis is Lys-143 and in mammals it is in Lys-120. Thus far, only monoubiquitinated H2B has been found.

Table 4. Enzymes involved in H2A and H2B ubiquitination/deubiquitination in different organisms

ubH2B duH2B ubH2A duH2A E2 E3 E2 E3 S. cerevisiae Rad6 Bre1 Ubp8 - - - Ubp10 (Dot4) Ubp3/4 (Doa4) S. pombe Rhp6 Brl1 Brl2 Drosophila Dhr6 Bre1 Nonstop dRing L(3)73h USP7 (Sce) Mouse mHR6A LASU1 mHR6B Human hHR6A/ RNF20 USP22 Ring1B Ubp-M hHR 6B USP3 2A-HUB 2A-DUB UbcH6 USP21 Mdm2 USP3 Arabidopsis HUB1 SUP32 Modified from (Weake and Workman, 2008; Zhang, 2003).

39 In addition to H2A and H2B, ubiquitination of H3 and H1 has also been reported (Chen et al., 1998; Pham and Sauer, 2000), but the sites for ubiquitination for these two proteins are still not known. The histone variants H2A.Z and macro H2A1.2 are also reported to be monoubiquitinated at Lys-120 or Lys-121 for H2A.Z and Lys-115 for macroH2A1.2 in mammals (Chu et al., 2006; Sarcinella et al., 2007). The monoubiquination of H2A.Z is associated with human X-chromosome inactivation, but the biological function of monoubiquitination of macroH2A1.2 is unclear.

Ubiquitin can be removed through hydrolysis of the peptide bond at Gly76 by deubiquitinating enzymes (DUB). These consist of C-terminal hydrolases (UCH) and ubiquitin-specific processing proteases (UBP) (D'Andrea and Pellman, 1998).

Enzymes involved in ubiquitination/deubiquitination in different species are listed in Table 4, and the biological functions of the histone ubiquitination/deubiquitination modifications are given in Table 5.

Table 5. The biological functions of histone ubiquitination/deubiquitination modifications.

Functions of ubiquitination and crosstalk with other histone modifications Transcriptional activation Transcriptional DNA Cell Silencing damage cycle

Initiation Early Late enlongation enlongation ubH2B √ √ √ H3K4me2/3, H3K79me2/3 duH2B √ √ ubH2A √ √ √ H3K4me2/3, H2AXpho H3K4dem3 H1 ubH2AX H3K27me3 duH2A √ √

40

Histone ubiquitination and deubiquitination play important roles in regulating gene transcription. They are based on the large amount of factors involved in addition to ubiquitin-conjugating enzymes and ubiquitin ligases that regulate ubH2A/duH2A and ubH2B/duH2B. The mechanism for transcription regulation by ubH2A/duH2A and ubH2B/duH2B is based on a crosstalk with other histone modifications. For example, in human HOX A7 gene silencing, H3K27me3 is a prerequisite for ubH2A and DNA methylation, whereas ubH2A and DNA methylation are interdependent (Wu et al., 2008). Interestingly, ubH2B can either activate transcription or silence gene activity. These dual functions depend on gene location: for genes located in euchromatin regions, ubH2B appears to result in gene activation, while for genes located in heterochromatic regions, ubH2B has a negative effect. Regulation of gene transcription by ubH2B involves also a crosstalk with other histone modifications, for instance, with H3K4me2/3 and H3K79me2/3. However, unlike ubH2A, ubH2B is upstream of H3K4me2/3 and H3K79me2/3 for initiation and the early stages of transcription elongation (Kao et al., 2004; Wood et al., 2003).

Sumoylation

Even though it was discovered only a decade ago, sumoylation of proteins have got great attention because of its vast ranges of target proteins. Similar to ubiquitination, sumoylation is a procedure of a Small Ubiquitin-like Modifier (SUMO) protein covalently attaching to the target protein. The SUMO proteins are about 12 kD (100- amino acids). As their name indicates, they are 8-15% identical to ubiquitin and have similar structure and mechanism of attachment to target proteins.

Since its identification in 1996 (Matunis et al., 1996), a large number of proteins (more than 50 proteins) have been shown to be post-translationally modified by SUMO. For example, the nuclear pore complex members RanGAP1 and RanBP2 (Joseph et al., 2002); the chromatin modifiers EZH2 and SUZ12 (Riising et al., 2008); Nuclear bodies

41 members PML, HIPK2; transcription factors p53 and CTCF (MacPherson et al., 2009); transcription co-factors HDAC1 , p300 and TIF1Mascle et al.; signal transduction proteins IKB, FAK (Gill, 2004). So, sumoylation is involved in various cellular processes, such as transcriptional regulation, nuclear transport, maintenance of genome integrity, and signal transduction, targeting a protein to specific subcellular locations (Johnson, 2004).

Here I will briefly describe some recently discoveries of the functions of sumoylation modification in histone and histone association factors.

First, Smt3 (the Drosophila homologue of SUMO-3) is expressed throughout development, more highly expresses during embryogenesis and in adult females (Huang et al., 1998). It has been observed in chromocentre in polytene chromosome in Drosophila (Lehembre et al., 2000). In humans, SUMO protein is also found restricted to the constitutive heterochromatin regions in pachytene spermatocytes (Metzler-Guillemain et al., 2008). Also, members involved in epigenetic regulators have been found can act either as sumoylation E3 ligase (for example: Pc2 in mammals and Suppressor of variegation 2-10 (Su(var)2-10) in Drosophila (Kagey et al., 2003; Mohr and Boswell, 1999)) or as target proteins (for example: SOP-2 (Zhang et al., 2004), SUZ12 and EZH2 (Riising et al., 2008)). Pc2 was first shown to act as a SUMO E3 ligase for repression of CtBP in mammals (Kagey et al., 2003). SUMO modification of histone 4 (H4) has been shown to relate to transcriptional repression (Shiio and Eisenman, 2003). SOP-2 (a PH (Polyhomeotic) and SCM (Sec comb on midleg) homolog in C. elegans) is also required to be modified by sumoylation to have the repression regulation of Hox gene. So, sumoylation modifications of histone and histone associate factors required for their proper repression functions.

Phosphorylation

Phosphorylation is the most well known in signaling transduction pathways. Histone 3 serine 10 (H3S10) was one of the first proteins found to be phosphorylated (Mahadevan et al., 1991). The phosphorylation of H3S10 by JIL-1 kinase can counteract with

42 heterochromatization and gene silencing (Deng et al., 2008). Studies of linker histone H1 in mouse cells revealed that phosphorylation of this histone may affect its binding to HP1 (Hale et al., 2006). H2A.X is another known histone which can be phosphorylated. Phosphorylation of H2A-X is an early event in double-strand break repair.

Limited by the space in this thesis, some other aspects which should be considered in epigenetic regulation of gene expression mechanisms have not been described. For example, the non-coding RNA mediates gene expression mechanisms; the TrxG regulation of gene activation mechanisms; the histone variation mechanisms……

The rapidly increasing data in the past few years strongly support the hypothesis that the epigenetic regulations of gene expression play important roles in development.

1.5 Regulation of gene expression by PcG proteins

1.5.1 Introduction

What makes our skin cell different from our liver cells, even though they have the same DNA sequence? The answer is simple: because they express different proteins. There are, however, much more complicated questions: how to decide which protein should be expressed or not; how cells maintain their determined state over many cell divisions (a process termed “cellular memory”). We know at least some parts of the answers to these questions. The Polycomb groups (PcG) and trithorax group (TrxG) are two gene families, that have antagonistic functions: PcG proteins are believed to be required to maintain repression Hox gene activity, while TrxG proteins maintain activated expression. PcG proteins were originally identified in Drosophila as factors necessary to maintain cell-fate decisions throughout embryogenesis by repressing Hox gene expression in a segment- specific manner. Now PcG is known to constitute a large chromatin associated family of proteins which is involved in many cellular memory processes: such as Hox gene regulation (body layout), X-inactivation in female mammals and stem cell regulation. Here, I shall first introduce PcG members and their functions, then I will describe PcG

43 mechanisms in genome programming. I shall also briefly discuss PcG genes in an evolutionary perspective and finish this chapter by describing the inheritance of the memory states.

1.5.2 Genetic and biochemical characterization of PcG proteins

The first PcG gene, Polycomb (Pc), was identified and analyzed by Pamela and Edward B. Lewis (Lewis, 1978). Heterozygous Pc mutant male flies have extra sex combs on the second and third legs. Homozygous mutants are embryonic lethals. Subsequent studies showed that Hox genes are similarly misexpressed in several other mutants, so this set of mutants that cause Hox gene derepression was named the Polycomb group (Duncan, 1982). More than 20 years ago, Jürgens (Jurgens, 1985) predicted that there are about 30- 40 PcG genes in the Drosophila genome based on an embryonic assay: embryos that are double homozygous for mutations in two different PcG genes express in general strongly enhanced homeotic transformation compared to single mutants. Until 2006, only 16 PcG genes had been identified. Alonso and co-workers (Gaytan de Ayala Alonso et al., 2007) tried to identify further PcG genes by a genome wide systematic genetic screen. When I am writing this thesis in late 2008, 33 PcG genes have been identified in Drosophila. Table 8 shows their biological functions and the mutant phenotypes.

Based on the genetic and the biochemistry studies of PcG regulation on Hox gene repression, so far three main PcG complexes have been identified: PRC1 (Polycomb repress complex 1), PRC2 (Polycomb repress complex 2) and PhoRC (Pho-repressive complex). Except these three complexes, there are some genes which are loosely related to major complexes or the ones with unknown function.

44 Table 6. Polycomb group members in Drosophila melanogaster.

Gene Name Location Protein Biochemical activity Mutant functional phenotype* domain Polycomb 3L N-terminal Binds to H3K27me3 I chromo- (Pc) domain

Interacts with dRing C-terminal Pc box Polycomb like 2R PHD Enhances H3K27me3 I (Pcl) Tudor Posterior sex 2R Ring finger Enhances ubiquitination I comb

(Psc) Polyhomeotic- X SAM Might promote the spreading III distal of PcG phd Oligomerization Polyhomeotic- X SAM Oligomerization III proximal php Sex comb 3R RING-type H2AK119ub ubiquitination I extra zinc finger (Sce/dRing) Pleiohomeotic 4 Zinc finger Sequence-specific DNA II binding; can interact with (pho) PRC1 and PRC2 components Pleiohomeotic- 3L Zinc finger Sequence-specific DNA III

45 like binding; can interact with PRC2 components (phol) Scm-related 2L MBT Binds to H3K9me1/2 and I gene H4K20me1/2 SAM containing four mbt domains

(dSfmbt) Enhancer of 3L SET H3K27 methyltransferase I zeste H1K26 methyltransferase (E(z)) Extra sex 2L WD-40 Contributes to H3K9me and I comb H3K27 methylation

(ESC) Extra sex 2L WD-40 Contributes to H3K9me and I comb like H3K27 methylation

(ESCL) Suppressor of 3L VEFS box Contributes to H3K27 I zeste (Su(z)12) methylation Zinc finger P55 3R Histone- Associates with HDAC, binding H3K27me. (Nurf-55, or

Caf1)) Sex comb on 3R MBT, SAM, Self-association (SAM midleg Zing finger domain), binds to mono- methylated histone (Scm)

46 super sex 2R Unknown III comb

(sxc) Additional sex 2R combs

(Asx) Su(z)2 2R Cramped X II Multi sex X II comb

(mxc) Pipsqueak 2L BTB-POZ DNA binding

(Psq) Dorsal Switch X HMG Single strand DNA binding Protein

(Dsp1) Calypso 2R I

Siren 5 2R Unknown III Siren 1/kto 3L Unknown III Siren 9 /skd 3L Unknown III Siren 2 3L Unknown III Siren 7 3L Unknown III Siren 8 3L Unknown III Siren 3 3R Unknown III Siren 4 3R Unknown III Siren 6 3R Unknown III Circe 3R Unknown III

47 *: Class I phenotype: Strong homeotic transformation; Homozygous lethal. Class II phenotype: Mild homeotic transformation. Class III phenotype: Weak homeotic transformation.

The PRC1 complex

A 2MD PRC1 (Polycomb repression complex 1) complex was purified from 0-12 hr transgenic Drosophila embryos. It contains five core proteins: Phd, Php, Psc, Pc and dRing (Francis et al., 2001; Saurin et al., 2001; Shao et al., 1999) (Table 9).

Table 7. PcG complex paralogs in different organisms

Proteins D. M. musculus A. C. elegans Melanogaster thaliana PRC1 complex (2Md) Polycomb Pc Cbx2/M33 Cbx4/MPc2 Cbx6 Cbx8/MPc3 Cbx7 polyhomeotic-dorsal phd Edr1/Mph1/Rae28 SOP-2 Edr2/Mph2 polyhomeotic- php proximal Posterior Sex Combs Psc Bmi1 Mel-18 NsPc1 PcGF3 PcGF5 MBLR dRing/Sex combs dRing/Sce Ring1/Ring1a extra Rnf2/Ring1b

48 PRC2 complex (core complex) Extra sex comb ESC Eed FIE MES-6

Enhancer of zeste E(z) Ezh1 CLF MES-2 Ezh2 MEA SWN

Supressor of zeste 12 Su(z)12 SU(Z)12 FIS2 VRN2 EMF2

Nurf55 p55 RbAp48 MSI1-5 RbAp46 Pho-RC complex Pleiohomeotic Pho YY1, YY2

Pleiohomeotic-like Phol YY1, YY2

Scm-related gene dSfmbt SFMBT containing four MBT domains Pcl-PRC2 Polycomb-like Pcl Enhancer of zeste E(z) Supressor of zeste 12 Su(z)12 Extra sex comb ESC Nurff55 Nurff55 Heat-shock cognate HSC70 protein 70

49 Scm was also purified but at a lower amount. In addition to that, 25 other associated gene products were also co-purified with these core components; for example: Zeste, six TAFs (TATA box binding protein-associated factors) proteins: TAFII250, TAFII110, TAFII85, TAFII62, TAFII42 and TAFII30. The large numbers of proteins associated with PRC1 implies that PRC1 may have multiple roles in gene regulation. An intriguing thing is the co-purification of dTAFII promoter factors, which indicate a direct interaction between PcG and promoter complexes. However, the main function of PRC1 is to maintain genes silenced via its functional components. The N-terminus of Pc contains a chromo-domain which preferentially binds to H3K27me3; while its C-terminus has a PC domain that can bind to dRing. dRing has an E3 ubiquitin ligase activity, which specifically catalyzes monoubiquitination of H2AK119. The H2AK119 monoubiquitination correlates well with transcriptional silence and is presumed to inhibit RNA polymerase II transcriptional elongation and DNA damage repair (Zhou et al., 2009; Zhou et al., 2008). In addition to ubiquitination of H2AK119, a mammalian homolog of PRC1 was recently shown also to act as E3 ubiquitin ligase for Geminin, an important factor for DNA replication (Ohtsubo et al., 2008). The C-terminus of Ph contains a SAM (sterile alpha motif) domain. The SAM domain is responsible for the polymerization of PcG complexes (Kim et al., 2002) and so is thought to promote the spreading of PcG complex along the chromatin. Psc is a ring finger-containing protein, but the function in Drosophila is still unclear except that it can enhance the ubiquitination of H2AK119.

The PRC2 complex

The Drosophila PRC2 complexes are multiple entities that change dynamically during development. Three distinct PRC2 complexes are found in Drosophila embryos (Table 10):

The first biochemically isolated PRC2 complex from 0-24 hr transgenic embryos has the size of 600 kD that contains three PcG proteins Enhancer of zeste [E(z)], Suppressor of zeste 12 (Su(z)12), Extra sex combs (ESC) and/or Extra sex combs-like (ESCL), and Nurf55 (Muller et al., 2002). This 600 kD complex is the most common one. The main function of this complex is the histone 3 lysine 27 methyltransferase activity carried out by E(z) via its SET domain. However, E(z) alone cannot catalyze the H3K27 methylation,

50 it needs Nurf55 to bind to nucleosomes, while Su(z)12 and ESC are needed for the enzyme activity. esc and escl are two homolog genes in Drosophila with a similar function. ESC protein level is high in 0-18 hr embryos, and decreases from 18-24 hour embryos to 2nd instar larvae. There is no detectable ESC in 3rd instar larvae, pupae and adult males, but it re-appears in adult females. In a fashion different from ESC, ESCL is expressed ubiquitously throughout development (the expression level is, however, weaker in pupae). Genetic and biochemically studies show that ESCL can substitute for ESC and functions as a backup capacity during development (Kurzhals et al., 2008; Ohno et al., 2008). Su(z)12 contains a VEFS domain which is conserved from plant through mammals. The biological function of Su(z)12 remains unclear. It is needed for all levels of methylation in H3K27 (H3K27me1,2,3) (Chen et al., 2008; Nekrasov et al., 2007).

Table 8. The PRC2 complexes. PRC2 Embryo Components stages E(z) ESC Su(z)12 Nurf55 Rpd3 Pcl HSC70

600 kD 0-24 hour √ √ √ √

1 MDa 0-12 hour √ √ √ √ √ √

Pcl-PRC2 16-18 hour √ √ √ √ √ √

The 2nd PRC2 complex isolated, except for the same core component as the 600 kD complex, contains Rpd3 and Pcl. The PHD domain in Pcl can directly bind to the histone de-acetylase Rpd3. These two complexes seem functionally distinct from each other: the 600 kD complex is present through the whole embryogenesis, while the 1 MD complex is there until the mid embryo stage (Furuyama et al., 2003; Tie et al., 2001; Tie et al., 2003).

The 3rd PRC2 complex, Pcl-PRC2, was purified from 16-18 hr embryo and contains Pcl and Heat-shock cognate protein 70 (HSC70) except for the 600 kD core components (Nekrasov et al., 2007). Pcl-PRC2 is required to generate high levels of H3-K27 trimethylation at Polycomb target genes but is not needed for the genome-wide H3-K27

51 mono- and dimethylation. Pcl mutant embryos show strong misexpression of Ubx and Abd-B, whereas en, another target gene is only misexpressed in a few rare cells (Nekrasov et al., 2007). However, another Pcl complex (about 1500 kD) which contains no E(z) was purified from third stage larvae. This larval Pcl complex (PCLC) functions upstream of E(z) and also bind to the Ubx PRE (Savla et al., 2008).

Pho-RC complex

The Pho-repressive complex (PhoRC) contains Pho (pleiohomeotic), Phol (pleiohomeotic-like) and dSfmbt (Scm-related gene containing four mbt domains). It is the only PcG complex that contains sequence-specific DNA binding proteins: Pho and Phol (Klymenko et al., 2006). Pho and Phol function redundantly to maintain Hox gene silencing (Brown et al., 2003). pho single mutant clones show slight Hox gene misexpression, and phol single mutant clones show no Hox gene misexpression. However, the double mutant clones of pho and phol show severe misexpression. The human homolog of Pho and Phol is Yin Yang 1 (YY1). YY1 is a multifunctional nuclear protein that can act as a transcriptional repressor or activator (Seto et al., 1991; Shi et al., 1991): for example, it can repress the activity of the adeno-associated viral P5 promoter in the absence of the oncoprotein E1A, but can also activate the promoter in the presence of E1A. It plays roles in cell proliferation, differentiation, survival and nervous system. Interestingly, YY1 regulated genes in the nervous system show contrasting behaviour: some genes are repressed by YY1: (i.e. Dynamin 1, Glast), while some are activated (i.e. Engrailed 2, Snail) (He and Casaccia-Bonnefil, 2008). Klymenko and co-workers purified the PhoRC complex of Drosophila (Nekrasov et al., 2007). They found that Pho actually exits in two complexes, one with dSfmbt (the Pho-RC complex which is associated with PcG silencing). The other complex, co-purified with INO80 (a Drosophila nucleosome-remodeling complex) is independent of Pho-RC function. While in mammals, Cai and co-workers studied the function of Pho-INO80 in HEK293 cells (Cai et al., 2007). They show that INO80 acts as an co-activator with YY1 to activate transcription. Incidentally, a study on even-skipped (eve) regulation in Drosophila shows

52 that Pho maintains both active and repressed transcriptional states through Eve PRE (Fujioka et al., 2008).

Figure 11. The biochemical activity of the PcG complex. Modified from (Schwartz and Pirrotta, 2008)

The other component in PhoRC complex is dSfmbt, which contains four copies of “malignant bran tumor” (MBT) and a “sterile a motif” (SAM) domain. As in other PcG lacking somatic clones, Ubx was de-repressed in dSfmbt mutant clones 72 hr after induction in disc. dSfmbt1 homozygous embryos from heterozygours mothers showed mild misexpression of Hox genes. However, dSfmbt1 homozygous embryos from mutant germ line cells fail to develop. The MBT domains contribute mainly to the binding of dSfmbt to mono- and di-methylated H3K9 and H4K20, which is in contrast to the well documented Pc preference in binding of tri-methylated H3K27.

The three main complexes have different contributions to epigenetic marks (Table 9) and are supposed to localize in the regulatory region of target gene (Fig. 11)

53 One may speculate that: dSfmbt “scans” PRE-flanking chromatin for nucleosomes that are only mono- or di-methylated at H3K9 and/or H4K20 and docks onto such nucleosomes through its MBT domain. Then the PRC2 bound to PREs interacts with Pho/Phol, methylates H3K27me3, which then facilitates PRC1 to bind to the target gene (Klymenko et al., 2006).

Table 9. PcG members contribute to the epigenetic marks. PcG H3K27me H3K9me H4K20me H2A119ub Me1 Me2 Me Me1 Me2 Me3 Me1 Me2 Me3 3 E(z) √ √ √ Su(z)12 √ √ √ ESC √ √ √ ESCL √ √ Pcl √ dSfmbt √ √ √ √ Pc √

In addition to the themes of PcG complex, there are some variation members that have looser relationships with the main complexes or belong to the complexes that have not yet been purified. For example: the Additional sex combs (Asx) gene of Drosophila is required for activation and repression of homeotic loci. It interacts specifically with Polycomb and super sex combs.

In Drosophila, E(z) is associate with Polycomb-like (PCL) protein since PCL shares polytene chromosome staining pattern with E(z). Indeed, a 1-MDa ESC-E(z) complex containing PCL and the histone deacetylase RPD3 during early embryogenesis. The different composition of the PRC2 complex may be involved in the silencing of different targets. PCL contains two plant homeodomain (PHD) domains, a motif present in many proteins involved in chromatin function, such as CHD3, Mi2, TRX, ASH1, and ASH2. It has recently been shown that the PHD domain link histone methylation to active chromatin remodeling. Multiple PCL homologs have been identified in mammals. For

54 example, there are three genes in the mouse genome that encode PCL homologs, namely mPcl1, mPcl2, and mPcl3 (Cao et al., 2008).

Nervous System Polycomb 1 (NSPc1), another Psc homolog in mammals, plays important roles in promoting H2A ubiquitination and cooperates with DNA methylation in Hox gene silencing (Wu et al., 2008). The Polycomb Group gene PCGF5 was identified as one of the genes most strongly downregulated upon Notch inhibition. Notch may be responsible for maintaining expression of a transcriptional repressor that suppresses p16 and p19ARF. Whether PCGF5 acts as an analogous to Bmi-1 in this cellular context and mediates p16/p19ARF repression is still under investigation.

MBLR (Mel18 and Bmi1-like RING finger protein) interacts with Ring1B and acts as a transcriptional repressor in transiently transfected cells. Serine 32 of MBLR is specifically phosphorylated during mitosis by CDK7 (Akasaka et al., 2002).

1.5.3 Mechanisms of PcG regulation of gene silencing

1.5.3.1 The Polycomb Response Element (PRE)

To understand the mechanism of how PcG complexes regulate gene expression, we have to first understand how PcG complexes are recruited to their target genes. In Drosophila, a functional analysis of the regulatory regions of several PcG target genes has led to the identification of some specific elements which are necessary and sufficient for PcG- mediated repression. All three PcG complexes bind to such regions that are in general several hundred base pair long. These sequences are called Polycomb response elements (PREs). So far, several studies have focused on PREs of a few genes in Drosophila: for example: the Abd-B PRE Fab7, the Ubx PRE, bxd and the engrailed PRE (Fig. 12).

55

Figure 12. Some PcG target gene PREs . Modified from (Ringrose and Paro, 2007).

Even though Pho/Phol are the only DNA binding proteins among the PcG complex components, they are not the only PRE binding proteins for PcG repression. Genetic and biochemical studies show that in Drosophila, PREs contain a cluster of GAGAG motifs, which are bound by the GAGA factor (GAF) and Pipsqueak (PSQ) (Horard et al., 2000; Huang et al., 2002). Further more, SP1/KLF (promoter-specific transcription factor / Krüppel-like factors) were found to bind to engrailed PRE (Brown et al., 2005); DSP1 (dorsal switch protein 1) is also involved in some PRE binding; the Zeste protein has long been known to be involved in both activation and silencing at PREs (Dejardin and Cavalli, 2004); and lastly, Grh (Grainyhead) and PSQ bind to similar sequences. These proteins are involved in homeotic gene regulation (Blastyak et al., 2006; Uv et al., 1994). Even though these proteins often bind at or near PREs, the number and distribution of the binding sites is quite different among different targets. None of these proteins so far has been shown to be present at all PREs. Since PREs contain so many protein binding motif sequences, it is not strange that, in general, PREs are not conserved sequences, but the motif sequences are conserved (Fig 12). Interestingly, some PRE binding proteins are

56 involved both in the regulation of activation and repression of gene activity: for example: Zeste, Pho/Phol, Dsp1, GAF/PSQ. The other two proteins, Grh and Sp1/KLF, are not expressed uniformly but show a tissue specific expression pattern (Ringrose and Paro, 2007). The recruitment of PcG to PRE is the first step for PcG silencing to work. Already a glimpse of what factors makes up PREs shows how complicated the system is. PREs have, however, been only found in Drosophila, mammalian PREs (if there are any) have not been identified.

1.5.3.2 PcG regulation of gene silencing at specific loci.

A stepwise model was proposed for PcG complex regulation of gene silencing (Fig, 13). The first step is the binding to DNA sequence (PRE) through DNA binding protein Pho(Phol); then PCL in PCL-PRC2 complex interacts with Pho/Phol, leading to recruitment of PRC2 to target genes and at the same time, E(z) establishes the silence mark by tri-methylation of H3K27me1/2/3, which, in turn, facilitates the binding of N- terminal Chromodomain of Pc and further recruits the PRC1 complex. The Ring-type zinc finger domain in dRing in the PRC1 complex contributes to H2AK119 monoubiquitination, thus further enhancing the silent state of the chromatin once the silent state has been established.

PRC2 and PhoRC may be abolished from a silenced site while PRC1 may still be present (Ohno et al., 2008). The staying of PRC1 at target gene loci while the H3K27me3 mark has gone means that PRC1 may have a binding facility other than H3K27me3. A possible explanation for PcG complex spreading along the chromosome and formation of a higher order chromatin structure is the SAM domain in Ph. Ph-SAM can form visible filaments and can enable PcG complexes to interact with weaker sites nearby (Kim et al., 2002).

57

Figure 13. Gene silencing induced by PcG binding to specific loci.

In this context, a question has to be considered: PRC2 complex has three variations that differ in size and components: the 600kD PRC2 complex, the 1 MDa PRC2 complex and the Pcl-PRC2 complex (Table 10). When considering the different representation of H3K27me2 and H3K27me3 in total nucleosomes (>50% and 5-10% respectively), Schwartz and Pirrotta (2008) proposed that the common core 600kD complex “probably carries out the much more general function of dimethylating H3K27” (Schwartz and Pirrotta, 2008).

Accordingly, PcG proteins inhibit transcription by changing the structure of the chromatin fiber: PhoRC and/or other PRE binding protein find the target, the PRC2

58 complex establishes (or writes) the silencing mark H3K27me3 and the PRC1 maintains (or reads) the silence state and decorates the chromatin with H2AK119ub (Fig 14).

Figure 14. PcG confers negative cis-regulation of target genes at the chromatin level.

1.5.3.3 PcG gene regulation at chromatin level

Genome wide analysis of PcG binding shows that PcG proteins binding to chromosomes is not always limited to PRE domains, but many of the proteins have a wider binding region, for example Pc, Ph and/or H3K27me3 (Negre et al., 2006; Schwartz et al., 2006; Tolhuis et al., 2006). The regions where Pc, Ph and/or H3K27me3 bind are called PcG domains (PcD). These domains may range from many kilobases to hundreds of kilobases up- and downstream of PREs. PcG proteins can not only regulate silencing of a gene in a cis configuration but can also mediate silencing of genes that are far away from PREs in the genome (a phenomenon called long-range gene silencing) (Fig 15). The regulation of BX-C (bithorax complex) by PcG proteins in Drosophila is one example. BX-C is a cluster of Hox genes (Ubx, Abd-A and Abd-B) which contain several PREs. Mcp is one of the PRE elements that controls Abdominal –B (Abd-B) expression and it is located 60 kb far away from its target (Busturia et al., 1997; Lewis, 1978).

59

Figure 15. PcG long range regulation of gene silencing in cis (A) and in trans (B).

Right now, there are two models to explain how these epigenetic marks extend around PREs and how the long-range gene silencing work. They are called the Chromatin walk and the Chromatin-probing models (Fig. 16) (Mateos-Langerak and Cavalli, 2008). Figure 16A, B show two possible walk mechanisms: in Figure 16A, nucleosome walk and PcG show the epigenetic marks. The Pc in a PRC1 complex first binds to the H3K27me3 from the neighboring nucleosome, bringing it to the proximity of the PRE, and then PRC2 tri-methylates H3K27me1 or H3K27me2. Another possibility is that Pho- RC binds to H3K9me1/2 or H4K20me1/2, forming hypomethylated

60

Figure 16. Models for PcG mediat long range silencing. Modified from (Mateos-Langerak and Cavalli, 2008) nucleosomes which facilitates de novo tri-methylation of H3K27. In figure 16B case, instead of nucleosome walk, the PcG complex walks along the chromatin. The de novo H3K27me3 serves as a platform for Pc binding, which then further recruits a PcG complex to methylate the neighboring nucleosomes, allowing the spreading of a silencing mark along the chromatin. Figure 16C shows the chromatin-probing model. Instead of

61 spreading along the chromatin, the PRE-bound PcG can methylate the nucleosomes that happen to interact with the enzymatic components of PcG.

1.5.3.4 PcG genes and the three-dimensional (3D) organization of the nucleus

The spatial organizations of chromatin (nuclear architecture) are important for the proper function of the cell (Cremer et al., 2004). In Drosophila, in the repressive state, PcG forms “PcG bodies” which are form by the PRES through long-range interactions. While in the active state, the active gene “move out” from the PRE-promoter interaction regions and become activated (Lanzuolo et al., 2007). In mammals, which PRE has not been defined, similar chromatin loops are also found. These chromatin loops are PcG-occupied and enriched of H3K27me3 (Tiwari et al., 2008b).

1.5.4 Evolution of PcG

PcG and TrxG proteins are often said to be evolutionary conserved. TrxG components are, indeed, found in fungi, plants and animals. Table 3 shows two features: Drosophila (an invertebrate) has in general single copies of the PcG proteins (except ph, esc and psc), but vertebrates have multiple paralogs of most PcG members. The other feature is the lack of PRC1 homologs in plants and C.elegans.

The components of the PRC2 complex are found in plants and animals, even though they are missing in S. cerevisiae and S. pombe, but another fungus, Neurospora crassa, possesses the H3K27me3 mark. Accordingly, PRC2 genes might have an ancient function in transcriptional repression.

The evolution of the PRC1 complex must be more complex: on one hand, there are no genes encoding core PRC1 proteins in fungi, plants (except for the Sce homolog) or C elegans (except for the Scm homolog), which may indicate that PRC1 genes have been added in the course of animal evolution. On the other hand, they might have been lost from plants and fungi.

During evolution some or all genes get duplicated and subsequently their coding sequences diverge by spontaneous mutations. For the PRC1 complex, Drosophila has one copy of Pc gene, but vertebrates have five Pc homologs with different domain

62 structure and biochemical properties (Whitcomb et al., 2007). For the other members in PRC1 there is also one gene copy of each in Drosophila, for example: psc and dRing, while vertebrates have 6 and 2 homologs respectively. The key domains of these homologs are highly conserved but regions outside the key domains diverge from their Drosophila counterparts and from their paralogs. For example, the highly conserved region of Pc is the chromodomain [chromobox (Cbx)] and Pc box with 75-88% amino acid identity between a vertebrate and Drosophila. The length and other motifs within each paralog differ, which may contribute to differential functions. In mammals, Cbx7 and Cbx8 can bypass replicative senescence due to repression of the INK4a-ARF locus. The binding of Cbx8 to INK4a-ARF locus depends on Bmi1 (Psc homolog), whereas Cbx7 bypasses senescence through a Bmi1-independent fashion (Bernard et al., 2005; Dietrich et al., 2007). The five Cbx proteins in mouse (Cbx2, 4, 6, 7 and 8) bind differentially to H3K27me3 and H3K9me3: Cbx7 and Cbx2 bind to H3K9me3 and H3K27me3, Cbx4 prefers H3K9me3, Cbx6 and Cbx8 do not bind to either modification.

With the exception of Cbx4 all localize to the Xi during female mouse embryonic stem cell differentiation and Cbx7 association with chromatin is developmentally regulated (Bernstein et al., 2006).

Psc homologs in vertebrates are another example of non-redundant paralogs. There are 6 homologs in a mammalian genome: Bmi1, Mel-18, NsPc1, PcGF3, PcGF5 and MBLR. Even though the encoded proteins have 63% amino acid identity, Mel-18 and Bmi1 mutant mice display similar but unique phenotypes. In addition, Mel-18 and Bmi1 have opposite effects on cell-cycle regulation: Bmi1 acts as an oncogene while Mel-18 behaves like a tumor suppressor (Guo et al., 2007a; Guo et al., 2007b). Both Bmi1 and Mel-18 can form a stable PRC1 complex, but only Bmi1 has been shown to positively regulate the H2AK119ub formation by Ring1B. A structural analysis shows that Bmi1 contains a potential H2A-binding surface, which is absent in Mel-18. Perhaps the different components in PRC1 contribute to its catalytic activity in vivo.

Mouse Ring1A and Ring1B are two homologs of dRing in mouse. They share 100% identity in the Ring domain with Drosophila dRing protein, 48% and 55% similarity to the full length of Drosophila dRing, respectively. Both Ring1A (-/-) and Ring1A (+/-)

63 mice show anterior transformations and other abnormalities of the axial skeleton (del Mar Lorente et al., 2000). Ring1B heterozygous mice show no skeletal phenotype but abnormal gastrulation (Voncken et al., 2003). At the molecular level, both Ring1A and Ring1B are needed for H2A ubiquitination during X-inactivation, showing an overlapping function of these two proteins. Ring1B is needed for H2A ubiquitination in embryonic stem cells. A genetic interaction between YY1 and Ring1A genes has been shown (Lorente et al., 2006).

Vertebrates have undergone a tetraploidization of the genome in the distant past and have, therefore, retained multiple copies of many genes. Furthermore, some of their PcG members have undergone multiple duplications, which have resulted in either related or antagonistic functions. Some PcG genes have probably been lost in the course of evolution, for example the plants may have lost PRC1 and the fungi and C. elegans have lost Su(z)12.

1.5.5 PcG and cell cycle

PcG dependent silencing is not only important for correct expression of homeotic genes during early embryogenesis, but also plays an important role in cell proliferation. PcG complexes regulate the self-renewal of various types of embryonic and adult stem cells of vertebrates. Many cancer types have abnormal PcG complex expression levels.

The cell cycle has been divided into four phases: mitotic (M) phase, synthesis (S) phase and two gap phases (G1 and G2), between the M phase to S phase and S phase to M phase, respectively (Fig. 17).

64

Figure 17. PcG regulation of cell cycle.

The progression through the cell cycle is highly regulated: at the transition from G1 phase to S phase and from G2 phase to M phase, there are two checkpoints: the G1checkpoint and the G2 one. In the middle of mitosis, there is one more checkpoint: the metaphase checkpoint. These checkpoints assess cell-intrinsic signals and are governed by two key protein families: cyclin-dependent protein kinases (Cdks) and cyclins. A total of 13 Cdks and many cyclins have thus far been identified in mammals. Cdk1 is supposed to govern the G2 checkpoint, while Cdk2 controls the G1 checkpoint. In addition, cell-extrinsic signals also regulate the processes from early G1 phase to late G1 phase. The transition is called restriction (R) point. The early G1 phase is mitogen-dependent; the late G1 phase is mitogen-independent. In the absence of mitogen stimulation, cells can enter a quiescent G0 phase which is characterized by small cell size and low metabolic activity. Transition from an early G1 phase to late G1 phase is regulated by Cdk4/6 and cyclin D through inactivation of Retinoblastoma tumor suppressor protein (RB) and activation of Cyclin E. Cdk 2 and cyclin E regulate the mitogen-independent late G1 phase.

65 Two cyclin-dependent kinase inhibitors (CDKI) regulate the transition through the G1 phase: Ink4a family and CIP/KIP family. Ink4a family members (p15INK4b and p16INK4a) directly inhibit Cdk4/6-cyclin D complexes; and members of CIP/KIP family (p21CIP. p27KIP1, p57KIP2) are direct inhibitors of the late G1 Cdk2-cyclin E complexes.

PcG members have been reported to regulate cell cycle in different ways. First, the indirect affects of cell cycle regulation via regular cell cycle regulators, for example, Hox genes. Several Hox genes are involved in the control of cell proliferation (Duboule, 1995; Morgan et al., 2007; Salser and Kenyon, 1996). Second, PcG members regulate chromatin condensation during mitosis. For example, Drosophila topoisomerase II and Barren proteins are required for proper chromatin condensation, and they are targeted by PcG complexes (Lupo et al., 2001). The centrosomal and chromosomal factor (ccf), another chromosome condensation regulator in Drosophila, colocalizes with Psc binding sites on polytene chromosomes (Kodjabachian et al., 1998). In addition, php, Pc and Psc mutant embryos show anaphase bridges during chromatid segregation and E(z) mutants show chromosome misalignment at metaphase (O'Dor et al., 2006). Finally, PcG complexes seem to directly regulate the cell cycle control genes cyclin A (Martinez et al., 2006), cyclin B (Oktaba et al., 2008) and cyclin G (Salvaing et al., 2008).

1.6 Epigenetic inheritance

I have mainly discussed the mechanisms of organization of chromatin by PcG for establishing and maintaining cellular identity. The next key question is how the epigenetic marks are transmitted to the daughter cells, and another, how the cells inherit the epigenetic marks to remember their specificity. This process is referred to as mitotic epigenetic inheritance. Many such phenomena have been studied in Drosophila. For example, in Drosophila embryos, undifferentiated cells responsible for the formation of adult structure can go through many cells divisions and still retain the ability to form the appropriate structure upon hormonal induction.

In many organisms (but not in Drosophila that has very weak DNA methylation or not at all), both DNA and histones can be modified. Both DNA and histone modifications are

66 thought to be involved in these epigenetic phenomena. I have discussed the mechanism for DNA methylation. Here, I will first focus on describing models on chromatin assembly pathways, the “piggy-back” and “bi-piggy-back” models (Martin and Zhang, 2007) (Fig 18), and then, from a PcG perspective, briefly describe the localization pattern of some PcG members during cell cycle (Fig 19).

The semiconservative model of histone replication states (Fig 18A) that during DNA replication, the nucleosome is split so that the old H3/H4 dimer go together with the old DNA strand and the new H3/H4 dimers are assembled into the daughter DNA strand (Tagami et al., 2004). The old histone modifications are recognized by histone modification binding proteins and these binding proteins further recruit histone modifying proteins, which then modify the new dimers. Positive support for this model comes from the study on genomic regions associated with gene activity (Ahmad and Henikoff, 2002). However, studies from the bulk chromatin indicate that most H3/H4 tetramers remain intact during DNA replication and the mono-nucleosomes isolated from the bulk chromatin are homogenous, containing either H3.1 or H3.3. A conservative model (Fig 18B) was proposed to explain this phenomenon: The old tetramers are intact and found on both daughter strand DNA, the nucleosomes assembled with newly synthesized tetramers. In semi-conservation model (Fig 18C), the histone binding modification proteins bound at the old nucleosomes, modifing the new adjacent nucleosomes. “Piggy-back” model (Fig 18D): The methylated copying DNA recruits CpG-methyl binding proteins, which then recruit histone modifying proteins to methylate histones. An example in support of this model is the recruitment of the SUV39H1 by methyl-CpG binding protein MeCP2 to the target genes, leading to H3K9 methylation and gene silencing (Fuks et al., 2003).

67

68

Figure 18. Chromatin replication models. (A) Semi-conservative model. (B) Conservative model. (C) DNA semi-conservative model. (D) “Piggy-back” model. (E) “Bi-piggy-back ”model. Modified from (Martin and Zhang, 2007).

“Bi-piggy-back” model (Fig 18E): As usual, there is always an exception for any model, in this case for the “piggy-back” model: EZH2 interacts with DNA methyltransferases and directs DNA methylation to target genes, and this function is necessary for gene silencing in mammalian cells (Vire et al., 2006). In this case, DNA methylation depends on the methylation of H3K27 by EZH2 and SUZ12 interacts directly with DNMT and then leads to gene silencing. Vire et al. (2006) did not, however, study the dependence of histone methylation on DNA methylation. So here I refer to this phenomenon as “bi- piggy-back” mechanism.

In Drosophila embryos, Pc, Ph and Psc dissociate from the chromatin before mitotic metaphase. Pc reassociates to chromatin after chromatin has decondensed at the next interphase, while Ph and Psc rebind to chromatin already at telophase (Fig. 19) (Buchenau et al., 1998; Dietzel et al., 1999).

69

Figure 19. The localization of PcG members during cell cycle.

1.7 Epigenetics and disease

The first evidence of a role for epigenetics in human disease came from the study of genomic imprinting (Reik, 1989). Now more and more evidence have shown that epigenetic mechanisms should also be considered in human diseases. Human epidemiological studies showed that prenatal and early postnatal environmental factors influence the adult risk of developing various diseases, such as cancer, chronic disease, diabetes, rheumatoid arthritis (Barker, 2004; Gluckman et al., 2007; Sanchez-Pernaute et al., 2008; Yajnik, 2004), and even schizophrenia (Malaspina et al., 2008; Quenstedt and Parshall, 1998; Reynolds, 2000; van Os and Selten, 1998). Here I will briefly describe the epigenetic alterations in cancers and the potential epigenetic targets for cancer therapy.

Cancer is the consequence of a combination of genetic disorder and aberrant epigenetic states. Genetic defects usually refer to DNA sequence deletion, insertion or point mutation. They are easy to visualize and the changes are usually permanently. Abnormal epigenetic alterations include changes of DNA methylation, improper histone modifications and the disruption of higher-level chromatin organization in nucleus (Feinberg, 2007; Jirtle and Skinner, 2007). Unlike genetic changes, epigenetic changes are reversible. Therefore epigenetic modifiers are attractive targets for epigenetic therapy.

70 2. AIM

The aims of this thesis were to investigate:

 the binding pattern of Su(z)12 protein to polytene chromosomes and compare to E(z) and H3K27me3.

 whether Su(z)12 binding to the white gene could explain the dominant effect of Su(z)12 mutants on z1 repression of white expression

 the effect of Su(z)12 on H3K27me3 methylation

 the expression pattern of Su(z)12 during Drosophila development

 the mechanism by which Su(z)12 regulates target gene silencing

 the role of Su(z)12 in cell cycle regulation.

71 3. RESULTS AND DISCUSSION

Suppressor of zeste 12 (Su(z)12) is needed for histone 3 lysine 27 tri-methylation (H3K27me3) in vivo (paper I)

This study investigates the in vivo mechanism of Su(z)12 in gene silencing. Su(z)12 mutant flies exhibit strong homeotic transformation caused by mis-expression of homeotic genes in embryos and larvae (Birve et al., 2001). Su(z)12 has also been suggested to be involved in position effect variegation (PEV) and in regulation of the white gene expression by interaction with zeste. To test the molecular mechanisms for these two phenomena, we stained salivary gland polytene chromosomes from wildtype, z1w+, z1w+UZ and z1wis larvae with Su(z)12 and heterochromatin protein 1 (HP1) antibodies. The eye pigments of the two latter phenotypes becomes completely suppressed in heterozygous Su(z)12 mutants. Surprisingly, we did not find a Su(z)12 binding site in the white locus from any of these larvae. A weak binding of HP1 at the white locus in polytene chromosome is found in the z1w+UZ larvae, indicating that the inserted p-element can recruit heterochromatic silencing proteins. We also stained polytene chromosomes of the same fly strains with a H3K27me3 antibody, but no binding site at the white locus was found. These data did not support the hypothesis that PcG and HP1 are involved in z1 dependent repression of the white gene in Drosophila.

Su(z)12 co-localizes well with E(z) on polytene chromosomes, but not always with H3K27me3. ESC protein has been shown to completely co-localize with E(z) (Tie et al., 1998), thus our data further support that the PRC2 complex subunits are present at the same sites on polytene chromosomes. We find that Su(z)12 binds mainly to interband regions and these sites do not always overlap with H3K27me3 nor with HP1. Our results confirm the chromatin immune-precipitation Chip analysis results reported by Schwartz and the co-worker (Schwartz et al., 2006).

In humans, SUZ12 is found to regulate H3K9me and HP1distribution (de la Cruz et al., 2007). Related to our observation that Su(z)12 mutants exhibit PEV, a characteristic which is unique among PcG members, we speculate that Su(z)12 may have some interaction with HP1. However, we did not observe co-staining of Su(z)12 and HP1 on

72 wildtype polytene chromosomes. So, from this point of view, mammals and Drosophila seem to have some differences in controlling gene silencing.

This study was first to show that Su(z)12 is needed for H3K27me3 in vivo. Intriguingly, over-expression of Su(z)12 increases H3K27me3 activity in wing discs. Since Su(z)12 does not have a methyltransferase activity of its own, we hypothesize that either Su(z)12 protein is rate limiting or that increased Su(z)12 levels induce positive feedback, activating other PRC2 components, which lead to high H3K27me3 levels.

When analyzing the polytene chromosome staining results, we also noticed that Su(z)12 exhibits a weak staining in the nucleolus region. The nucleolus is composed of densely packed fibrils and granules. It is the site of ribosomal RNA synthesis, which forms the major part of the ribosomes. Recent studies show that sumoylation is important for nucleolus organization (Heun, 2007), and that SUZ12 and EZH2 are sumoylated in vivo and in vitro (Riising et al., 2008). We could thus speculate that Su(z)12 may have other functions in addition to silencing.

Su(z)12 regulates the engrailed (en) gene both directly and indirectly (paper II)

The engrailed gene, is a target of PcG silencing, and has been well studied. In all those studies the main focus have been on the regulatory role of the engrailed PRE (Devido et al., 2008) and the PRC1 complex, i.e. Pc and Ph (Busturia and Morata, 1988; Randsholt et al., 2000). As expected, engrailed is up-regulated in the anterior regions in wing discs (where En is not expressed) in PRC1 somatic mutant clones due to the resulting de- repression, but loss of PRC1 function has no effect in the posterior region (where En usually is expressed).

In this study, we discovered a novel phenomenon. Instead of being up-regulated in the anterior region in Su(z)12 knock-out clones in wing discs, we found down-regulation of En in the posterior region. This could be interpreted as Su(z)12 acting as an engrailed activator. However, we found that this “activator” role was caused by an indirect regulatory loop. Loss of Su(z)12 leads to up-regulation of Ubx and Abd-A, which then

73 suppresses en expression. We also tested engrailed expression pattern in E(z) knock out clones in wing discs. The result was the same as with Su(z)12. In addition to engrailed, we also detected that other PcG target genes, like wingless and cut, behaved similarly in Su(z)12 knock out clones.

Recently, Oktaba and co-workers found a similar phenomenon (Oktaba et al., 2008). They tested the expression of en and other PcG target genes in different PcG gene knock- out clones, like in dsfmbt, ph, pho, scm, Su(z)12 PcG genes, and some double knock out clones (i.e Scm; BXC). They found that en and Dll were down-regulated in dSfmbt and Scm knock-out clones, respectively, but in the double knock-out of Scm and BXC clones, strong up-regulation of Dll was observed. This is in complete agreement with our results.

Our results emphasize that each gene has to be regarded as a unique entity. Their regulation can differ between developmental stages, tissues and between compartments within tissues.

The role of Su(z)12 in regulating cell proliferation and apoptosis (paper III)

The human homolog of Su(z)12 (SUZ12/JJAZ1/KIAA0160) was first reported to be up- regulated in endometrial stromal tumors. The two zinc finger protein encoding genes JAZF1 and JJAZ1 are located at the sites of the 7p15 and 17q21. Frequent fusion events between the JAZF1 and JJAZ1 genes was found in translocation (t(7;17)(p15;q21)) in such tumors. Since then, many reports have shown that PcG are involved in various tumors (Ballestar and Esteller, 2008). In this study, we investigate the localization pattern of Su(z)12 during the cell cycle and its role in cell proliferation.

Unlike the uniform localization pattern of Su(z)12 in S-phase cells, Su(z)12 has very weak staining in metaphase chromosomes in mitotic embryonic cells. Similar patterns have been reported for Ph, Psc and Pc in Drosophila (Buchenau et al., 1998; Dietzel et al., 1999; Paro and Zink, 1993). This indicates that PcG proteins are dynamically regulated during mitotic cell division in Drosophila.

74 In meiotic sperm cells the localization patterns are even more complicated: Staining Drosophila testes for Su(z)12 protein showed that there are three types of Su(z)12 localization patterns. In spermatocytes, Su(z)12 protein is localized in nuclear foci. At the rounded spermatid stage, Su(z)12 has a ubiquitous distribution, which is quite similar to the distribution pattern for SUZ12 at the ovulated oocyte stage in mouse (Solter et al., 2004). At the spermatid stage, Su(z)12 has totally left the needle shape sperm nuclei. One could speculate that the complicated dynamic localization of Su(z)12 during Drosophila spermatogenesis is essential to prevent the spermatids from undergoing differentiation. In other words, Su(z)12 may be essential for keeping the germ cell lineage to retain its pluripotency.

In this study, we also show that Su(z)12 are involved in regulation of cell proliferation and apoptosis. The most interesting discovery here is the different phenotypes obtained from over-expression of Su(z)12. When driven by ey-gal4 or vg-Gal4 drivers, no overgrowth was observed, while when Su(z)12 was driven by Hem-Gal4 , the hemocyte cell numbers increased significantly. We think that the reason for the different results between wing discs and hemocyte stem cells are probably due to that the different tissues have different regulatory networks.

Su(z)12 encodes different isoforms which are functionally different (paper IV)

We found that Su(z)12 encodes two isoforms and that they are developmentally and dynamically regulated. Only Su(z)12-B and the human homolog can rescue Su(z)12 mutants. The specific neuron expression pattern indicates that Su(z)12 may involve in regulation of neuron development.

75

4. CONCLUSIONS

 Su(z)12 has 90 binding sites in polytene chromosomes. These binding sites are overlaping well with E(z), but do not completely overlap with H3K27me3.

 Su(z)12 is essential for H3K27me3 in vivo.

 Su(z)12 regulate the engrailed gene both directly and indirectly via Ubx.

 Su(z)12 is dynamically regulated during cell division.

 Lack of Su(z)12 may decrease cell proliferation and increase apoptosis.

 There are two different isoforms of Su(z)12 which are developmentally and/or tissue specifically regulated.

76

ACKNOWLEDGEMENTS

For a long time before writing this thesis I have been thinking about what to write in this part. Many people have encouraged and helped me on my way. I do not think for a second that I ever could have finished this work without their support. I deeply appreciate it. It is, therefore, my self-evident obligation to try to express thanks first to some of you:

First and foremost, my deepest gratitude to my supervisor Åsa Rasmuson-Lestander, not only for your extensive knowledge of genetics, but also for your ability to encourage a truth-seeking approach to science, and for criticizing my manuscripts and this thesis. You deserve my deepest respect.

Jan Larsson, my co-supervisor, you are the kind of a person that people first ask for help when they have a problem (or at least I have always done so). You are reliable, helpful and wise. I am deeply indebted to you.

Anssi Saura for all your support! Without your support and encouragement I would not have done this job. I am awed by your wide knowledge and your kindness. I respect you as a great person.

David Robert for suggestions to this project.

Jurg Muller (EMBL, Heidelberg, Germany) for contributing with useful discussions.

I should also like to thank the folks from the Genetics of the old days:

Stefan Andersson Escher, my first genetics teacher in Sweden and a great party organizer. Kerstin Kristiansson for showing me how to use the pipette; Per Stenberg for the computer and sequence support; Anna Birve, for your friendship and support. Bettan, for showing how to fish in Umeå; Astrid for our good friendship and the annual Christmas cards! Karin E, for your kindness, Gunilla and Agneta, for always being patient with my “late order” fly food; and other “Genetic colleagues”: Jesper, Magnus, Markus, Tor-Mikael, Anna W, Maggan and Marianne, for creating a friendly environment in my first few years of studying in Sweden.

77 I should also like to thank all the colleagues in the Department of Molecular Biology, in particular the PhD students in Åsa´s group: Linn and Anna L, you have a warm heart, you are kind, honest and considerate, I have been very happy and lucky to work together with you; Erik, for nice JC articles and discussion; John, impressed by your peaceful and efficient work. Thank you for showing me the beautiful tradition of a Swedish wedding party; and Anna S, I thank you all for making a friendly working atmosphere. I wish you all good luck.

My room mates: David, for your understanding and help, people should be happy for having a friend which can trusted, and even better if he or she is helpful. Regina, you have been like a sister, you always want to help people, sometimes you are simply too nice. I am so glad to have friends like you.

Jonas and Lisa for the discussion of cell cycle, Linus and Chaz, Sara, Andreas and Sanna, for your friendship.

Victoria Shingler and Martin Gullberg, for your kindness, Roland Rosqvist, for help in microscopy, Roland, Matt, Bernt Erik, Sun Nyunt, Iwa, Gunnar, Sven, Mikael, Christina G, Eleonora for your support.

Edmund, for the friendly discussions and showing me the RNA stuff.

Berit, Ethel, Maria W, and in particular Helena Östbye for always help me with many administration things and friendly smile.

I should like to thank the present or past “Fly people”:

Ruth Palmer, I am impressed by your intelligent and positive attitude; Camilla Sjögren, for your kindness; Dan Hultmark, for giving us the driver strains; and all the colleagues in the fly floor: Christina, Caroline, Therese, Margret, Fredrik, Jana, Gaura, Anna- Mia, Lina, Malin, Calle, Ines, Michael, Anna-Karin, Karin B, Peter, Ingrid, Martin, Jens-Ola, Mazen, Torbjörn, Shenhua, Lixiao. I wish you all the best.

For those people outside the work, I should also like to thank:

78 My dear friends Eleonora and Caroline, I am glad to have friends like you who can share the happiness and also sadness. Take care!

My Chinese friends: Aihong Li, Good luck with everything. Tong Li, for always the tea support from Beijing. Lu Li, for our discussion from the western blot to the opinion of marriage. Kui Liu and Yan Shen, Yunshen Zhao and Yafan Mei, Yu Chen and Ou Tang, Yu Jun and Liming Gu, Xiaoru Wang, Yan Xin and Xiaohong Lu, Zhiyun Zhen and Chunglian Shi, Wei Jian and Weigang Gu, Jingxia Liu and Jiguo Yu, Junfa Liu, Bo Xu and Shaojun Xiong, Ming Yuan and Jian Lu, Bo Huang, Changchun Chen, Tianyan Song, Nu Huang (黄女), for all your friendship!

My lovely neighbors: Susan och Fredrik, Terese och John, Bigitta, Christina och Håkan, Susan och Åke, Lotta och Jonas. Vilken tur har vi som bor granne med er. Speciellt tack till Siv och Sigg, man säger att barn är en produkt av Gud, men att gamla människor är en produkt av sig själva. Vi känner oss alltid välkomna hos er. Det värmer mitt hjärta när jag ser hur ni behandlar varandra och andra. Tusen tack!

Finally, I would like to thank my family: Mama, for supporting and encouraging me all my life, I love you, mom! 妈妈，我爱你! Brother, Weiming, for love! I wish you the best!

I thank my husband, Wenchao, for all these years of support in general, the computer support in particular. I am not sure if I shall ever graduate from your computer course. Bobby and Oliver, my lovely sons, you are the sun in my life. You give me the strength to meet each day as it comes; I love you, my sons! Thank you for taking care of your mom!

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