Polycomb Group (PcG) proteins are responsible for post-transcriptional modifications in histone tails leading to chromatin condensation and changes in gene expression. In Arabidopsis thaliana, curly leaf (CLF) is a member of the Polycomb Reporssive Complex 2 (PRC2), which cnfers a repressive epigenetic mark, namely trimethylation of histone H3 at lysine 27 (H3K27me3). In the clf mutant, the expression of the floral organ identity gene AGAMOUS (AG) is derepressed in vegetative stages and coincides with loss of H3K27me3 at the AG locus. Recent whole genome prfiling studies have suggested that PcG genes regulate mang more developmental regulators than AG (about 15% of Arabidopis genes). However, it remains unclear what the relevance of PcG regulation of these targets is for plant development; in addition, it is not known how changes in J3K27me3 casue gene repression in plants. To unravel the role of CLFcin A. thaliana, a T-DNA mutagenesis in the clf background was performed to identify mutations enhancing or suppressing the Clf- phenotype, as these may identify additional PcG genes and targets. Firstly, I screened an A. thaliana T-DNA mutagenized population and identified four mutations suppressing the Clf- phenotype: suppressor of polycomb 1 to 4 (sop1, sop2, sop3 and sop4). Secondly, I characterized these four mutants. The sop1 mutant had normal flowering time and the suppressed phenotype is due to a loss of function mutation in SEPALLATA3 (SEP3). I establied the SEP3 is an activator and a co-factor of AG. Also, I found that SEP3 is stronlgy mis-expressed in clf mutants and SEP3 chromatin is enriched the H3K27me3, which stronly suggests that SEP3 is a direct target of CLF. In addition, I showed that a mutation in Flowering Locus T (FT), which is a positive regulator of SEP3, suppreesed the Clf- phenotype suggesting the FT is also a target of CLF. Suppressors sop3, sop3 and sop4 are late flowering, unlike sop1, and show increased expression of Flowering Locus C (FLC), a MADS-box transcription factor gene that represses flowering. I found that the sop4 mutation in likely casued by disruption of FPA, a predicted RNA binding protein that promotes flowering time by repressing FLC. Consistent with this, sop4 mutants show hight levels of FLC. Unexpectedly, fpa clf (sop4) mutatns are much later flowering than clf FRI mutants, which have similarly high levels of FLC. This suggests that FPA may regulate other genes controlling flowering thant FLC. The genes involved in sop2 and sop3 mutants remain to be identified. In this thesis I brought genetic and molecular evidence showing that CLF, though the PRC2, control floral induction (FLC), floral integration (FT) and floral organ formation (SEP3 and AG) in A. thaliana.

The Polycomb group (Pc-G) and trithorax group (trx-G) genes play crucial roles in development by regulating expression of homeotic and other genes that control cell fate. Both groups catalyse modifications in chromatin, including histone methylation, leading to epigenetic changes in gene activity. The trx-G antagonises the function of Pc-G genes by activating Pc-G target genes, and consequently trx-G mutants suppress Pc-G mutants. The trx-G genes are relatively poorly characterised in plants. We identified a novel trx-G candidate SUPRESSOR OF POLYCOMB 12 (SOP12) by a genetic screen for suppressors of mutants for the Arabidopsis Pc-G gene CURLY LEAF (CLF). Thus sop12 mutations have no discernible phenotype in wild type backgrounds but partially suppress the leaf curling and early flowering phenotypes of clf mutants. Molecular cloning shows that SOP12 encodes a Harbinger transposase nuclease-like protein which is conserved in green plants, although key residues required for the catalytic activity of the nuclease domain are not conserved. In sop12 clf double mutants, many CLF target genes are down-regulated relative to clf mutant, which suggests SOP12 is a general activator of Pc-G target genes instead of a target of CLF or a late flowering suppressor. The CLF gene encodes an H3K27me3 histone methyltransferase, however chromatin immunoprecipitation (ChIP) analysis indicates that SOP12 does not antagonise Pc-G by removing H3K27me3 methylation, which is consistent with the fact that sop12 suppresses mutants for another Pc-G gene, LIKE HETEROCHROMATIN PROTEIN 1 (LHP1), which is not involved in H3K27me3 deposition. . Rather, genetic analysis shows that sop12 enhances the phenotype of mutants of EARLY FLOWERING IN SHORT DAYS (EFS), a trx-G gene involved in deposition of H3K36me3, and of ULTRAPETALA 1 (ULT1), a plant specific trx-G gene. The enhancement indicates SOP12 may act together with ULT or EFS proteins, or at least regulate the same targets in synergistic ways. For example, SOP12 activates AP3 expression, a role which overlaps with EFS. Yeast two hybrid screening and imunoprecipitation followed by Mass spectrometry were performed to identify numerous potential SOP12 interacting proteins but await further validation. One protein (SUP1) identified through yeast two hybrid screens was independently identified by another group as a Pc-G suppressor, suggesting that SOP12 and SUP1 may act in a common complex to regulate Pc-G targets. Collectively, my data suggests that SOP12 represents a domestic transposase that has acquired a role as a novel, plant specific trx-G members.

Although all cells in a multicellular organism contain the same set of genes, the spatiotemporal expression of these genes needs to be dynamically regulated for morphogenesis and life cycle transitions to take place. Polycomb-group (PcG) proteins are evolutionarily-conserved epigenetic regulators that function – via epigenetic marks such as H3K27me3 and modifications to chromatin structure – to maintain the repression of developmentally-important genes so that these genes are only expressed in the appropriate cells at the appropriate times. This repressive activity of the PcG is antagonised by the trithorax-group (trxG) of proteins. Although they maintain specific patterns of gene repression, PcG proteins are ubiquitously expressed. How their activity is regulated is largely unknown. To identify such regulatory pathways, a genetic screen for modifiers of PcG activity in Arabidopsis was carried out previously using the PcG mutant curly leaf (clf), which has moderately-severe developmental defects due to the ectopic or untimely expression of developmental regulators such as floral homeotic genes and the important flowering time regulator FLOWERING LOCUS T (FT). I characterised three novel potential regulators identified in this genetic screen: the chromatin-associated protein AT-HOOK MOTIF NUCLEAR LOCALISED PROTEIN 22 (AHL22), the 26S proteasome and the novel trithorax-group members ANTAGONIST OF LHP1 1 and 2 (ALP1 and ALP2). I found that the dominant sop-11D mutation is caused by over expression of AHL22 which suppresses the phenotype of clf by reducing FT expression. However, genetic analysis suggests that AHL22 may act in a parallel pathway to the PcG. I showed that mutations affecting diverse subunits of the 26S proteasome reduce the mis-expression of CLF targets and suppress the phenotypes of clf although they do not restore H3K27me3 levels at these targets. Pharmacological inhibition of the proteasome also alleviated the mis-expression of target genes found in clf mutants. Analysis of PcG protein levels following proteasome inhibition suggests that the 26S proteasome antagonises the PcG by degrading the key PcG member EMBRYONIC FLOWER 1 (EMF1), which is likely to be important for implementing target gene repression. Surprisingly, my proteomic analysis showed that the novel trxG members ALP1 and ALP2 are accessory components of a core PcG complex – the Polycomb Repressive Complex 2 (PRC2) – in vivo, suggesting that that ALP1 and ALP2 may antagonise PcG repression by preventing the association of core PRC2 components with accessory components EMF1, LIKE HETEROCHROMATIN PROTEIN 1 and the PHD finger proteins VERNALISATION5 and VIN3-LIKE 1. My results reveal a previously unknown role for 26S proteasomal degradation in the regulation of PcG activity during vegetative development and identify novel in vivo associators of the core PRC2 and point to their role in modulating PcG activity. These results thereby increase our understanding of how the PcG is regulated and serve as a starting point to discover how specificity is given to the PcG mediated repression, either by targeted degradation of EMF1 by various E3 ligases or by different combinations of PRC2 associators.

Inheritance of gene expression patterns through multiple rounds of cell division is crucial for the normal development of multi-cellular organisms and is mediated by epigenetic mechanisms. Many epigenetic mechanisms are believed to involve heritable changes to chromatin structure. This includes maintenance of transcriptional repression by Polycomb Group (PcG) proteins. It is unknown how PcG-dependent repression is maintained during or re-established after mitosis, a process that involves many physical and biochemical changes to chromatin. Understanding the behavior of PcG proteins during mitosis is key to answering this question: if PcG proteins remain bound in mitosis they may constitute the memory themselves, or else transcriptional memory must reside elsewhere, such as in the altered chromatin structures induced by PcG proteins. PcG protein association with chromosomes in mitosis in Drosophila S2 cells was examined by immunofluorescence and cellular fractionation. PcG proteins are associated with mitotic chromosomes, which is consistent with a role in carrying information about transcriptional repression through mitosis. Localization of PcG proteins to specific sites in the genome was assessed by chromatin immunoprecipitation (ChIP) followed by genome-wide sequencing (ChIP- SEQ) on mitotic cells. A method for isolating pure populations of mitotic cells was developed to access PcG protein localization in mitosis unambiguously. PcG proteins were not detected at well-characterized PcG targets including Hox genes on mitotic chromosomes, but a covalent modification of histone H3 associated with PcG- dependent repression, trimethylation of lysine 27 (H3K27me3), is retained at these sites. Two PcG proteins Posterior Sex Combs (PSC) and Polyhomeotic (PH) remain at about 10% of their interphase binding sites in mitosis. PSC and PH are preferentially retained in mitosis at sites that overlap recently described borders of chromatin domains (1), including sites that overlap domain borders flanking Hox gene clusters. These persistent binding sites may serve to nucleate re-establishment of PcG binding at target genes upon mitotic exit, perhaps with assistance of H3K27me3. Thus PcG proteins may form part of the transcriptional memory carried through mitosis, but perhaps not by persistent association at the targets of repression. Retention of elements at chromatin boundaries in mitosis may serve as a general mechanism for epigenetic memory.

Neurons in the central nervous system are typically not replaced and must therefore maintain their choice of fate and their synaptic connections throughout the life of an organism. I have used Drosophila genetics to analyze genes that prevent neurons from switching fates and allow them to form synapses onto target neurons. The Drosophila fly eye is composed of approximately 750 ommatidia, each comprising eight photoreceptor neurons (R1-R8) surrounded by non-neuronal accessory cells. These photoreceptor neurons undergo a well-defined developmental specification process and form synapses at defined locations in the brain. I have taken advantage of this system to investigate two questions: 1) how do neurons maintain their fate after specification? and 2) how do neurons form stable synapses? For the first half of my dissertation, I have focused my research on a gene, Sce, that I have shown is essential to prevent R7 neurons from undergoing a late switch in cell fate. Sce is an integral component of the Polycomb Group (PcG) complex that is essential for maintaining repression of multiple genes throughout the genome. I found that PcGs are required to prevent R7s from derepression of the R8-specific transcription factor Senseless. For the second half of my dissertation, I focused on the gene syd-1 that was identified to be required for proper presynaptic formation of R7 neurons. Previous studies in Caenorhabditis elegans suggested that Syd-1 acts upstream of Liprin-α and that Liprin-α promotes presynaptic development by binding the kinesin Kif1a to promote axon transport. I used live image analysis to show that, unlike Liprin-α, Syd-1 is not necessary to promote axon transport. Instead, we show that in R7s, Syd-1 acts upstream of Trio, and our results suggest that Syd-1's function is to promote Trio activity. This dissertation includes both my previously published and co-authored materials. / 10000-01-01

Polycomb-group (Pc-G) proteins regulate homeotic gene silencing associated with the repressive covalent histone modification, trimethylation of histone H3 lysine 27 (H3K27me3). Pc-G mediated silencing is believed to remodel chromatin, rendering target genes inaccessible to transcription factors. Pc-G mediated silencing might result in irreversible changes in chromatin structure, however, there has been little analysis addressing whether Pc-G mediated silencing is reversible. In this work we focused on CURLY LEAF (CLF), the first Pc-G homologue discovered in Arabidopsis. CLF mediated repression of the floral homeotic gene AGAMOUS (AG) was challenged during early and late leaf development. AG was activated by the late leaf promoter, revealing that Pc-G mediated silencing can be overcome in old leaves in the presence of CLF. AG was also activated in young leaf primordia, yet did not persist in older leaves, revealing that transient activation of a Pc-G target is not epigenetically stable. To address the mechanism of Pc-G action within an endogenous environment, the histone dynamics at the APETALA1 (AP1) locus were characterized by Chromatin Immunoprecipitation. Unexpectedly, we found that the activation of AP1 in leaves did not require the removal of H3K27me3, questioning whether H3K27me3 is sufficient to silence. The roles of CLF in leaf and flower development are masked due to partial redundancy with SWINGER (SWN). clf- swn- mutants form a callus-like mass on sterile-tissue culture with no distinguishable plant organs. The role of CLF in regulating flowering time in natural populations of A. thaliana was investigated by complementing clf- mutants with CLF alleles from two accessions. We found that natural variation in CLF did not affect flowering time. To dissect the roles of CLF and SWN in late leaf and flower development, two approaches were developed for targeted expression. Firstly, CLF was introduced into the LhG4/ pOp transactivation system to provide CLF during early plant development. For mosaic analysis, CLF was introduced into the CRE lox recombination system in order to create clf- sectors surrounded by CLF+ SWN+ and CLF+ swn- cells.

Our lab has recently found that E2F3, an essential cell cycle regulator, regulates the self-renewal capacity of neural precursor cells (NPCs) in the developing mouse brain. Chromatin immunoprecipitation (ChIP) and immunoblotting techniques revealed several E2F3 target genes, including the polycomb group (PcG) protein, EZH2. Further ChIP and immunoblotting techniques identified the neural stem cell self-renewal regulators p16INK4a and Sox2 as shared gene targets of E2F3 and PcG proteins, indicating that E2F3 and PcG proteins may co-regulate these target genes. E2f3-/- NPCs demonstrated dysregulated expression of EZH2, p16INK4a, and SOX2 and decreased enrichment of PcG proteins at target genes. Restoring EZH2 expression to E2f3+/+ levels restores p16INK4a and SOX2 expression levels to near E2f3+/+ levels, and also partially rescues NPC self-renewal capacity toward E2f3+/+ levels. Taken together, these results suggest that E2F3 controls NPC self-renewal by modulating expression of p16INK4a and SOX2 via regulation of PcG expression, and potentially PcG recruitment.

Our lab has recently found that E2F3, an essential cell cycle regulator, regulates the self-renewal capacity of neural precursor cells (NPCs) in the developing mouse brain. Chromatin immunoprecipitation (ChIP) and immunoblotting techniques revealed several E2F3 target genes, including the polycomb group (PcG) protein, EZH2. Further ChIP and immunoblotting techniques identified the neural stem cell self-renewal regulators p16INK4a and Sox2 as shared gene targets of E2F3 and PcG proteins, indicating that E2F3 and PcG proteins may co-regulate these target genes. E2f3-/- NPCs demonstrated dysregulated expression of EZH2, p16INK4a, and SOX2 and decreased enrichment of PcG proteins at target genes. Restoring EZH2 expression to E2f3+/+ levels restores p16INK4a and SOX2 expression levels to near E2f3+/+ levels, and also partially rescues NPC self-renewal capacity toward E2f3+/+ levels. Taken together, these results suggest that E2F3 controls NPC self-renewal by modulating expression of p16INK4a and SOX2 via regulation of PcG expression, and potentially PcG recruitment.

Our lab has recently found that E2F3, an essential cell cycle regulator, regulates the self-renewal capacity of neural precursor cells (NPCs) in the developing mouse brain. Chromatin immunoprecipitation (ChIP) and immunoblotting techniques revealed several E2F3 target genes, including the polycomb group (PcG) protein, EZH2. Further ChIP and immunoblotting techniques identified the neural stem cell self-renewal regulators p16INK4a and Sox2 as shared gene targets of E2F3 and PcG proteins, indicating that E2F3 and PcG proteins may co-regulate these target genes. E2f3-/- NPCs demonstrated dysregulated expression of EZH2, p16INK4a, and SOX2 and decreased enrichment of PcG proteins at target genes. Restoring EZH2 expression to E2f3+/+ levels restores p16INK4a and SOX2 expression levels to near E2f3+/+ levels, and also partially rescues NPC self-renewal capacity toward E2f3+/+ levels. Taken together, these results suggest that E2F3 controls NPC self-renewal by modulating expression of p16INK4a and SOX2 via regulation of PcG expression, and potentially PcG recruitment.

<p>In multicellular organisms all cells in one individual have an identical genotype, and yet their bodies consist of many and very different tissues and thus many different cell types. Somehow there must be a difference in how genes are interpreted. So, there must be signals that tell the genes when and where to be active and inactive, respectively. In some instances a specific an expression pattern (active or inactive) is epigenetic; it is established and maintained throughout multiple rounds of cell divisions. In the developing <i>Drosophila</i> embryo, the proper expression pattern of e.g. the homeotic genes <i>Abd-B</i> and <i>Ubx</i> is to be kept active in the posterior part and silenced in the anterior. Properly silenced homeotic genes are crucial for the correct segmentation pattern of the fly and the Polycomb group (Pc-G) proteins are vital for maintaining this type of stable repression.</p><p>As part of this thesis, <i>Suppressor of zeste 12 (Su(z)12)</i> is characterized as a <i>Drosophila</i> Pc-G gene. Mutations in the gene cause widespread misexpression of several homeotic genes in embryos and larvae. Results show that the silencing of the homeotic genes <i>Abd-B</i> and <i>Ubx</i>, probably is mediated via physical binding of SU(Z)12 to Polycomb Response Elements in the BX-C. <i>Su(z)12</i> mutations are strong suppressors of position-effect-variegation and the SU(Z)12 protein binds weakly to the heterochromatic centromeric region. These results indicate that SU(Z)12 has a function in heterochromatin-mediated repression, which is an unusual feature for a Pc-G protein. The structure of the <i>Su(z)12</i> gene was determined and the deduced protein contains a C2-H2 zinc finger domain, several nuclear localization signals, and a region, the VEFS box, with high homology to mammalian and plant homologues. <i>Su(z)12 </i>was originally isolated in a screen for modifiers of the zeste-white interaction and I present results that suggests that this effect is mediated through an interaction between <i>Su(z)12 </i>and <i>zeste</i>. I also show that <i>Su(z)12</i> interact genetically with other Pc-G mutants and that the SU(Z)12 protein binds more than 100 euchromatic bands on polytene chromosomes. I also present results showing that SU(Z)12 is a subunit of two different E(Z)/ESC embryonic silencing complexes, one 1MDa and one 600 kDa complex, where the larger complex also contains PCL and RPD3. </p><p>In conclusion, results presented in this thesis show that the recently identified Pc-G gene, <i>Su(z)12</i>, is of vital importance for correct maintenance of silencing of the developmentally important homeotic genes.</p>