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The cell's nucleus contains DNA in the form of chromosomes, which are the hereditary content of the organism. The proper transmission of DNA from one generation to the next is critical. Along with this crucial process, cells will also need to transcribe the DNA, silence certain genes (or whole chromosomes) during development and regulate other chromosome dynamics that are still being identified. The molecular components responsible for these processes are starting to be identified. However, the regulation of these components and how they interact with each other is not well understood.The condensin complex is one component that has been identified to play a role in chromosome dynamics. Activity of the complex has been studied in vitro but in vivo activity has been difficult to measure. Similarly, understanding the regulation of the complex has been difficult given the lack of assays and that the complex is essential for cell survival. In this dissertation, I have identified and characterized a regulator of condensin II function using Drosophila melanogaster. The chromo-domain protein Mrg15 interacts with condensin II to inhibit homologous chromosome interactions.Lastly, I look at the role of condensin II in female meiosis. Meiosis involves pairing and subsequent segregation of homologous chromosomes. The process of the initial pairing has remained elusive but specialized structures have evolved to maintain this pairing. Condensin II can antagonize a basal level of homologous pairing and also removes the specialized structure that pair meiotic chromosomes. This dissertation will add to the growing knowledge of the regulation of the condensin II complex and its role in female meiosis.

One of the most important outstanding questions in biology involves the precise spatial and temporal regulation of gene activity, which enables different cell types to express the specific set of genes required for their function and is therefore a cornerstone for complex biological life. Cis-regulatory elements, such as gene promoters and enhancers, play a key role in controlling gene activity. These elements physically interact with the genes they regulate within structural chromatin domains. The organisation of chromosomes into these domains is critical for specific regulation of gene expression and disruption of these structures underlies common human disease. However, it is not understood how chromatin domains form, how interactions between the cis-regulatory elements contained within them are established, or how such interactions influence gene expression. The major hurdles in addressing these questions are to determine chromatin structures with high resolution and sensitivity and to examine their dynamic nature within single cells. To overcome these, I have developed Tri-C, a new chromosome conformation capture assay that can analyse concurrent chromatin interactions at single alleles at high resolution. By combining Tri-C with conventional chromosome conformation capture techniques, I have analysed the three-dimensional regulatory landscapes of the well-characterised murine globin loci at unprecedented depth. Additionally, to examine the roles of cis-regulatory elements in establishing chromatin architecture, I have analysed how engineered deletions in enhancers and CTCF-binding elements in the murine alpha-globin locus disrupt its chromatin landscape. These analyses reveal that the chromatin domains containing the globin genes represent compartmentalised structures, which are delimited by CTCF boundaries. The heterogeneity of interactions in these domains between individual cells is indicative for a dynamic process of loop extrusion underlying their formation. Within chromatin domains, preferential structures are formed in which multiple enhancers and promoters interact simultaneously. These complexes provide a structural basis for understanding how multiple cis-regulatory elements cooperate to establish robust regulation of gene expression. Importantly, these complex, tissue-specific structures, cannot be explained by loop extrusion alone and indicate other, independent mechanisms contributing to chromosome architecture, likely involving interactions mediated by multi-protein complexes. Together, these analyses show that the current view of genome organisation, in which chromosomes are organised by stable loops and domains that self-assemble into hierarchical structures, is not correct. Rather, chromatin architecture reflects a complex interplay between distinct molecular mechanisms contributing to the formation of regulatory landscapes that facilitate precise, robust control of gene expression.

Made available in DSpace on 2018-08-01T22:57:27Z (GMT). No. of bitstreams: 1 tese_10051_Dissertação Final Lucas Mesquita Barreto.pdf: 1926946 bytes, checksum: 21145320a1a7d898eb87b86e37b7dd01 (MD5) Previous issue date: 2016-07-18 / Previous cytogenetic studies in Dorstenia mention that the species may have 24 to 72 chromosomes, and suggested a conserved chromosome number 2n = 32 for the Neotropic species. However, some information reported in the literature are dubious or insufficient to assess the potential of cytogenetic data to the better understand of systematics and evolution issues within this genus. Here, eight species of Neotropical Dorstenia had their karyotypes characterized, and the nuclear DNA content measured. Dorstenia bahiensis, D. cayapia, D. grazielae, D. hirta and D. turnerifolia had their karyotypes characterized and the DNA nuclear content measured for the first time. Morphological plant characters and morphometric data were submitted to cluster analysis, followed by a test of group sharpness, and ordination analysis, aiming to support the discussion about the potential of cytogenetic data to infrageneric systematic of Dorstenia. The species showed chromosome number of 2n = 32, varying in chromosomes morphology. The karyotypes least asymmetric were observed in Dorstenia elata, and the more asymmetric were registered in D. bahiensis and D. bonijesu. The 2C value ranged from 3.21 picograms (pg) D. bahiensis to 5.47 pg in D. arifolia. Morphologically similar species, like D. hirta and D. turnerifolia, grouped together based on morphometric data. The sharp groups based on morphometric data correspond to species circumscribed under the sections Dorstenia, Lecania and Emygodia, previously established based on the plant morphology. Our results supports that the chromosome number 2n = 32 is possible conserved in the Neotropical species of Dorstenia, and indicate the potential of cytogenetic data to the systematics of this genus.

In order to equally segregate their genetic material into daughter cells during mitosis, it is essential that chromosomes undergo major restructuring to facilitate compaction. However, the process of transforming diffuse, entangled interphase chromatin into discrete, highly organised chromosomal structures is extremely complex, and currently not completely understood. The complexes involved in chromatin compaction and sister chromatid decatenation in preparation for mitosis include condensins I and II. Mutations in condensin subunits have been identified in human tumours, reflecting the importance of accurate cell division in the prevention of aneuploidy and tumour formation. Most mutations described in TCGA (The Cancer Genome Atlas) and COSMIC (Catalogue of Somatic Mutations in Cancer) are missense, and therefore likely to only partially affect condensin function. Most functional genetic studies of condensin, however, have used loss of function systems, which typically cause severe chromosome segregation defects and cell death. Mice carrying global hypomorphic mutations within the kleisin subunit of the condensin II complex develop T cell lymphomas. The Caph2nes/nes mouse model is therefore a good system for understanding how condensin dysfunction can influence tumourigenesis. However, little is known about which cellular processes are affected in mutant cells before transformation. I therefore set out to use the Caph2nes/nes mouse model to study the consequences of the condensin II deficiency on cell cycle regulation in several different hematopoietic lineages. The Caph2nes/nes mice are viable and fertile, with no obvious abnormalities other than the thymus, which is drastically reduced in size. Previous studies reported greater than a hundred-fold reduction in the number of CD4+ CD8+ thymocytes. I set out to understand why the alteration of a ubiquitously expressed protein which functions in a fundamental cellular process would result in such a cell-type specific block in development. To achieve this, I investigated the possibility that condensin II is involved in interphase processes as well as in mitosis. In addition, I studied the aspects of T cell development that may make this lineage particularly vulnerable to condensin II deficiency. Finally, I carried out a preliminary investigation into the biochemical properties of the condensin complexes. During my PhD., I found strong evidence to suggest that the Caph2nes/nes T cell-specific phenotype arises due to abnormal cell division. However, I was unable to find any evidence to support the hypothesis that the phenotype is a consequence of abnormal interphase processes. Upon systematic analysis of several stages of hematopoietic differentiation, I found that at a specific stage of T cell development, the mutation results in an increased proportion of cells with abnormal ploidy, followed by a drastic reduction in cell numbers. Erythroid cells revealed a similar increase in the frequency of hyperdiploid cells, but no reduction in cell numbers. B cells and hematopoietic precursors did not reveal an increase in hyperdiploidy, or a reduction in cell numbers in wildtype relative to mutant. Subsequently, I found preliminary evidence to suggest that the T cell-specificity may be due to more rapid progression of CD4+ CD8+ T cells from S phase to M phase, relative to other hematopoietic stages. Finally, a preliminary investigation into the biochemical properties of the condensin complex revealed apparent imbalances in the expression of condensin subunits in T, B and erythroid cells. The sedimentation profile of CAP-H2 from whole-thymus extract did not exclude the possibility that condensin subunits might be forming heavier-weight complexes with non-SMC proteins. Further work must be carried out to determine whether this sedimentation pattern is unique to T cells.

Nuestro grupo ha estudiado la estructura de la cromatina de núcleos de eritrocitos de pollo (Bartolomé et al., 1994; Bartolomé et al., 1995; Bermúdez et al., 1998). La consecuencia de estos estudios ha sido la elaboración de un modelo para el plegamiento de la fibra de cromatina con una elevada concentración local del DNA (Daban y Bermúdez, 1998; Daban, 2000). Sin embargo, el nivel máximo de condensación en la cromatina, se encuentra en el interior de los cromosomas metafásicos. Aunque la bibliografía ha planteado diferentes modelos para el plegamiento de la cromatina en el interior de éstos, existe un conocimiento muy escaso acerca de la estructura molecular de la cromatina en los cromosomas condensados.Se ha realizado un estudio exhaustivo de microscopía electrónica de transmisión sobre la estructura de los cromosomas metafásicos de células HeLa. Se han estudiado un total de 4410 micrografías de cromosomas metafásicos, que en su mayor parte han sido tratados con diversos medios parcialmente desnaturalizantes, para poder analizar su estructura interna.Morfológicamente, los cromosomas estudiados en este trabajo pueden agruparse en tres tipos diferentes: compactos, granulados y fibrilados. La morfología más abundante es la compacta y se observa en presencia de cationes monovalentes y divalentes a concentración similar a la presente en la cromatina metafásica (Mg2+ 1.7-40 mM). Estos cromosomas tienen las cromátidas muy densas y en sus bordes se aprecian una serie de estructuras planas superpuestas. En condiciones de menor concentración de cationes (Mg2+£ 1.7 mM), la morfología dominante es la granular. Estos cromosomas están compuestos principalmente por gran cantidad de cuerpos circulares de 30-40 nm de diámetro. Únicamente en condiciones de fuerza iónica extremadamente baja podemos encontrar la morfología fibrilar, la cual se caracteriza por la abundancia de fibras de 30-40 nm.Los resultados obtenidos con cromosomas parcialmente desnaturalizados nos permiten concluir que existen tres elementos estructurales en el interior de los cromosomas metafásicos: la fibra, el gránulo y la placa.Las fibras gruesas con diámetros que oscilan entre los 100 y los 500 nm son el resultado de la deformación plástica de las cromátidas durante los diferentes procesos de preparación de las muestras. En función de las condiciones iónicas del medio las fibras gruesas muestran gránulos o placas en su interior. Las fibras delgadas están formadas por una sucesión de cuerpos de 30-40 nm de diámetro unidos irregularmente mediante interacciones cabeza-cola. Las fibras delgadas se observan dominantemente en condiciones de concentración salina extremadamente baja.Los gránulos son unos cuerpos circulares compactos de unos 30-40 nm de diámetro. Estos cuerpos compactos descritos previamente por nuestro grupo y se interpretaron como una forma de plegamiento solenoidal de la fibra de 30 nm (Daban y Bermúdez, 1998). Se encuentran presentes en todas las condiciones estudiadas en este trabajo, siendo especialmente abundantes en presencia de iones divalentes a concentración baja y en muestras tratadas con nucleasa micrococal. La placa es un elemento estructural característico de los cromosomas cuando éstos se encuentran en su forma más compacta, en presencia de concentraciones elevadas de cationes divalentes. Esta estructura no había sido descrita previamente por otros laboratorios. Es una estructura cromatínica de gran regularidad y con una superficie muy lisa. Hemos estimado la altura de estas placas a través de muestras sombreadas unidireccionalemente con platino. El promedio de los valores obtenidos es de 6.7 ± 1.4 nm.En conjunto los resultados obtenidos en esta tesis permiten sugerir que el componente principal de la cromatina en los cromosomas metafásicos es el gránulo de 30-40 nm. Dependiendo de las condiciones iónicas, este elemento estructural fundamental se agrega a través de uniones cabeza-cola para formar fibras (fuerza iónica muy baja), o bien se agrega mediante interacciones laterales para formar placas (condiciones salinas próximas a las de la cromatina metafásica). / Our group has studied the chromatin structure in the chicken erythrocyte nuclei (Bartolome et al., 1994; Bartolomé et al., 1995; Bermúdez et al., 1998). The consequences of this studies has been the elaboration of a folding model of the chromatin fiber with a high local concentration of DNA. However, the maximum level of chromatin condensation, is found in the metaphase chromosomes. Although the bibliography has proposed different models to explain the chromatin folding inside the chromosomes, there is a low knowledge about the molecular structure of chromatin in the condensed chromosomes. In this thesis, we have carried out an exhaustive electron microscopy study about the HeLa cells metaphase chromosomes. We have studied a large number of chromosome electron micrographs (4410). Chromosomes were partially denaturated under a wide variety of conditions in order to observe some chromatin structural element inside them.Our studies indicate that chromosomes can adopt three global structural forms in function of the ionic conditions: compact, granular and fibrillar.The compact form is the most frequent and we can observe it in the presence of monovalent and divalent cations in similar concentrations than the ones found in metaphase chromatin (Mg2+ 1.7-40 mM). These chromosomes have highly condensed chromatids and we can appreciate overlapped chromatin plates around the chromosomes edges. When the chromosomes are incubated with solutions containing lower cations concentration (Mg 2+£ 1.7 mM) they become granular. The granular structures seen inside these chromosomes show a diameter of about 35 nm. Fibrillar chromosomes are observed only at very low ionic strength. The fibers seen emanating from the chromatids have a diameter of 30-40 nm.Our results obtained from partially denaturated chromosomes show that there are three structural elements inside the metaphase chromosomes: the fiber, the 30-40 nm chromatin granule and the plate.The largest fibers with a diameter of 100-400 nm, presumably are produced by mechanical deformation of chromosomes during the preparation processes. Depending of the ionic conditions these fibrillar structures are composed by plates or granules. The thinnest fibers are formed by face to face association of the 30-40 nm chromatin granules. These kind of fibers are usually found only at very low ionic strength.The chromatin granules are compact bodies with 35 nm of diameter. These compact bodies were previously described in our laboratory and were modeled as compact solenoids of nucleosomes forming (Daban and Bermúdez, 1998). They are usually seen at low divalent cation concentrations and in chromosome samples treated with micrococal nuclease.The plate is the most frequent structural element when the chromosomes are in their compact form (high ionic strength, similar to physiological conditions). This element has not been described by any group. It is a chromatin element with a regular structure and very smooth surface. We have estimated the height of the steps between layers in unidirectional shadowing experiments. The value obtained is 6.7 ± 1.4 nm.Our results suggest that the fundamental component inside the metaphase is the 30-40 nm chromatin granules. Depending of the ionic conditions, this basic structural element forms fibers through face to face interactions (very low ionic strength) or form plates through side to side interactions (high ionic strength similar to metaphase chromatin).

Yes / Abstract The budding yeast genome contains regions where meiotic recombination initiates more frequently than in others. This pattern parallels enrichment for the meiotic chromosome axis proteins Hop1 and Red1. These proteins are important for Spo11-catalyzed double strand break formation; their contribution to crossover recombination remains undefined. Using the sequence-specific VMA1-derived endonuclease (VDE) to initiate recombination in meiosis, we show that chromosome structure influences the choice of proteins that resolve recombination intermediates to form crossovers. At a Hop1-enriched locus, most VDE-initiated crossovers, like most Spo11-initiated crossovers, required the meiosis-specific MutLγ resolvase. In contrast, at a locus with lower Hop1 occupancy, most VDE-initiated crossovers were MutLγ-independent. In pch2 mutants, the two loci displayed similar Hop1 occupancy levels, and VDE-induced crossovers were similarly MutLγ-dependent. We suggest that meiotic and mitotic recombination pathways coexist within meiotic cells, and that features of meiotic chromosome structure determine whether one or the other predominates in different regions.

Im Rahmen dieser Arbeit wurde ein neuartiges 3D Fluoreszenz Mikroskop, OMX gennant, entworfen und gebaut. Ein umfassender Design-Neuansatz erlaubt es den neuen Anforderungen der aktuellen Biologie bezüglich erhöhter Auflösung in Zeit und Raum Rechnung zu tragen. Mit Ausnahme vom Auflegen des Objektträgers sind alle Aspekte des Mikroskops Computer-gesteuert. Einen Großteil der Software floß in ein neues, eigenständiges Open-Source Projekt ein. Es erlaubt die Verarbeitung sehr großer, mehrdimensionaler Bilddaten, und die Entwicklung neuartiger Algorithmen in einer flexiblen Oberfläche. OMX hat zwei Betriebsarten: Im ersten Modus können bis zu 100 Bilder pro Sekunde mit optischer Auflösung in mehreren Farbkanälen aufgenommen werden. Dies entspricht etwa 10 3D Bildern pro Sekunde. Im zweiten Modus können mit der Structured Illumination Mikroskopie fixierte Präparate mit eine Auflösung unterhalb des Abbe-Limits untersucht werden. Im zweiten Teil dieser Arbeit, stelle ich erste Forschungsergebnisse von OMX vor. Drosophila X-chromosomen markiert mit GFP-MSL3 wurden in situ im sub-Sekunden Bereich beobachtet. Mit Hilfe neuentwickelter Algorithmen konnte ich die Chromosomendynamik analysieren. Das Falten und Entfalten von Bereichen eines Chromosoms wurde als Funktion der Zeit darstellen. Chromosomenstrukturen wurden mit Hilfe der SIM an fixierten primären embryonalen Kulturen untersucht. Unterstrukturen von 100-200nm sind erkennenbar. Viele Bilder zeigen eine DNA-reiche Hülle die einen DNA-armen Chromosomenkern umgibt. Ausserdem habe ich polytäne Chromosomen mit SIM aufgenommen. Bandstrukturen zeigen sich mit deutlich erhöhter Detailklarheit, und Längsfasern sind sichtbar, die ansonsten nur vom Elektronenmikroskop her bekannt sind. Als weiteres Beispiel der verbesserten Auflösungsfähigkeit habe ich Kernporen untersucht. In mit DAPI gefärbten Mauszellen zeigen diese sich als dunkle Punkte mit einer Größe von etwa 120nm. / A novel fluorescence 3D wide-field light microscope called OMX, was designed and implemented. The novel design addresses improved speed and resolution requirements of current biology research. After designing and building the microscope body I designed and implemented the needed computer software for the eight computers required to operate OMX. Over the course of the project I also designed and implemented a new Open-Source software platform for algorithm development and image analysis. It focuses on very large multi-dimensional image data handling and visualization in general. OMX can operate in two modes: In the first mode a live specimen can be observed at optical resolution (approx. 250nm) at speeds up to 100 sections per second simultaneously in multiple wavelength channels. This equals about 10 3D images per second. The second mode is for observing fixed preparations at resolutions below the Abbe diffraction limit using Structured Illumination Microscopy (SIM). This produces 3D volumetric image data with lateral resolution near 100nm and axial resolution of about 200nm. In the second part of this thesis I show first results achieved using the OMX microscope. Chromosome dynamics was analyzed using various newly developed image analysis algorithms. Sub-second motion was observed for in situ Drosophila X-chromosomes tagged with GFP-MSL3. Parts of the chromosome can be traced within the nucleus and time-series data shows its folding and unfolding as a function of time. Chromosome structure was imaged using SIM on formaldehyde fixed primary embryonic cultures stained with DAPI. Features of the sub-structure with sizes around 100-200nm were apparent. Many chromosomes show an outer layer along the chromatin axis appearing persistently denser in DNA than the central core. Polytene chromosomes were imaged using SIM. Band patterns are visible in much more detail than in conventional deconvolution microscopy and longitudinal fibers known only from electron microscopy were visible.

The intimate relationship between the higher-order chromatin organisation and the regulation of gene expression is increasingly attracting attention in the scientific community. Thanks to high-resolution microscopy, genome-wide molecular biology tools (3C, ChIP-on-chip), and bioinformatics, detailed structures of chromatin loops, territories, and nuclear domains are gradually emerging. However, to fully reveal a comprehensive map of nuclear organisation, some fundamental questions remain to be answered in order to fit all the pieces of the jigsaw together. The underlying mechanisms, precisely organising the interaction of the different parts of chromatin need to be understood. Previous work in our lab hypothesised and verified the “transcription factory” model for the organisation of mammalian genomes. It is widely assumed that active polymerases track along their templates as they make RNA. However, after allowing engaged polymerases to extend their transcripts in tagged precursors (e.g., Br-U or Br-UTP), and immunolabelling the now-tagged nascent RNA, active transcription units are found to be clustered in nuclei, in small and numerous sites we call “transcription factories”. Previous work suggested the transcription machinery acts both as an enzyme as well as a molecular tie that maintains chromatin loops, and the different classes of polymerases are concentrated in their own dedicated factories. This thesis aims to further characterise transcription factories. Different genes are transcribed by different classes of RNA polymerase (i.e., I, II, or III), and the resulting transcripts are processed differently (e.g., some are capped, others spliced). Do factories specialise in transcribing particular subsets of genes? This thesis developed a method using replicating minichromosomes as probes to examine whether transcription occurs in factories, and whether factories specialise in transcribing particular sets of genes. Plasmids encoding the SV40 origin of replication are transfected into COS-7 cells, where they are assembled into minichromosomes. Using RNA fluorescence in situ hybridisation (FISH), sites where minichromosomes are transcribed are visualised as discrete foci, which specialise in transcribing different groups of genes. Polymerases I, II, and III units have their own dedicated factories, and different polymerase II promoters and the presence of an intron determine the nuclear location of transcription. Using chromosome conformation capture (3C), minichromosomes with similar promoters are found in close proximity. They are also found close to similar endogenous promoters and so are likely to share factories with them. In the second part of this thesis, I used RNA FISH to confirm results obtained by tiling microarrays. Addition of tumour necrosis factor alpha (TNF alpha) to human umbilical vein endothelial cells induces an inflammatory response and the transcription of a selected sub-set of genes. My collaborators used tiling arrays to demonstrate a wave of transcription that swept along selected long genes on stimulation. RNA FISH confirmed these results, and that long introns are co-transcriptionally spliced. Results are consistent with one polymerase being engaged on an allele at any time, and with a major checkpoint that regulates polymerase escape from the first few thousand nucleotides into the long gene.