Dissertations / Theses on the topic 'Chromatin-remodelling'

Plants selectively deplete incident light of red (R) wavelength, relative to far-red (FR) wavelength light. Consequently, the relative proportions of R and FR (R:FR ratio) act as an indicator of surrounding vegetation and plants, via the phytochrome photoreceptors, are capable of detecting this. Low R:FR ratio is interpreted as surrounding competition and results in plants eliciting, what is known as, the shade avoidance response. This involves a host of both phenotypic and molecular changes, including increased hypocotyl growth, earlier flowering time and changes in gene expression. The fundamental mechanisms underlying R:FR-mediated changes in gene expression are not fully characterised. Given that increasing evidence in Arabidopsis suggests that the structure of chromatin is integral in the regulation of gene expression in response to environmental stimuli, it was investigated to see if this was the case in R:FR ratio signalling. Here, DNaseI sensitivity assays demonstrate that the shade avoidance genes ATHB2, PIL1 and XTH15 all undergo gross changes in chromatin structure in plants grown in light/dark cycles and treated with low R:FR ratio. These low R:FR induced changes in DNaseI sensitivity are conspicuously absent when plants are grown in continuous light, suggesting an involvement of the circadian clock. Complementary to this, the use of ChIP has identified the coding region of PIL1 to show increased association with hyper-acetylated H3K9 and possibly H3K14 in response to low R:FR ratio. Together, this work demonstrates that changes in R:FR ratio induce changes in gene expression that are correlated with changes in chromatin structure and histone modifications and that these changes could be regulated by the circadian clock. In addition, the identification and construction of multiple mutant and transgenic lines expressing altered levels of chromatin modifying protein was attempted.

Plants - including commercially important crops - are exposed to numerous pathogens often resulting in significant loss of yield. Understanding the underlying mechanisms of pathogen recognition and defence strategies is key in successfully ensuring food security. Research on plant-pathogen interactions has mainly focused on the gene networks after pathogen perception as well the identification of resistance genes. Latest research suggests that chromatin remodelling, including nucleosome displacement and DNA or histone-modifying enzymes are important in plant immunity. This thesis focuses on chromatin remodelling as the mechanism by which plants mount an effective immune response. The thesis also investigates the role of histone acetylation as one of several chromatin remodelling mechanisms. Histone acetyltransferases (HATs) and histone deacetylases (HDACs) are two classes of histone modifying enzymes that antagonistically govern the acetylation levels of histones in gene promoters and gene bodies ultimately affecting gene expression. HAG1 was identified as an important positive regulator of plant immunity in the interaction with Pst DC3000. A proteomic approach allowed the identification of TOPLESS family members as HAG1 interactors. Considering that chromatin remodelling is an important aspect of plant immunity, it was hypothesised that pathogens have evolved mechanisms to interfere with such processes. To this end, this thesis will present a comprehensive approach towards identifying Pst DC3000 Type-III effectors with the ability to interfere with chromatin remodelling. HopO1-1 was initially identified as an effector with chromatin binding properties, however, further experiments pointed more strongly towards this effector’s involvement in processes such as translation and photosynthesis. Overall, this thesis contributes towards a better understanding of the roles of histone acetylation and HAG1 histone acetyltransferase in plant immunity and sheds light into which Pst DC3000 effectors could be potentially involved in chromatin remodelling processes.

Drosophila nucleosome remodelling factor (NURF) is one of the founding members of the ISWI family of ATP-dependent chromatin remodelling enzymes and mediates energy-dependent nucleosome sliding leading to transcription regulation. In previous work (Wysocka et al., 2006), NURF was shown to be recruited to gene targets by binding specific histone modifications. The largest subunit of NURF, NURF301, contains a bromodomain and three PHD finger domains that have the ability to recognize specific histone modifications. Here we determine the histone binding-specificities of these domains, and how NURF histone binding is influenced by histone modification "cross-talk". This has been analyzed by histone peptide library array assays and our study shows that the PHD2 domain specifically recognizes the histone H3K4me3 mark. This binding can be inhibited by phosphorylation of H3 Thr 3, while enhanced by acetylation of H3 Lys 9 and phosphorylation of Ser 10. The binding specificities of bromodomain, PHD and PHD1 domains were also determined. These data were confirmed by peptide pull-down, Biacore and immunofluorescence microscopy assays. Moreover, two different NURF301-A/B and NURF301-C isoforms were CTAP-tagged by recombineering, and we used chromatin immunoprecipitation coupled sequencing (ChIP-Seq) to profile the genome-wide distribution of NURF in vivo. Therefore, our results identify regulatory mechanisms of histone modifications directing recruitment of ATP-dependent chromatin remodelling enzymes.

The thesis describes the analysis of the function of the small ubiquitin-like modifier (SUMO) in Drosophila melanogaster, and a potential effector of such modification, the chromatin remodelling factor dATRX. A substrate of SUMO modification, the transcriptional repressor Tramtrack 69 was verified, and the site of SUMO modification narrowed to a single lysine residue. Mutation of this prevented SUMOylation, and the functional consequence of this was analysed in a variety of assays, including transcriptional repression, subcellular localisation, degradation and overexpression in fly tissues. The ETS-domain transcription factor Pointed was also identified as a potential SUMO substrate and the functional consequences of such a modification analysed in a similar manner. The role of SUMOylation in the intact fly was analysed through clonal loss of function and overexpression phenotypes in both adult and larval fly tissues, implicating it in wing and eye development and melanotic tumour formation. The similarities to the phenotypes of mutation in dISW1, another chromatin remodelling factor are discussed. SUMO was shown to be a strong suppressor of position effect variegation (PEV) and potential mechanisms for its action are discussed. The mechanism by which SUMOylation acts is addressed through analysis of SUMO interacting proteins using a yeast two-hybrid screen. This identified the chromatin remodelling complexes NuRD and dATRX and the histone methyltransferase pR-Set7 as potential effectors of SUMO modification, which were verified by GST-pulldown assay. They were shown to interact with wild type SUMO but not a mutant form that is unable to repress transcription, suggesting a possible role in transcriptional repression by SUMO.

RSC is a member of the multi-subunit SWI/SNF family of ATPase-dependent chromatin remodellers and it is implicated in transcriptional regulation and DNA repair in Saccharomyces cerevisiae. The central ATPase subunit, Sth1, translocates nucleosomes in vitro and mutations in human RSC sub-unit orthologues are implicated in human disease. RSC is found in two isoforms, defined by the presence of either the Rsc1 or Rsc2 subunits, and these appear to confer distinct remodelling functions in different genomic contexts. At the MAT locus, Rsc1 and Rsc2 appear to mediate different forms of nucleosome positioning which are required for efficient mating type switching. Elsewhere in the genome, it has been suggested that RSC can create partially un-wrapped nucleosomes in order to facilitate transcription factor binding. This thesis uses indirect-end-label analysis and chromatin-sequencing technologies to dissect the chromatin remodelling functions of RSC and to determine the roles of Rsc1, Rsc2 and their subdomains. The work presented here suggests that four chromatin-remodelling outcomes arise from RSC activity. Firstly, RSC alters the positions of a tract of nucleosomes abutting HO endonuclease-induced double-strand DNA breaks both at MAT and non-MAT loci in a Rsc1-dependent manner. This activity can be transferred from Rsc1 to Rsc2 by swapping BAH domains. Secondly, RSC can aggregate nucleosomes into a large nuclease-resistant structure, termed an alphasome, in a Rsc2- and Rsc7-dependent manner. Thirdly, RSC positions nucleosomes at tRNA genes in a manner that requires both Rsc1 and Rsc2. Finally, chromatin particles consistent with previously described un-wound nucleosomes are confirmed to be present in specific promoter regions. Although Rsc1- and Rsc2- dependent subsets of these promoters could be identified, and associations with binding motifs for particular transcriptions factors were discovered, it was ultimately not possible to unambiguously define why some gene promoters depend on one RSC sub-unit rather than the other.

Compaction of chromosomes in the eukaryotic cell is due to interactions between DNA and proteins and interactions between proteins. These two types of interaction form a dynamic structure, known as "chromatin". The condensation of chromatin must be carefully regulated, since the structure is an obstacle for factors that need access to the DNA. An extensive range of components, one group of which is the ATP-dependent chromatin remodel-ling complexes, controls the accessibility of DNA. These complexes have been studied in a variety of eukaryotic systems, and their functions in major events in the cell, such as replication, DNA-repair and transcription have been established, as have their roles in the assembly and maintenance of chromatin. All of the complexes contain a highly conserved ATPase, which belongs to the SWI2/SNF2 family of proteins, one group of which is known as the ISWI proteins. There are two forms of ISWI in human, known as "SNF2h" and "SNF2l".

We have identified a human SNF2h-assembly, B-WICH, that consists of SNF2h, William’s syndrome transcription factor (WSTF), nuclear myosin (NM1), and a number of additional nuclear proteins including the Myb-binding protein 1a (Myb bp1a), SF3b155/SAP155, the RNA helicase II/Guα, the proto-oncogene Dek, and the Cockayne Syndrome protein B (CSB). The 45S rRNA, 5S rRNA and 7SL RNA are all parts of the B-WICH assembly. The formation of B-WICH depends on active transcription, and is implicated in the regulation of both RNA transcription by both pol I and pol III. The B-WICH provides a link between RNA and the chromatin structure.

Neutrophils have traditionally been studied in the context of acute infection, forming the first line of defence against invading pathogens, but with a restricted set of proinflammatory functions. More recently, it has become appreciated that neutrophils are, in fact, more complex cells that exhibit heterogeneity and functional plasticity, and capable of a wide array of specialised functions that contribute to adaptive immune responses and pathology of chronic inflammatory conditions. These later roles of neutrophils arise largely as a result of changes in their transcriptional activity in response to changes in the microenvironment in vivo and in vitro. However, relatively little is known of the mechanisms regulating this gene expression. Recent work has shown that, in response to long-term exposure to the TLR8 agonist R848, human neutrophils undergo chromatin remodelling and de novo synthesis of TNFa to express the cytokine, IL-6, expression of which by human neutrophils was previously controversial. In this thesis, the effect of R848 on global gene expression and on changes in the chromatin architecture that may control gene expression of neutrophils, alone or in combination with the proinflammatory factors TNFa or GM-CSF was investigated. Neutrophils were freshly-isolated from healthy individuals and treated with R848, either alone or in combination with TNFa or GM-CSF for different time periods. Histone post- translational modifications (e.g. pan and residue-specific acetylation and citrullination) were investigated by western blotting. RNA-seq was also performed on ultrapure neutrophils (>99%) stimulated with the above agents for 6 h, followed by computational and bioinformatics analysis to predict activation mechanisms. All major findings were validated by qRT-PCR at the mRNA level, and, in some cases, at the protein level. Further functional assays, such as apoptosis, ELISA and measurement of ROS, were performed as appropriate. R848, alone or in combination with the cytokines, induced acetylation of H3 after 6 h of treatment while it decreased H3 citrullination. These effects were more pronounced after 18 h of stimulation. Acetylation and transamination correlated with activation of gene expression in response to R848, but the effects of R848 used together with the cytokines were not simply additive. Unlike the cytokines, R848 induced the expression of interferon- response genes (IRGs) in an interferon-independent, but endogenous TNFa-dependent, manner. Exogenous TNFa failed to have any further effect on R848-induced expression of IRGs, while GM-CSF inhibited their induction. In contrast, R848 plus TNFa synergistically increased the expression of members of the IL-12 family cytokines. IL-23 was found, for the first time, to be expressed and secreted by neutrophils under such co-stimulation. On the other hand, R848 plus GM-CSF synergistically induced the expression of chemokines of the CC family and this effect was entirely dependent on endogenous TNFa. Finally, R848 and the cytokines had a pro-survival effect on neutrophils, but this effect was the outcome of different and complementary patterns of regulation of the anti-apoptotic proteins Mcl-1 and Bfl-1. The absence of an additive effect in apoptosis delay in response to the combined treatments excluded the possibility that TNFa or GM-CSF further increased gene expression in R848-treated neutrophils, simply because they further enhance neutrophil survival. In conclusion, these data show, for the first time, that chromatin remodelling agents, such as R848, can induce substantial changes in the patterns of gene expression of human neutrophils, when used alone or in combination with pro-inflammatory cytokines. The data predict that such chromatin re-modelling may occur in vivo in certain inflammatory conditions, substantially changing how neutrophils can respond to local cytokines. The data also show the importance of endogenous TNF expression and how this may result in autocrine signalling to regulate neutrophil function in human disease.

ATP-dependent chromatin remodellers represent a class of proteins that restructure chromatin through the action of a conserved helicase-like ATPase domain. Remodellers typically have several accessory binding domains alongside the ATPase. These confer target specificity and most commonly recognise histone post-translational modifications. SMARCAD1 is a ubiquitous chromatin remodeller involved with DNA replication and re- pair. It binds directly to PCNA at the site of DNA replication and recruits co-repressor KAP1 in order to silence newly produced chromatin. In contrast to most other chromatin remodellers, SMARCAD1 does not contain several different types of accessory domains. Only two CUE do- mains have been identified in addition to the SMARCAD1 core ATPase domain. CUE domains are a type of helical ubiquitin-binding domain. This thesis presents the findings of an investigation into the structure and function of the SMARCAD1 double CUE domain. The solution NMR structure is presented with results from NMR binding experiments mapped onto the structure. Each CUE domain was found to be an independent helix bundle connected by a dynamic flexible linker. The N-terminal CUE domain, CUE-1, binds ubiquitin and has an adjacent SUMO (a ubiquitin-like protein) binding motif on a protruding extended helix. The C-terminal CUE domain, CUE-2, has a very similar structure to several published CUE domains but does not bind ubiquitin due to a charged substitution at a highly conserved CUE consensus position. The SMARCAD1 double CUE domain binds KAP1 from nuclear extract and is likely to mediate the interaction between SMARCAD1 and KAP1. SMARCAD1 double CUE domain is not involved with PCNA binding.

Transcription of the ribosomal genes accounts for the majority of transcription in the cell due to the constant high demand for ribosomes. The number of proteins synthesized correlates with an effective ribosomal biogenesis, which is regulated by cell growth and proliferation. In the work presented in this thesis, we have investigated the ribosomal RNA genes 45S and 5S rRNA, which are transcribed by RNA Pol I and RNA Pol III, respectively. The focus of this work is the chromatin remodelling complex B-WICH, which is composed of WSTF, the ATPase SNF2h and NM1. We have studied in particular its role in ribosomal gene transcription. We showed in Study I that B-WICH is required to set the stage at rRNA gene promoters by remodelling the chromatin into an open, transcriptionally active configuration. This results in the binding of histone acetyl transferases to the genes and subsequent histone acetylation, which is needed for ribosomal gene activation. Study II investigated the role of B-WICH in transcription mediated by RNA polymerase III. We showed that B-WICH is essential to create an accessible chromatin atmosphere at 5S rRNA genes, which is compatible with the results obtained in Study 1. In this case, however, B-WICH operates as a licensing factor for c-Myc and the Myc/Max/Mxd network. Study III confirmed the importance and the function of the B-WICH complex as an activator of ribosomal genes. We demonstrated that B-WICH is important for the remodelling of the rDNA chromatin into an active, competent state in response to extracellular stimuli, and that the association of the B-WICH complex to the rRNA gene promoter is regulated by proliferative and metabolic changes in cells. The work presented in this thesis has confirmed that the B-WICH complex is an important regulator and activator of Pol I and Pol III transcription. We conclude that B-WICH is essential for remodelling the rDNA chromatin into a transcriptionally active state, as required for efficient ribosomal gene transcription.

At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 2: Manuscript. Paper 3: Manuscript.

 

In embryonic stem cells (ESCs), the SWI/SNF, CHD, and INO80 families of ATP-dependent chromatin remodellers have been implicated in maintaining pluripotency-associated gene expression, however the involvement of ISWI family remodellers has yet to be defined. Here, we explore the importance of the mammalian ISWI homologue SNF2H (Smarca5) by deriving a conditional knockout mouse ESC line and observing the consequences of SNF2H depletion on the pluripotent state. Cre-mediated deletion of Snf2h disrupts hallmark characteristics of pluripotency, resulting in distinct morphological changes; reduced expression of the master transcription factors Oct4, Sox2, and Nanog; and reduced alkaline phosphatase activity. To understand the mechanisms of SNF2H-mediated regulation, we mapped SNF2H-bound nucleosomes genome-wide. SNF2H is broadly distributed across the genome, but is preferentially enriched at active regulatory regions and transcription factor binding sites. These findings demonstrate the importance of SNF2H in ESCs and shed light on genome-wide mechanisms of transcriptional regulation.

The development of the nervous system is a complex task that involves precise connections between billions of neurons. This is achieved, at least in part, by the overproduction of neurons and the survival of a select few that compete for limited survival and growth promoting factors, such as neurotrophic factors. The neurotrophin brain-derived neurotrophin factor (BDNF) has been shown to play an important role in proliferation and differentiation of cortical neuronal precursors (Bartkowska et al 2007). Moreover, BDNF induces binding of the transcription factor CREB to gene promoters in a nitric oxide (NO)-dependent manner (Riccio et al 2006). NO positively regulates a large number of transcription factors and genes in the nervous system (Hemish et al 2003; Dhakshinamoorthy et al 2007). I have demonstrated that NO achieves this broad level of gene regulation by influencing chromatin remodelling. My data also show that NO accumulates within the nucleus of cortical neurons upon BDNF stimulation, thereby inducing Snitrosylation of a wide array of nuclear proteins. S-nitrosylation of histone deacetylase 2 (HDAC2) decreases its affinity for chromatin, leading to increased histone acetylation levels. This NO-dependent regulation of HDAC2 promotes changes in endogenous gene expression and affects the dendritic length of cortical neurons.

The INO80 complex is a large ATPase chromatin remodeller which contains 15 accessory subunits in S.cerevisiae. Its subunits include the highly conserved ATPases Ruvb1 and Ruvb2, the actin-related proteins Arp5, Arp8, Act1 and Arp4, Actin, and a number of IES (I̱noE̱ighty S̱pecific) subunits Ies1, Ies2, Ies3, Ies4, Ies5 and Ies6, in addition to subunits Nhp10 and Taf14. All 15 of the accessory subunits are assembled around a catalytic core component known as Ino80. The INO80 complex has roles in transcription, DNA repair, replication, and chromosome segregation. These roles are in addition to its traditional nucleosome remodelling activities and the dispacement of H2A.Z from chromatin. Recent studies in S. cerevisiae have identified the subunit Ies6 as a critical component of the INO80 complex. Deletion of IES6, which encodes the small accessory subunit, clearly mimics the deletion f the gene encoding the catalytic subunit, INO80. Surprisingly, only one domain within Ies6 has been formally identified based on sequence analysis. This domain belongs to the L1_C class of domains. Such domains are commonly associated with DNA binding activity and transcription factors. This stud has further characterised the Ies6 subunit both genetically and biochemically. Genetically, it has demonstrated that single point mutations at regions of proposed subunit-subunit interaction between the Arp5 or Rvb2 subunits, or within the YL1_C are not sufficient to disrupt Ies6 function. However, expression of a double point mutation, ies6(K114E/Y125A), in combination with rad50 deletion, caused a sensitivity to replication inhibition, but not chromosome segregation inhibition, indicating a potential separation of function in this utant due to the loss due of only one of the biological functions of Ies6. Biochemically, we have confirmed that DBA binding capacity of Ies6 resides within the YL_C domain. In addition, although it has been demonstrated that the removal of H2A.Z acetylation exacerbates the increase in cellular ploidy observed in ies6 null cells, we found that overall levels of H2A.Z acetylation were not influenced by the loss of Ies6. This indicates that the role of H2A.Z acetylation in chromosome segregation may only affect ploidy status upon the loss of Ies6. In addition, work on the R2TP complex (which contains the INO80 APases Ruvb1/Ruvb2, and subunits Tah1 and Phi1) has revealed the recruitment mechanism for the molecular chaperone, Hsp90, and the telomere length regulation protein, Tel2. Together, the R2TP complex, Hsp90 and Tel2 promote the stabilisation and maturation of multi-protein complexes. These include Phosphatidylinositol 3-kinase-related kinases (PIKKs, a family of kinases involved i Serine and Threonine phosphorylation), subunits of the INO80 complex and subunits of the SWR1 chromatin remodelling complex (a partner comlex to INO80 that incorporates H2A.Z into chromatin).

Epigenetic modifications of DNA and histones play a key role in governing the expression of genes essential during neural development. Histone deacetylase (HDAC) enzymes exert their functions by directly promoting changes in neuronal gene expression through the interaction with chromatin remodelling proteins and specific transcription factors. In cortical neurons, S-nitrosylation of HDAC2 results in transcriptional activation of genes that support neuronal survival and dendritogenesis (Nott et al., 2008). Bead array analysis identified brahma (Brm), a subunit of the ATP-dependent chromatin remodelling complex Brm/Brg-associated factor (BAF), as one of the genes regulated by HDAC2 S-nitrosylation. Expression of mutant form of HDAC2 that cannot be S-nitrosylated decreased Brm protein levels, leading to defects of radial neuronal migration and cortical laminar pattern. Thus, HDAC2 S-nitrosylation is necessary for the correct neuron radial migration during cortical development. HDACs are recruited to chromatin and exert their transcriptional regulatory functions via interaction with other nuclear factors, as part of multiprotein complexes. To identify the binding partners of HDAC2 in cortical neurons, whole sample mass spectrometry analysis was performed. Our analysis revealed a direct interaction of HDAC2 with nucleosome remodelling and histone deacetylase (NuRD) repressor complex. The core ATPase subunit of the NuRD complex includes a family of mutually exclusive chromodomain helicase DNA binding (CHD) proteins, named CHD3, CHD4 and CHD5. In embryonic cortex CHD4 is expressed in proliferating neuronal progenitors and maintained in postmitotic neurons, whereas homologous CHD3 and CHD5 are confined to postmitotic neurons. CHD3, CHD4 and CHD5 are associated with the promoters of neuronal genes indicating that they directly regulate transcriptional programs during neural development. Importantly, down-regulation of CHD3 subunit specifically expressed in postmitotic neurons, results in cortical radial migration defects, confirming the role of NuRD in cortical development. These studies highlight the importance of HDAC2 S-nitrosylation and ATP-dependent chromatin remodelling complexes in cortical development and cortical radial migration.

Recent studies have uncovered de novo mutations of the gene encoding the chromatin remodelling protein Snf2l in patients with schizophrenia, Rett-like syndrome and intellectual disability. Snf2l and its closely related protein, Snf2h, play a critical role in embryonic and post-natal brain development. Murine models lacking functional Snf2h or Snf2l point to complementary activities of these remodelers; Snf2h cKO mice present with a significantly reduced cerebellum, while Snf2l Ex6DEL (exon 6 deleted) cerebella are larger than their wild-type counterparts. Granule neuron progenitors (GNPs) isolated from Ex6DEL cerebella display delayed cell cycle exit and hindered terminal differentiation compared to wild-type controls. Moreover, loss of Snf2l activity results in widespread transcriptome shifts which underlie the Ex6DEL GNP differentiation phenotype. In particular, key transcription factors are differentially expressed without Snf2l remodelling activity. We confirm that ERK pathway activation is misregulated in Ex6DEL GNPs, possibly in response to elevated fibroblast growth factor 8 (Fgf8) expression in these cultures. We find that Snf2l activity maintains the chromatin landscape throughout GNP differentiation, as Ex6DEL cultures have a global increase in chromatin accessibility. We suggest that Snf2l-mediated chromatin condensation is responsible for proper regulation of gene expression programs in GNP differentiation.

Background: The higher-order structure of chromatin changes in response to extracellular and environmental signals. We observed nuclear morphological changes in biopsied cancer tissue after chemotherapy. Since chromatin structure dictates gene expression, and therefore function, further investigation of this phenomenon may increase our understanding of therapeutic responses. I hypothesised that nuclear morphological changes in cancer in response to DNA-damage by chemotherapy are mediated by histone deacetylases (de Ruijter, van Gennip et al.). Methods: Ovarian cancer cell lines PEO1/PEO4 (platinum sensitive/resistant) were selected as in vitro models, and primary ovarian cancer xenografts OV1002 and HOX424 as in vivo models. Expression levels of HDACs, heterochromatin protein 1 (HP1), and DNA damage response (DDR) proteins were profiled by Western blot analysis after treatment with cisplatin. Immunofluorescence imaging was undertaken using confocal microscopy, and nuclear texture and γH2AX foci were measured in Image J. Cell cycle and apoptosis were detected by flow cytometry. Thirty eight different ovarian cancer biopsies and 175 xenograft samples were assessed for HDAC and HP1 expression in response to chemotherapy by quantitative immunofluorescence. HDAC2 expression was modulated by interfering RNAs (siRNA). Results: I demonstrated nuclear morphological changes in clinical tumours, xenografts, and cell lines in response to platinum chemotherapy by robust measurement of nuclear texture. Expression of HDAC2 increased in PEO1 cells treated with cisplatin at 24h, and this was accompanied by high expression of HP1s. Expression of components of both HDACs and DDR pathways (pBRCA1, γH2AX, pATM, pATR) showed time dependent changes after cisplatin treatment. Knockdown of HDAC2 reduced the expression of HP1, induced DNA double strand breaks (DSB) measured by γH2AX, and interfered with the activation of DDR induced by cisplatin. Furthermore, HDAC2 depletion affected γH2AX foci formation, cell cycle distribution, and apoptosis triggered by cisplatin, and was additive to the inhibitory effect of cisplatin in cell lines. By inhibiting expression of HDAC2, I observed reversible alteration of chromatin patterns during cisplatin treatment to some degree. In clinical ovarian cancer specimens, expression of HDAC4, HDAC8 and HP1γ significantly increased after chemotherapy in sensitive patients, with enhanced heterogeneity in chromatin pattern. HDAC2, HDAC8, and HP1 expression were also increased after carboplatin treatment in carboplatin-sensitive xenografts. Conclusion: These results demonstrate alterations in nuclear morphology after chemotherapy, and implicate HDACs in having a role in higher order chromatin changes and in cellular DNA damage responses in ovarian cancer both in vitro and in vivo.

The centromere binding factor 1, Cbf1, of Saccharomyces cerevisiae is a bHLH/ZIP protein which has been described as a determinant of specific chromatin structures and as a tethering factor for activators of transcription at the promoters of genes of the Methionine Biosynthesis Pathway. Deletion mutants show various phenotypes, among them methionine auxotrophy, an increased rate of chromosome loss, modifications in the growth rate and modification of the chromatin structure at MET genes. Meiosis competence also becomes greatly reduced in cbf1 cells. The sequence motif (RTCACRTG) to which Cbf1p binds is found at multiple loci through the yeast genome. This thesis shows that the chromatin structure is reorganised at multiple Cbf1p binding sites in vivo, when yeast cells are starved to enter meiosis. Extensive remodelling occurs at the MET16, MET17(25), DRS2 and GDH3 loci and at the YAL060W open reading frame, as detected by in vivo digestion of chromatin with micrococcal nuclease and indirect end-labelling. The same kind of analysis showed that the remodelling of chromatin at Cbf1p binding sites is not specific for meiosis, it occurs also in similarly starved haploid cells. The lack of methionine is a key trigger of these changes. This reorganisation of chromatin is dependent on Cbf1p, since starved cbf1 cells do not display any modification in nuclease accessibility patterns at or around Cbf1p binding sites. Mutational analysis revealed that a negative charge at a putative phosphorylation site (serine residue 226) and the DNA-bindmg activity of Cbf1p are both required for the chromatin reorganisation to occur in response to starvation. CBF1 mutants which do not reorganise chromatin were also shown to be unable to enter meiosis, suggesting that the remodelling of chromatin at multiple Cbf1p binding sites may be required to enter pre-meiotic DNA replication, since such cells arrest before the initiation of this process. In summary, the results presented in this thesis are compatible with a model in which Cbf1p plays an active role as part of a mechanism sensing the nutrient availability and regulates the reorganisation of chromatin, at multiple loci through the yeast genome, in response to starvation conditions.

Plant cells require considerable transcriptional reprograming to mount an effective response to pathogens. Plant responses to pathogens have to be finely balanced with other vital biological processes such as development and growth. A major mechanism controlling the modulation of gene expression is chromatin remodelling. Chromatin remodelling requires histone covalent modifications and/or the action of ATP- dependent remodelling complexes. The combined action of these determine the accessibility of transcription factors and the basal transcription machinery to DNA and therefore greatly impact gene expression. There are several examples of histone modifying and chromatin remodelling enzymes previously shown to regulate plant development and immunity. This thesis explored the role of chromatin in plant defences, and how chromatin remodelling forms an integral part of the defence response. Chapter 1 aimed to discover a “hidden” signal of chromatin marks in plant defence-responsive genes using an array of bioinformatics techniques. Subsequently, histone H3K27 tri- methylation (H3K27me3) was identified as a mark associated with gene repression at defence-related loci. The role of histone H3K27me3 and its associated histone demethylase enzymes REF6 and ELF6 were empirically characterised. Chapter 2 is dedicated to a reverse genetics screening investigating the role of the chromatin remodelling ATPases Arabidopsis family in plant defences, and describes the most prominent phenotypes. And lastly, Chapter 3 dissects in greater detail the role of the chromatin remodelling ATPase EDA16 in plant defence. Pathogen assays, RNA-seq and other molecular techniques suggest that EDA16 is a negative regulator of immunity induced upon pathogen perception to regulate the amplitude of defence responses.

The role of chromatin structure on viral gene expression during HCMV infection of a permissive cell type was investigated. Experiments demonstrated that the MIEP was associated with chromatin throughout infection and was subject to modification consistent with the known temporal transcription of IE genes. However, immediately upon infection, the MIEP was associated with a repressed chromatin structure. This repression was relieved through use of the histone deacetylase (HDAC) inhibitor Trichostatin-A (TSA), confirming that the regulation of IE gene expression at this time was mediated by an inhibitory chromatin structure. Furthermore, down-regulation of the ND10-associated transcriptional repressor, hDaxx, which is known to interact with HDACs, led to similar relief from repression. Further analysis of promoters of prototypic viral early and late genes, UL44 and pp28 respectively, also revealed that all classes of viral genes are subject to chromatin-mediated regulation throughout productive infection. Although differentiation-dependent regulation of MIEP activity underpins viral latency and reactivation, down-regulation of the intrinsic anti-viral repressor hDaxx in in vitro latent model systems did not permit reactivation of IE gene expression. Consequently, repression of IE gene expression by hDaxx does not appear to be involved in regulation of viral latency and reactivation. In conclusion, immediately upon infection of permissive cells, intrinsic repression of HCMV IE gene transcription occurs through the establishment of an inhibitory chromatin structure around the MIEP, mediated by hDaxx and HDACs. This repression is overcome by viral factors before full productive infection can begin. Full lytic infection then involves regulation of viral gene expression through remodelling of chromatin structure at all classes of HCMV promoters.

The S.cerevisiae Ino80 chromatin remodelling complex is known to be involved in the DNA damage response at double strand breaks and has more recently also been shown to play a role at stalled replication forks. The many functions of this remodellor are likely to be mediated by different subunits of the complex. Interestingly, strains harbouring a deletion of the IES6 subunit are hypersensitive to hydroxyurea and fail to stabilise stalled replication forks, phenocopying strains lacking the catalytic subunit, INO80, indicating a role for Ies6 within the complex's response to DNA damage. Although largely uncharacterised, Ies6 contains a YL1 domain, which is a putative DNA binding domain. In vitro DNA binding gel shift assays with recombinant Ies6 showed that this protein does possess DNA binding activity. Recombinant Ies6 bound both Holliday Junction and Y-fork DNA, as well as linear duplex DNA, displaying a small but reproducible preference for the two branch-structured DNAs. Recombinant Ies6 containing mutations in the protein's C-terminal YL1 domain were generated and a quadruple mutant, ies6-T119A/K122A/S127A/T129A, exhibited significantly reduced DNA binding activity compared to the wild-type protein. The importance of the DNA binding activity was investigated in vivo, and, in contrast to the wild-type strain, the DNA binding mutant of IES6 failed to complement the deletion strain's HU-hypersensitivity. Interestingly, overexpression of Top3 or Cdk1, but not Top2 or Clb2 also rescued the ies6 deletion strain's HU-hypersensitivity to near wild-type levels. Further investigation revealed that Ies6 is also required for the maintenance of correct cellular ploidy, as in the absence of IES6, cellular ploidy is seen to increase prior to a drift towards unregulated ploidy and aneuploidy, which are hallmarks of cancer. Notably, the protein's ability to bind DNA correlated with its ability to maintain cellular ploidy. We therefore propose that the Ino80 chromatin remodelling complex Ies6 subunit plays an important role in the maintenance of genomic stability.

SWI/SNF chromatin remodelling complexes are one of the well-characterized cellular machineries capable of regulation of gene expression. Numerous lines of evidence indicate that SWI/SNF complexes are involved in a wide range of cellular processes and the maintenance of homeostasis whereas aberrant expression of those proteins contributes towards cancer development. Colorectal cancer remains one of the most clinically significant cancers due to its high incidence in developed countries and previous studies have demonstrated that SWI/SNF complexes are aberrantly regulated in a significant proportion of patients with this disease. However, whilst the sequence of molecular events leading to CRC has been well-established, the role of SWI/SNF chromatin remodelling complex ATPase subunits Brm and its paralogue Brg1 in the colorectal tumorigenesis remains elusive. The chromatin remodelling catalytic subunit Brm has been found to interact with the Notch pathway effectors ICD-22 and CBF-1 and also to be necessary for expression of the Wnt target gene CD44 and for Rb-mediated cell cycle arrest. In this PhD thesis, the potential of Brm to modulate Wnt-driven intestinal tumorigenesis was addressed. Initially, a murine model carrying constitutively deleted Brm was used to assess the consequences of this loss on homeostasis of the small intestinal and colonic epithelia. The effects of Brm deficiency were also examined in the context of Wnt-activated epithelium via conditional loss of Apc. Additionally, the effect of concomitant loss of Brm and Brg1 was addressed in the contexts of both normal homeostasis and aberrant Wnt signalling. The results presented here demonstrate that Brm plays an important role in the small intestine by regulating the distribution of proliferating cells and cell fate decisions mediated through Notch pathway effectors. Furthermore, Brm deficiency was found to modulate intestinal phenotype of Wnt activation through the attenuation of the Wnt transcriptional programme and the suppressed expression of the intestinal stem cell marker Olfm4. Thus while Brg1 has been widely characterized as a bone fide tumour suppressor, the function of Brm continues to remain elusive especially in the light of contrasting effects co-mediated by Brm on proliferation, differentiation and gene expression. Taken together, these results elucidate the tissue-specific role of Brm, the catalytic subunit of SWI/SNF chromatin remodelling complex, on both normal intestinal homeostasis and acute activation of Wnt pathway while the extent of these Brm-dependent effects depend upon the gradient of Wnt signalling throughout the epithelium of small and large intestine.

Repo-Man is a chromatin-associated PP1 targeting subunit that coordinates chromosome re-organisation and nuclear envelope reassembly during mitotic exit. At the onset of mitosis, Repo-Man association with the chromosomes is very dynamic; at anaphase, Repo-Man targets to the chromatin in a stable manner and recruits PP1 to de-phosphorylate histone H3 at Thr3, Ser10 and Ser28. Previous studies have suggested that CDK1 and AuroraB are the kinases responsible for the inactivation of the complex and for its dispersal at the onset of mitosis respectively. We have previously shown that the binding of Repo-Man to PP1 is decreased in mitosis and we have identified a region adjacent to the RVTF motif that contains multiple mitotic phosphosites (RepoSLIM). This region is conserved only in another PP1 targeting subunit: Ki-67. In order to understand the importance of this region for the complex formation and stability, we have conducted mutational analyses on several residues, and addressed their contribution towards Repo-Man chromosome targeting and PP1 binding in vivo. We have identified new sites in Repo-Man that, when phosphorylated, contribute to the weakening of the binding between Repo-Man and PP1. Interestingly, our results also indicate that several kinases are involved in the mitotic regulation of the complex. We have also identified Lamin A/C as a Repo-Man substrate and introduced a new model for Lamin A/C regulation at interphase. Furthermore, we identified Repo-Man as a marker of malignancy in tripe egative breast cancer, which controls cell movement and levels of important oncogenic markers Aurora A and C-Myc, and propose Repo-Man/PP1 complex as a therapeutic target for the treatment of triple negative breast cancer through the newly identified RepoSLIM.

Currently, the quality of oocytes matured in vitro is still not satisfactory. In fact, only 5-10% of oocytes were evaluated to be intrinsically capable to produce in vitro embryos with full developmental competence. Recent studies have highlighted the importance of epigenetics, a mechanism controlling activation and repression of gene expression, in embryonic development. Indeed, several proteins involved in histone modifications including histone deacetylase 1 (HDAC-1), lysine specific demethylase 1 (LSD-1), and brahma related gene 1 (BRG-1) were shown vital to early development. We described the presence of these proteins during porcine oocyte maturation and embryo development and investigated whether these proteins were also involved in the regulation of developmental competence. We detected the three proteins in all stages of development, but nuclear localization was not always confirmed. We noticed that these three proteins followed the same general trend before activation but while the fluorescence signal of HDAC-1 increased after activation those of LSD-1 and BRG-1 decreased. Moreover, oocytes and embryos of lower developmental competence were observed to have differences in the fluorescence signal of HDAC-1, LSD-1, and BRG-1. The occurrence of targeted LSD-1 histone modifications, histone 3 lysine 4 mono- (H3K4me1) and di- (H3K4me2) methylation, were also found to be different in embryos of different developmental competence. This study suggests that HDAC-1, LSD-1 and BRG-1 are developmentally regulated during early development of swine embryos and are potentially involved in the acquisition of the embryonic developmental competence.
Aujourd'hui, la qualité des ovocytes maturés in vitro n'est toujours pas satisfaisante. En effet, seulement 5 à 10% des ovocytes peuvent intrinsèquement engendrer in vitro des embryons avec entière compétence développementale. Récemment, des études ont montré l'importance qu'a l'épigénome, mécanisme qui active ou inhibe l'expression des gènes, dans le développement embryonnaire. Effectivement, plusieurs protéines reconnues capables de modifier les histones telles que l'histone déacétylase 1 (HDAC-1), la déméthylase spécifique aux lysines 1 (LSD-1), ainsi que le gène brahma related 1 (BRG-1), ont été démontrées vitales aux premiers stades développementaux. Nos expériences ont pour buts de décrire par immuno-détection la présence des ces protéines pendant la maturation méiotique et le développement d'ovocytes et d'embryons porcins et de savoir si ces protéines sont aussi impliquées dans la régulation de la compétence développementale de ces-derniers. Nous avons décelé la présence des trois protéines à tous les stades de développement bien que leurs localisations nucléiques n'aient pas été toujours confirmées. De plus, nous avons observé que l'intensité du signal de fluorescence des trois protéines fluctue similairement au cours de la maturation des ovocytes. Cependant, après l'activation, la fluorescence de HDAC-1 augmente alors que celles de LSD-1 et BRG-1 diminuent. D'autre part, nous avons pu constater que les ovocytes et embryons de moindre compétence développementale présentent des différences de fluorescence pour les trois protéines étudiées. En outre, nous avons également détecté des différences dans la présence de mono- (H3K4me1) et di- (H3K4me2) méthylations sur la 4e lysine de l'histone 3, cibles de LSD-1, dans des embryons de différentes compétences développementales. Cette étude suggère que les protéines HDAC-1, LSD-1 et BRG-1 sont régulées aux premiers stades de développement po

DNA is compacted in the nucleus of eukaryotic cells into chromatin and the fundamental subunit is the nucleosome that consists of an octamer of histones surrounded by 1.67 superhelical turns of 146-147 bp DNA. Nucleosomes will therefore invariably affect the regulation of gene expression by restricting the access of regulatory proteins to DNA. The retinoid receptors are class II nuclear receptors that form heterodimers and bind to retinoic acid response elements (RAREs), found in the promoters of retinoid responsive genes.

Der pRB/E2F-Signalweg ist ein zentraler Regulator der Proliferationskontrolle in Säugerzellen, der in fast allen auftretenden Tumoren dereguliert ist. Durch unterschiedliche Mutationen in Komponenten dieses Signalwegs kommt es letzten Endes zu einer erhöhten Aktivität der E2F-Transkriptionsfaktoren und somit zu einer verstärkten Transkription von E2F-Zielgenen in diesen Tumoren. Um die molekularen Mechanismen der Rolle von E2F3 in der Zellzykluskontrolle und der Tumorigenese besser zu verstehen, wurden in dieser Arbeit per GST-Pulldown mit anschließender Massenspektrometrie neue potenzielle Interaktions-partner von E2F3 identifiziert. Ein identifizierter Interaktionspartner war die SNF2-ähnliche Helikase HELLS. HELLS interagiert in vitro und in vivo spezifisch mit der Marked Box-Domäne von E2F3, aber nicht mit anderen untersuchten E2F-Transkriptionsfaktoren, wie durch GST-Interaktionsstudien und Ko-Immunpräzipi-tationsexperimente demonstriert werden konnte. Durch Chromatin-Immunpräzipitation konnte zusätzlich gezeigt werden, dass E2F3 für die Rekrutierung von HELLS an E2F-regulierte Promotoren wie z. B. CDC6 oder p107 verantwortlich ist. Die shRNA-vermittelte Depletion von HELLS führte zu einer stark verminderten Induktion von allen untersuchten E2F-Zielgenen nach Serumstimulation und einem verspäteten Eintritt in die S-Phase der HELLS-depletierten Zellen, was zeigt, dass HELLS essenziell für die Induktion von E2F-Zielgenen ist. Bei der immunhistochemischen Untersuchung der E2F3- und HELLS-Expression in humanen Prostatakarzinomen zeigte sich, dass sowohl E2F3 als auch HELLS in späten aggressiven Stadien dieser Tumore sehr stark exprimiert sind, jedoch nur sehr schwach in den weniger aggressiven Tumoren. Diese Versuche zeigen, dass es sich bei HELLS um einen neuen Bestandteil des pRB/E2F-Signalwegs handelt, der eventuell in der Entstehung gewisser Tumorarten eine Rolle spielt und somit ein neues potenzielles Ziel für neuartige Krebstherapien darstellt.
The pRB/E2F pathway is a key regulator of proliferation in mammalian cells and is commonly mutated in human tumors. These mutations in the components of the pRB/E2F pathway lead to deregulated activity of the E2F transcription factors resulting in increased expression of E2F target genes. To further understand the molecular mechanisms of E2F3 in cell cycle control and its role in tumorigenesis new interaction partners for E2F3 were identified in the course of this thesis with the help of a GST-Pulldown approach coupled to mass spectrometric analysis. One of the identified interaction partners was the SNF2-like helicase HELLS. With the help of GST-interaction studies and Co-Immunoprecipitation assays it could be demonstrated that HELLS interacts specifically with E2F3 via its Marked Box domain but does not bind to the other investigated E2F transcription factors. HELLS could be detected at E2F target genes like p107 and CDC6 in vivo with the help of Chromatin-Immunoprecipitation assays. Furthermore, the forced recruitment of E2F3 to E2F target genes led to an enhanced binding of HELLS to these promotors suggesting that HELLS is recruited to E2F target genes via protein-protein interaction with E2F3. The shRNA-mediated depletion of HELLS led to a strongly reduced induction of E2F target genes and a delay in S-phase entry, showing that HELLS is essential for the induction of E2F target genes. During the immunohistochemical analysis of human prostate cancer specimens it became evident that both E2F3 and HELLS are strongly expressed in the more aggressive late stages but only weakly expressed in the early stages of this tumor type. These findings demonstrate that HELLS is a new component of the E2F/pRB pathway which might play a role in the development of certain tumors and might represent a new target for novel cancer therapies.

Non-melanoma skin cancer (NMSC) is the most common human cancer worldwide. Squamous cell carcinoma (SCC) and basal cell carcinoma (BCC) make up almost all NMSC. SCC usually arises from actinic keratosis (AK) as a result of exposure to sunlight. SCC and AK provide a useful clinical model to investigate changes involved in the progression of NMSC. This project examines the expression of Brm, Brg-1, Snail 1 and Snail 2 in the progression of NMSC. Brm and Brg-1 are subunits of the SWI/SNF chromatin-remodelling complex which is involved in regulating the access of cell machinery to DNA by altering the structure of chromatin. It has been suggested that loss of this function is involved in carcinogenesis as the cell is unable to access to DNA normally in order to repair mutations or activate apoptosis. The loss of Brm or Brg-1 has been described in several human cancers. Snail 1 and Snail 2 are zinc-finger transcription factors that are known for their role in epithelial to mesenchymal transition (EMT), a process vital to embryological development. Increased expression of these factors leads to a loss of cell-cell adhesion and a migratory phenotype and has been described in some human cancers. In this project, double-label immunohistochemistry was used to determine the relative expression of these proteins in human SCC, BCC, AK and normal skin. The expression of Snail was unable to be determined due to poor specificity of the antibodies used. The expression of both Brm and Brg-1 proteins was found to be dramatically and consistently decreased in SCC and BCC when compared to normal skin and AK. This loss of Brm and Brg-1 occured as the tumour progressed from benign AK to malignant SCC. This finding suggests that the loss of either Brm or Brg-1 constitutes a key step in carcinogenesis. The results of this study identify Brm and Brg-1 as putative tumour suppressors involved in the progression of non-melanoma skin cancer from benign to malignant.

Master of Medicine
Non-melanoma skin cancer (NMSC) is the most common human cancer worldwide. Squamous cell carcinoma (SCC) and basal cell carcinoma (BCC) make up almost all NMSC. SCC usually arises from actinic keratosis (AK) as a result of exposure to sunlight. SCC and AK provide a useful clinical model to investigate changes involved in the progression of NMSC. This project examines the expression of Brm, Brg-1, Snail 1 and Snail 2 in the progression of NMSC. Brm and Brg-1 are subunits of the SWI/SNF chromatin-remodelling complex which is involved in regulating the access of cell machinery to DNA by altering the structure of chromatin. It has been suggested that loss of this function is involved in carcinogenesis as the cell is unable to access to DNA normally in order to repair mutations or activate apoptosis. The loss of Brm or Brg-1 has been described in several human cancers. Snail 1 and Snail 2 are zinc-finger transcription factors that are known for their role in epithelial to mesenchymal transition (EMT), a process vital to embryological development. Increased expression of these factors leads to a loss of cell-cell adhesion and a migratory phenotype and has been described in some human cancers. In this project, double-label immunohistochemistry was used to determine the relative expression of these proteins in human SCC, BCC, AK and normal skin. The expression of Snail was unable to be determined due to poor specificity of the antibodies used. The expression of both Brm and Brg-1 proteins was found to be dramatically and consistently decreased in SCC and BCC when compared to normal skin and AK. This loss of Brm and Brg-1 occured as the tumour progressed from benign AK to malignant SCC. This finding suggests that the loss of either Brm or Brg-1 constitutes a key step in carcinogenesis. The results of this study identify Brm and Brg-1 as putative tumour suppressors involved in the progression of non-melanoma skin cancer from benign to malignant.

lnterleukin-6 (IL-6) is a pro-inflammatory cytokine produced by macrophages, endothelial cells, fibroblasts, vascular smooth muscle cells, T lymphocytes and adipocytes. IL-6 is regulated at the level of transcription. There are multiple regulatory elements in the IL-6 promoter. Three single nucleotide polymorphisms at position -597, -572 and -174 and a variable region of A's and T's at -373 have been identified in the IL-6 proximal promoter. The THP-1 macrophage-like cell line was induced to express IL-6 mRNA in response to IFN-? and LPS. Interferon-gamma induced IL-6 mRNA expression was inhibited by treatment with either atorvastatin or pravastatin. Monocytes extracted from the blood of healthy volunteers of known IL-6 promoter haplotype (+GG9/11G and -GG9/11G) were differentiated to macrophages ex vivo. These cells were cultured in the presence of IL-1beta, LPS or IFN-gamma, to induce IL-6 mRNA. Quantitative PCR was used to quantitate the level of IL-6 mRNA. IL-6 mRNA in macrophages from individuals with the IL-6 promoter haplotype +GG9/11G was significantly induced in response to IL-1beta when compared to -GG9/11G individuals. A similar though non significant effect was detected in response to IFN-gamma. No significant IL-6 promoter haplotype-specific difference was observed in response to LPS. Chromatin remodeling of the IL-6 promoter occurred within 30 minutes of stimulation and was specific to the region of the IL-6 promoter and the stimulus applied to the cells. A DNase I hypersensitive site was identified in the region of the polymorphic AnTn variable tract and a potential DNA cruciform structure in the IL-6 promoter was resolved by T7 Endonuclease I. Atomic force microscopy was used to visualise DNA cruciform structure in IL- 6 promoter haplotype constructs. This work contributes to the understanding of the molecular mechanisms underlying the normal inter-individual differences in the macrophage IL-6 inflammatory response.

The CCAAT box is a frequent promoter element bound by NF-Y, a trimer with H2A-H2B-like subunits. We developed a MNase I-based ChIP protocol on homogeneous cell populations to study cell-cycle promoters at the single nucleosome level. We analyzed histone acetylations and methylations and the association of enzymatic activities. This thesis presents different novel findings. The first finding was that H3-H4 take part of core promoters under active conditions, with the expected cohort of “positive” modifications, while H2A-H2B are removed and substituted by NF-Y. Through the use of a dominant negative mutant we show also that NF-Y is important for H3K36me3 deposition and for Pol II elongation. The second finding was that H3K4 methylations are highly dynamic and H3K4me1 is a crucial positive mark. Both functional and pharmacological inactivation led to state that KDM1 plays a positive role in transcription of G2/M genes. It requires CoREST, which is recruited on active promoters through direct interactions with NF-Y. Therefore, these preliminary data are the first in vivo indication of a crucial interplay between core histones and “deviant” histone-fold such as NF-Y, leading to fine tuning of histone methylations. The third finding was that NF-Y is not involved in histone acetyl-marks deposition as well as in histone methyl-marks: in fact, histone acetylation status of active cell cycle genes was only slightly perturbed after NF-Y removal. Moreover, this work proposed a special histone acetylation pattern typical of cell cycle gene cluster, characterized by H2BK120ac and H3K9,18,36ac deposited on H3 in repressive conditions. And finally, by in vivo analysis we showed GCN5 and PCAF involvement in the acetyl-marks deposition, and the probable NF-Y dependent recruitment of multisubunit complexes responsible of the chromatin remodelling, like STAGA and ATAC.

Lo stato di cellula pluripotente può essere raggiunto con l espressione ectopica di alcuni fattori di trascrizione che possono riprogrammare le cellule somatiche. (Takahashi, K., e Yamanaka, S., 2006; Takahashi, K., e Yamanaka, S., 2007).

Rubinstein–Taybi syndrome (RSTS) is a rare malformation disorder caused by mutations in the closely related CREBBP and EP300 genes, accounting respectively for up to 60 and 3% of cases. About 10% of CREBBP mutations are whole gene deletions often extending into flanking regions. Using FISH and microsatellite analyses as a first step in the CREBBP mutation screening of 63 Italian RSTS patients (pts), 6 deletions were identified, 3 of which were in a mosaic condition that has not been previously reported in RSTS. The clinical presentation was typical in all cases, but more severe in the three pts carrying constitutional deletions, raising a question about the possible underdiagnosis of a few cases of mild RSTS. The use of region-specific BAC clones and small CREBBP probes allowed to assess the extent of all of the deletions by mapping their endpoints to genomic intervals of 5–10 kb. Four of five intragenic breakpoints cluster at the 5' end of CREBBP, where there is a peak of breakpoints underlying rearrangements in RSTS pts and tumours. The search for genomic motifs did not reveal any low-copy repeats (LCRs) or any greater density of repetitive sequences. The FISH analysis extended to the EP300 genomic region did not reveal any deletions. Searching for mutations of CREBBP gene in 56 patients revealed 23 different mutations. In addition, one deletion and two amplifications were identified by a-CGH in 20 RSTS pts.

Changes in the epigenetic landscape are widespread in neoplasia, with de novo methylation and histone repressive marks commonly occurring in association with gene silencing. However, understanding the dynamics of epigenetic changes is often hindered due to the absence of adequate in vitro model systems that accurately reflect events occurring in vivo. Human mammary epithelial cells (HMECs) grown under standard culture conditions enter a growth arrest termed selection, but a subpopulation is able to escape from arrest and continue to proliferate. These cells, called post-selection cells, have many of the hallmarks seen in the earliest lesions of breast cancer, including transcriptional silencing and hypermethylation of the p16INK4A tumour suppressor gene. The overall aim of my thesis was to use post-selection HMECs as model system to identify and dissect the mechanism involved in early epigenetic aberrations. Firstly, using a microarray approach, I found that multiple members of the TGF-β signalling pathway were concordantly suppressed in post-selection cells, and this was associated with functional disruption of the TGF-β pathway. Interestingly, concordant gene suppression was not associated with aberrant DNA methylation, but with repressive chromatin remodelling. Secondly, to further understand the mechanism underpinning epigenetic silencing, I demonstrated using laser capture technology, that p16INK4A silencing is a precursor to DNA methylation and histone remodelling. Thirdly, I found that individual post-selection HMEC strains during the early passages shared a common 'wave' pattern of regional-specific methylation within the p16INK4A CpG island. Interestingly, the 'wave' pattern of early de novo methylation correlated with the apparent footprint of nucleosomes within the p16INK4A CpG island. Lastly, to further characterise the properties of the HMEC culture system, I demonstrated that post-selection cells do not possess a natural tumour-inducing property when transplanted into the mammary fat pad of immunocompromised mice. However, post-selection HMECs were associated with high expression of a variety of stem/progenitor markers, as well as stem/progenitor associated polycomb genes, thus demonstrating that these cells share some common features of stem/progenitor cells. The research presented in this thesis demonstrate that epigenetic changes occur early in the growth of post-selection HMECs and many of these changes are common in breast cancer.

ISWI is a member of a versatile family of ATP-dependent chromatin remodelling complexes involved not only in transcription regulation (initiation, elongation and termination), but also in other cellular functions like maintenance of higher order chromatin structure and DNA replication. TbISWI, a novel ATPase of the ISWI family in Trypanosoma brucei, is involved in the transcriptional repression of silent VSG expression sites (ESs) in both bloodstream form (BF) and procyclic form (PF) life cycle stages of the parasite. Using in silico analysis, I have found that TbISWI is well conserved across the eukaryotic lineage, including those members of the order Kinetoplastida that do not exhibit antigenic variation. Compared to the ISWIs of higher eukaryotes, TbISWI has greater representation of random coils within its structure, an indicator of more structural fluidity and flexibility of interaction with multiple protein partners. Using an eGFP reporter based assay, I have studied the role of TbISWI in transcriptional repression of silent areas of the T. brucei genome. TbISWI was found to be involved in preventing inappropriate transcription of the silent VSG repertoires. TbISWI was also found to downregulate transcription in RNA pol I, but not pol II, transcription units. These results argue for the presence of at least two functionally distinct TbISWI complexes in T. brucei. Using DNA staining and fluorescence in situ hybridisation (FISH), I have investigated the potential effect of TbISWI depletion on cell cycle progression and minichromosome segregation. I did not find any evidence for the role of TbISWI in the maintenance of centromeric heterochromatin in T. brucei.

During tissues development multipotent progenitor cells establish tissue-specific gene expression programmes, leading to differentiation into specialized cell types. It has been previously shown that the transcription factor p63, a master regulator of skin development, controls the expression of adhesion molecules and essential cytoskeleton components. It has also been shown that p63 plays an important role in establishing distinct three-dimensional conformations in the Epidermal Differentiation Complex (EDC) locus (Fessing et al., 2011). Here we show that in p63-null mice about 32% of keratinocytes showed altered nuclear morphology. Alterations in the nuclear shape were accompanied by decreased expression of nuclear lamins (Lamin A/C and Lamin B1), proteins of the LINC complex (Sun-1, nesprin-2/3) and Plectin. Plectin links components of the nuclear envelope (nesprin-3) with cytoskeleton and ChIP-qPCR assay with adult epidermal keratinocytes showed p63 binding to the consensus binding sequences on Plectin 1c, Sun-1 and Nesprin-3 promoters. As a possible consequence of the altered expression of nuclear lamins and nuclear envelope-associated proteins, changes in heterochromatin distribution as well as decrease of the expression of several polycomb proteins (Ezh2, Ring1B, Cbx4) has been observed in p63-null keratinocytes. Moreover, recent data in our lab have showed that p63 directly regulates Cbx4, a component of the polycomb PRC1 complex. Here we show that mice lacking Cbx4 displayed a skin phenotype, which partially resembles the one observed in p63-null mice with reduced epidermal thickness and keratinocyte proliferation. All together these data demonstrate that p63-regulated gene expression program in epidermal keratinocytes includes not only genes encoding adhesion molecules, cytoskeleton proteins (cytokeratins) and chromatin remodelling factors (Satb1, Brg1), but also polycomb proteins and components of the nuclear envelope, suggesting the existence of a functional link between cytoskeleton, nuclear architecture and three dimensional nuclear organization. Other proteins important for proper epidermal development and stratification, are cytokeratins. Here, we show that keratin genes play an essential role in spatial organization of other lineage-specific genes in keratinocytes during epidermal development. In fact, ablation of keratin type II locus from chromosome 15 in epidermal keratinocytes led to changes in the genomic organization with increased distance between the Loricrin gene located on chromosome 3 as well as between Satb1 gene located on chromosome 17 and keratin type II locus, resulting in a more peripheral localization of these genes in the nucleus. As a possible consequence of their peripheral localization, reduced expression of Loricrin and Satb1 has also been observed in keratins type II-deficient mice. These findings together with recent circularized chromosome conformation capture (4C) data, strongly suggest that keratin 5, Loricrin and Satb1 are part of the same interactome, which is required for the proper expression of these genes and proper epidermal development and epidermal barrier formation. Taken together these data suggest that higher order chromatin remodelling and spatial organization of genes in the nucleus are important for the establishment of lineage-specific differentiation programs in epidermal progenitor cells. These data provide an important background for further analyses of nuclear architecture in the alterations of epidermal differentiation, seen in pathological conditions, such as psoriasis and epithelial skin cancers.

Cell growth and proliferation are processes in the cell that must be tightly regulated. Transcription of ribosomal RNA and ribosomal biogenesis are directly linked to cell growth and proliferation, since the ribosomal RNA encodes for the majority of transcription in a cell and ribosomal biogenesis influences directly the number of proteins that are synthesized. In the work presented in this thesis, we have investigated the ribosomal RNA genes, namely the ribosomal DNA genes and the 5S rRNA genes, and their transcriptional regulation. One protein complex that is involved in RNA polymerase I and III transcription is the chromatin remodelling complex B‑WICH (WSTF, SNF2h, NM1). RNA polymerase I transcribes the rDNA gene, while RNA polymerase III transcribes the 5S rRNA gene, among others. In Study I we determined the mechanism by which B‑WICH is involved in regulating RNA polymerase I transcription. B‑WICH is associated with the rDNA gene and was able to create a more open chromatin structure, thereby facilitating the binding of HATs and the subsequent histone acetylation. This resulted in a more active transcription of the ribosomal DNA gene. In Study II we wanted to specify the role of NM1 in RNA polymerase I transcription. We found that NM1 is not capable of remodelling chromatin in the same way as B‑WICH, but we demonstrated also that NM1 is needed for active RNA polymerase I transcription and is able to attract the HAT PCAF. In Study III we investigated the intergenic part of the ribosomal DNA gene. We detected non-coding RNAs transcribed from the intergenic region that are transcribed by different RNA polymerases and that are regulated differently in different stress situations. Furthermore, these ncRNAs are distributed at different locations in the cell, suggesting that they have different functions. In Study IV we showed the involvement of B‑WICH in RNA Pol III transcription and, as we previously had shown in Study I, that B‑WICH is able to create a more open chromatin structure, in this case by acting as a licensing factor for c-Myc and the Myc/Max/Mxd network. Taken together, we have revealed the mechanism by which the B‑WICH complex is able to regulate RNA Pol I and Pol III transcription and we have determined the role of NM1 in the B‑WICH complex. We conclude that B‑WICH is an important factor in the regulation of cell growth and proliferation. Furthermore, we found that the intergenic spacer of the rDNA gene is actively transcribed, producing ncRNAs. Different cellular locations suggest that the ncRNAs have different functions.

At the time of the doctoral defence the following papers were unpublished and had a status as follows: Paper 2: Manuscript; Paper 3: Manuscript

ATP-dependent chromatin remodelers have been proposed to act sequentially, and to a certain extent non-redundantly, in the priming stages of the DNA Damage Response pathways by establishing chromatin in lesion sites ready to act as a scaffold for repair factors or to be displaced in order to allow DNA repair. Among remodeling factors proposed to play a role in DNA repair is SMARCAD1, a poorly characterized, non-canonical member of the SWR1-like family of SNF2/SWI2 superfamily of ATPases, which has recently been identified as a potential target for ATM/ATR phosphorylation at canonical and non-canonical sites upon DNA damage. The actual mechanism for SMARCAD1 recruitment and involvement in DNA remodeling is still unknown, and unlike most other chromatin remodelers, SMARCAD1 does not contain DNA- or histone-binding domains frequently accompanying such proteins. Instead, in addition to the core ATPase domain, only two CUE domains (a type of helical ubiquitin-binding domain) have been identified. This thesis presents the findings of an investigation intended to structurally characterize SMARCAD1 by dissecting and identifying its domain architecture, and examining the activity and ligand selectivity of its binding domains in the functional context of DNA damage repair. The solution NMR structure of the CUE1 domain is presented, describing a triple helix bundle consistent with other members of the family. Furthermore, a novel SUMO interacting motif was identified and through a combination of NMR titrations and phospho-proteomics analysis, shown to be constitutively phosphorylated which excludes the possibility of DNA damage dependent ATM targeting as the recruitment mechanism for DNA repair. Additionally, it is demonstrated that both CUE domains are poor binders of mono-ubiquitin, however CUE1 specifically mediates the high affinity binary interaction with the transcriptionally repressive master regulator KAP1. This interaction was shown to be independent of post-translational ubiquitylation but rather sustained through direct interaction with the dimeric RBCC domain of KAP1. Finally, mass spectrometry profiling of domain-dependent interactions (based on differential abundance relative to changes due to chemically induced DNA damage) suggests SMARCAD1 may be involved in p53 transcriptional regulation through interactions maintained with CUE1 prior to DNA damage, whereas the SIM domain selectively targets protein interactions upon DNA damage that simultaneously activate p53 transcriptional control and recruit SMARCAD1 to DNA damage repair pathways.

La línia germinal conté el passat i el futur d’una espècie, en la qual la informació genètica parental es recombina mitjançant la meiosi i és transmesa a la descendència. Així, entendre com el genoma s’organitza i es regula en l’espai nuclear durant la formació de cèl·lules germinals és essencial per comprendre les bases de la fertilitat i el seu impacte en la diversitat genètica. En cèl·lules somàtiques, el genoma s’organitza en territoris cromosòmics que estan formats per compartiments de cromatina plegats en dominis d’associació topològica (TADs) i bucles d’ADN. No obstant això, es coneix poc sobre l’organització de l’genoma en la línia germinal i com les reorganitzacions cromosòmiques poden modular-la. En aquest context, aquesta tesi té com a objectius: (i) entendre l’organització tridimensional de l’genoma durant l’espermatogènesi de ratolí i la seva relació amb la funció gènica i la localització de proteïnes aïllants (CTCF i cohesines), (ii) investigar les implicacions de les fusions Robertsonianes (Rb) en el plegament del genoma i la recombinació, i (iii) caracteritzar la variabilitat de PRDM9 en poblacions naturals de ratolins Rb, incloent el sistema Rb de Madeira i el de Barcelona (BRbS). Per això, hem combinat anàlisis citològics amb tecnologies de seqüenciació massiva i desenvolupat un protocol de citometria per obtenir poblacions cel·lulars germinals enriquides, incloent espermatogonis, espermatòcits primaris, espermàtides rodones i espermatozoides. Els nostres resultats revelen que l’estructura d’ordre superior del genoma és extremadament dinàmica durant l’espermatogènesi, on els espermatogonis presenten compartiments i TADs que desapareixen durant la meiosi i es restableixen posteriorment en cèl·lules post-meiòtiques. A més, hi ha una correlació entre la transcripció i els compartiments A, amb gens actius específics de tipus cel·lular relacionats amb la progressió de l’espermatogènesi, la fecundació i el desenvolupament embrionari. Addicionalment, hem trobat una correlació entre la localització de cohesines i transcripció activa tant en cèl·lules meiòtiques com post-meiòtiques, suggerint que les cohesines regulen la transcripció. Les fusions Rb reorganitzen la localització espacial dels cromosomes i en espermatòcits primaris, augmenten les interaccions heteròlogues, promovent nous entorns de regulació. En espermàtides rodones, les fusions afegeixen restriccions mecàniques que redueixen les interaccions inter-cromosòmiques. A més, les fusions Rb afecten tant a el nombre com a la distribució cromosòmica dels punts de recombinació, especialment en els metacéntrics fusionats en homozigosi, mentre que la presència de metacéntrics heterozigots asinapsats indueix una heterocromatinizació de la vesícula sexual. La reducció de la recombinació també es detecta en les anàlisis de desequilibri de lligament basats en SNPs, detectant alta divergència genètica en poblacions Rb comparades amb estàndard. Addicionalment, hem caracteritzat una gran variabilitat de PRDM9, sent especialment alta al sistema Rb insular de Madeira en comparació amb el sistema continental BRbS. Aquestes diferències es poden atribuir a la combinació de diferents factors: (i) la història evolutiva de cada sistema Rb, (ii) la prevalença de fusions Rb afectant la diversitat genètica, i en menor grau, (iii) restriccions funcionals meiòtiques (per exemple, asimetria en els hotspot de recombinació). En conjunt, aquesta tesi mostra que la cromatina pateix una remodelació profunda durant l’espermatogènesi específica de tipus cel·lular, en la qual l’activitat transcripcional es correlaciona amb l’estat de la cromatina i la localització de les cohesines. Addicionalment, les fusions Rb alteren l’organització del genoma en la línia germinal, afectant a la recombinació meiòtica i la diversitat genètica.
La línea germinal contiene el pasado y el futuro de una especie, en la que la información genética parental se recombina mediante la meiosis y es transmitida a la descendencia. Así, entender como el genoma se organiza y se regula en el espacio nuclear durante la formación de células germinales es esencial para comprender las bases de la fertilidad y su impacto en la diversidad genética. En células somáticas, el genoma se organiza en territorios cromosómicos que están formados por compartimentos de cromatina plegados en dominios de asociación topológica (TADs) y bucles de ADN. Sin embargo, se conoce poco acerca de la organización del genoma en la línea germinal y como las reorganizaciones cromosómicas pueden modularla. En este contexto, esta tesis tiene como objetivos: (i) entender la organización tridimensional del genoma durante la espermatogénesis de ratón y su relación con la función génica y la localización de proteínas aislantes (CTCF y cohesinas), (ii) investigar las implicaciones de las fusiones Robertsonianas (Rb) en el plegamiento del genoma y la recombinación, y (iii) caracterizar la variabilidad de PRDM9 en poblaciones naturales de ratones Rb, incluyendo el sistema Rb de Madeira y el de Barcelona (BRbS). Por ello, hemos combinado análisis citológicos con tecnologías de secuenciación masiva y desarrollado un protocolo de citometría para obtener poblaciones celulares germinales enriquecidas, incluyendo espermatogonias, espermatocitos primarios, espermátidas redondas y espermatozoides. Nuestros resultados revelan que la estructura de orden superior del genoma es extremadamente dinámica durante la espermatogénesis, donde las espermatogonias presentan compartimentos y TADs que desaparecen durante la meiosis y se reestablecen posteriormente en células post-meióticas. Además, hay una correlación entre la transcripción y los compartimentos A, con genes activos específicos de tipo celular relacionados con la progresión de la espermatogénesis, la fecundación y el desarrollo embrionario. Adicionalmente, hemos hallado una correlación entre la localización de cohesinas y transcripción activa tanto en células meióticas como post-meióticas, sugiriendo que las cohesinas regulan la transcripción. Las fusiones Rb reorganizan la localización espacial de los cromosomas y en espermatocitos primarios, aumentan las interacciones heterólogas, promoviendo nuevos entornos de regulación. En espermátidas redondas, las fusiones añaden restricciones mecánicas que reducen las interacciones inter-cromosómicas. Además, las fusiones Rb afectan tanto al número como a la distribución cromosómica de los puntos de recombinación, especialmente en los metacéntricos fusionados en homocigosis, mientras que la presencia de metacéntricos heterocigotos asinapsados induce una heterocromatinización de la vesícula sexual. La reducción de la recombinación también se detecta en los análisis de desequilibrio de ligamiento basados en SNPs, detectando alta divergencia genética en poblaciones Rb comparadas con estándar. Adicionalmente, hemos caracterizado una gran variabilidad de PRDM9, siendo especialmente alta en el sistema Rb insular de Madeira en comparación con el sistema continental BRbS. Tales diferencias se pueden atribuir a la combinación de distintos factores: (i) la historia evolutiva de cada sistema Rb, (ii) la prevalencia de fusiones Rb afectando a la diversidad genética, y en menor grado, (iii) restricciones funcionales meióticas (por ejemplo, asimetría en los hotspot de recombinación). En conjunto, esta tesis muestra que la cromatina sufre una remodelación profunda durante la espermatogénesis específica del tipo celular, en la que la actividad transcripcional se correlaciona con el estado de la cromatina y la localización de las cohesinas. Adicionalmente, las fusiones Rb alteran la organización del genoma en la línea germinal, afectando a la recombinación meiótica y a la diversidad genética.
The germline holds the past and the future of a species, as parental genetic information is recombined through meiosis and transmitted to the offspring. Thus, understanding how the genome is organized and regulated in the nuclear space during the formation of germ cells is essential to comprehend the bases of fertility and its impact on genetic diversity. In the last twenty years, many studies have shown that in somatic cells, the genome is organized in chromosome territories which are formed by chromatin compartments folded into topological associated domains (TADs) and DNA loops. However, little is known about how the genome is organized in the germline and how chromosomal reorganizations modulate genome architecture. In this context, this thesis aims to: (i) understand the three-dimensional organization of the genome during mouse spermatogenesis and its interplay with gene function and occupancy of insulator proteins (CTCF and cohesins), (ii) investigate the implications of Robertsonian (Rb) fusions in genome folding and meiotic recombination, and (iii) characterize the variability of PRDM9 in natural house mouse populations with Rb fusions: the Madeira Rb system and the Barcelona Rb system (BRbS). We combined cytological analysis with next generation sequencing technologies, and we developed an efficient cell sorting protocol to obtain enriched germ cell fractions including spermatogonia, primary spermatocytes at early and late prophase I, round spermatids and sperm. Our results revealed that the higher-order structure of the genome is extremely dynamic during spermatogenesis, where spermatogonia presents somatic-like compartments and TADs, that disappear during meiosis to be re-established later on in post-meiotic cells. Moreover, transcription correlates with A compartments throughout spermatogenesis, with cell-specific active genes involved in spermatogenesis progression, fertilization and embryonic development. In addition, we found a correlation between cohesin occupancy and active transcription in both meiotic and post-meiotic cells, suggesting a transcription-regulating role of meiotic cohesins. Although germ cells with Rb fusions presented the main features of genome architecture, Rb fusions reorganize the spatial chromosome occupancy. In primary spermatocytes, Rb fusions increase heterologous interactions, promoting the formation of novel regulatory environments. In round spermatids, Rb fusions reduce inter-chromosomal interactions as a result of mechanistic constrains. The cytological data shows that the increase in heterologous interactions is concomitant with the presence of asynapsed heterozygous metacentrics, which induce the full heterochromatinization of the sex body. Furthermore, Rb fusions affect the number and chromosomal distribution of crossovers in primary spermatocytes, especially in the case of fused metacentrics in homozygosis. The reduction in recombination was also observed in the analysis of linkage disequilibrium based on SNP genotyping, which translated into high levels of genetic divergence in Rb populations when compared to standard mice. In addition, our characterization of PRDM9 variability detected an unprecedented variability in natural house mouse populations, being especially high in the insular Madeira Rb system when compared to the continental BRbS. Such differences could be attributed to the combination of different factors: (i) the evolutionary history of each Rb system, (ii) the prevalence of Rb fusions affecting genetic diversity, and to a lesser extent (iii) meiotic functional constrains (i.e., recombination hotspot asymmetry). Taken together, this thesis shows that chromatin undergoes profound remodeling during spermatogenesis in a cell-specific way, where transcriptional activity correlates with the chromatin state and cohesin occupancy. In addition, Rb fusions alter genome organization in the germline, having an impact on meiotic recombination and genetic diversity.