Tip60 is a 60 kDa nuclear protein which exists in three isoforms, belongs to the MYST/HAT family of proteins and was discovered after its interaction with the Human HIV-1 Tat. As a nuclear protein, Tip60 can act as a coactivator or repressor. To understand the HAT action of Tip60, two possible catalytic models exist; the ping-pong and the ternary complex formation models. In correlation with the exploration of HAT catalytic action, mutations of a Cys to Ala and a Glu to Gln on Esa1 (yeast homolog of Tip60 and MYST/HAT prototype), was reported to show wild type-like and decreased acetylating properties, respectively. In this work, Tip60 HAT action was explored. In Tip60, the Cys in the active site is important for acetylation of the H4(1-20) substrate and the Glu showed semi loss in acetylating the H4(1-20) peptide substrate. These data highlight a unique mechanism of Tip60 catalysis.

No description available.

No description available.

La chromatine est plus qu’un système d’empaquetage de l’ADN ; elle est le support de toutes les réactions liées à l’ADN dans le noyau des cellules eucaryotes et participe au contrôle de l’accès de l’ARN polymérase II (ARNPolII) à l’ADN. Responsable de la transcription de tous les ARNm des cellules eucaryotes, l’ARNPolII doit, suivant son recrutement aux promoteurs des gènes, transcrire l’ADN en traversant la matrice chromatinienne. Grâce au domaine C-terminal (CTD) de sa sous-unité Rpb1, elle coordonne la maturation de l’ARNm en cours de synthèse ainsi que les modifications de la chromatine, concomitantes à la transcription. Cette thèse s’intéresse à deux aspects de la transcription : la matrice, avec la localisation de la variante d’histone H2A.Z, et la machinerie de transcription avec le cycle de phosphorylation du CTD de l’ARNPolII. Suivant l’introduction, le chapitre 2 de cette thèse constitue un protocole détaillé et annoté de la technique de ChIP-chip, chez la levure Saccharomyces cerevisiae. Cette technique phare dans l’étude in vivo des phénomènes liés à l’ADN a grandement facilité l’étude du rôle de la chromatine dans les phénomènes nucléaires, en permettant de localiser sur le génome les marques et les variantes d’histones. Ce chapitre souligne l’importance de contrôles adéquats, spécifiques à l’étude de la chromatine. Au chapitre 3, grâce à la méthode de ChIP-chip, la variante d’histone H2A.Z est cartographiée au génome de la levure Saccharomyces cerevisiae avec une résolution d’environ 300 paires de bases. Nos résultats montrent que H2A.Z orne un à deux nucléosomes au promoteur de la majorité des gènes. L’enrichissement de H2A.Z est anticorrélé à la transcription et nos résultats suggèrent qu’elle prépare la chromatine pour l’activation des gènes. De plus H2A.Z semble réguler la localisation des nucléosomes. Le chapitre suivant s’intéresse à la transcription sous l’angle de la machinerie de transcription en se focalisant sur le cycle de phosphorylation de l’ARN polymérase II. Le domaine C-terminal de sa plus large sous-unité est formé de répétitions d’un heptapeptide YSPTSPS dont les résidus peuvent être modifiés au cours de la transcription. Cette étude localise les marques de phosphorylation des trois résidus sérine de manière systématique dans des souches mutantes des kinases et phosphatases. Nos travaux confirment le profil universel des marques de phosphorylations aux gènes transcrits. Appuyés par des essais in vitro, ils révèlent l’interaction complexe des enzymes impliqués dans la phosphorylation, et identifient Ssu72 comme la phosphatase de la sérine 7. Cet article appuie également la notion de « variantes » des marques de phosphorylation bien que leur étude spécifique s’avère encore difficile. La discussion fait le point sur les travaux qui ont suivi ces articles, et sur les expériences excitantes en cours dans notre laboratoire. / Chromatin is more than just the eucaryotic DNA packaging system; it is the substrate of all reactions involving DNA in eukaryotic cells and actively regulates RNA Polymerase II (RNAPolII) access to DNA. Responsible for all mRNA transcription in eucaryotes, the RNAPolII must, following its recruitment to the pre-initiation complex, overcome the chromatin barrier in order to transcribe genes. The RNAPolII CTD allows for the co-transcriptional coordination of mRNA maturation and chromatin modifications. The work covered in this thesis addresses two aspects of transcription: the chromatin substrate, with the localization of H2A variant, H2A.Z, and the transcription complex with the phosphorylation cycle of the RNAPolII CTD. Following the introduction, chapter 2 constitutes a detailed and annotated Saccharomyces cerevisiae ChIP-chip protocol, from the culture to the hybridization of the array, with an emphasis on the proper controls required for chromatin study. This technique, extremely powerful for the in vivo study of all DNA transactions, leads to a better understanding of chromatin function in nuclear phenomena, thanks to the localization of histone variants and modifications. The third chapter maps the H2A.Z variant across the yeast genome at ~300 base pairs resolution using ChIP-chip. Our data shows that H2A.Z is incorporated into one or two promoter-bound nucleosomes at the majority of genes. H2A.Z enrichment is anticorrelated with transcription, and the results suggest that it configures chromatin structure to poise genes for transcriptional activation. Furthermore, we have shown that H2A.Z can regulate nucleosome positioning. The next chapter focuses on the transcription machinery and, more precisely, on the phosphorylation cycle of RNAPolII. The CTD contains repetitions of a heptapeptide (YSPTSPS) on which all serines are differentially phosphorylated along genes in a prescribed pattern during the transcription cycle. Here, we systematically profiled the location of the RNAPII phospho-isoforms in wild-type cells and mutants for most CTD modifying enzymes. The results provide evidence for a uniform CTD cycle across genes. Together with results from in vitro assays, these data reveal a complex interplay between the modifying enzymes, identify Ssu72 as the Ser7 phosphatase and show that proline isomerization is a key regulator of CTD dephosphorylation at the end of genes. Moreover, it reinforces the notion of variants of the phosphorylation marks, even though the exact nature of the variant is still difficult to identify. The discussion introduces the studies that followed this work, including new projects conceived in our lab.

The replication of chromosomes can be challenged by endogenous and environmental factors, interfering with the progression of replication forks. Therefore, cells have to coordinate DNA synthesis with mechanisms ensuring the stability and the recovery of halted forks. Homologous recombination (HR) is a universal mechanism that supports DNA repair and the robustness of DNA replication. Nonetheless, mechanisms regulating HR pathways, such as ectopic versus allelic recombination, remain poorly understood. Another essential pathway for genome stability is the wrapping of newly replicated DNA around nucleosomes, leading to the constitution of a chromatin fibre, which allows the structural organization of the genetic material. In Saccharomyces cerevisiae, deficiencies in chromatin assembly pathways lead to replication forks instability and consequent increase in the rate of HR. Histone chaperones play a crucial role during chromatin assembly, thus I decided to focus on the H3-H4 histone chaperone Chromatin Assembly Factor 1 (CAF-1), to study its role in HR processes in Schizosaccharomyces pombe. Indeed, HR includes a DNA synthesis step and little is known about the associated chromatin assembly. My data excluded a role for CAF-1 in allelic recombination and in the maintenance of forks stability. However, CAF-1 was found to play an important role during ectopic recombination, in promoting chromosomal rearrangements induced by halted replication forks. My data support a model according to which CAF-1 allows the stabilization of early recombination intermediates (D-loop), via nucleosome deposition during the elongation of these intermediates. Doing so, CAF-1 counteracts the dissociation of early recombination intermediates by the helicase Rqh1. Therefore, CAF-1 appears to be part of an equilibrium that regulates stability/dissociation of early steps of recombination events. Importantly, I found that the role of CAF-1 in this equilibrium is of particular importance during non-allelic recombination, revealing a novel regulation level of HR mechanisms and outcomes by chromatin assembly.

Transcription has turned out to be a discontinuous process when imaged at a single cell level. This observation is referred to as transcriptional bursting or pulsing and has been detected in a variety of organisms ranging from bacteria to mammalian cells. The dynamics of transcriptional pulsing are influenced by the properties intrinsic to the transcriptional process, as well as by upstream factors: chromatin environment, signalling molecules, cell cycle stage etc. In the first part of this thesis, we focused on the regulation of transcriptional pulsing in the nucleolus. Using imaging of living cells, we detected pulsatile transcription of a transgene with nucleolar localization whose expression was mediated by RNA polymerase II. In the second part of the thesis, we investigated the relationship between chromatin decondensation and transcriptional dynamics. We used hyperosmotic medium to induce global condensation of chromatin and revealed that upon chromatin decondensation, a transient spike in transcriptional intensity occurs in induvial living cells. Next, we analysed expression of TFRC and POLR2A genes in several cell cycle stages using single molecule RNA FISH. We detected increase in both frequency and size of transcriptional pulses during a limited time window which coincided with chromatin...

The glial cells of the brain and the peripheral nervous system retain the capacity to divide and proliferate throughout the lifespan of an individual and thereby have the propensity to give rise to the most adult neurological tumours. Among them, the tumours which arise from different kinds of glial cells are referred to as gliomas. Of the various types of gliomas, astrocytomas are the most common central nervous system neoplasms which make upto 60% of all the primary brain tumours. Being the most prevalent type, the WHO classifies them into grades ranging from I to IV based on their intensity of malignancy. Grade IV astrocytoma or Glioblastoma (GBM) is considered to be the most malignant form with a median survival of 14.6 months, in spite of all therapeutic modalities. GBM is further classified as primary and secondary GBM. Primary GBM manifests de novo without any early history of pre-malignant lesions, on the other hand secondary GBM arises progressively from lower grades over a period of 5-10 years. Like other malignancies, GBM also arises from various genetic and epigenetic variations. Epigenetic variations include all such mitotically and meiotically heritable traits that do not involve changes in DNA sequence. There are three major areas of epigenetics - DNA methylation, histone modifications and non-coding RNAs which are known to have profound effects on gene expression. A lot being known about the genetic derailments in GBM, in this study we looked into the epigenetic aspects of GBM. In our lab, we have carried out various high throughput studies, which unveiled the distorted landscape of DNA methylation and miRNA expression in GBM. This indicates that, in addition to the genetic mechanisms of gene alterations like mutations, copy number aberrations, protein coding genes are also affected by changes in methylation as well as by miRNA misregulation. The study has been divided into two parts. Part one of the study deals with the identification of chromobox homolog 7 (Cbx7), as a hypermethylated and downregulated gene in GBM. More importantly, Cbx7 is a member of the polycomb repressive complex and brings about its function through chromatin modifications. Here we have investigated the role of Cbx7 in gliomagenesis, and why it has to be silenced by methylation for tumorigenesis to ensue. In part two, we elucidated two unique ways of miRNA regulation in GBM. In the first section, we identified miR-326 as a PI3 kinase regulated miRNA and demonstrated its tumour suppressive role in GBM. In the other section, we analysed the copy number aberration data from TCGA and identified miR- 4484 as a miRNA subjected to deletion in GBM. We further went ahead to demonstrate its growth suppressive role in GBM. Part 1: Epigenetic regulation of the chromatin modifier Cbx7; chromobox homolog 7 DNA methylation is involved in the normal cellular control of expression and thereby plays a crucial role in maintaining the homeostasis of the cell. The phenomenon of DNA methylation keeps the various loci of the genome such as the germline specific genes and the repetitive transposable elements silenced, whereas the tumour suppressors and other growth modulator genes are spared from the methylation induced gene repression. One of the important steps that promote tumorigenesis is aberrant hypermethylation, which leads to the silencing of tumour-suppressor genes. Another important epigenetic phenomenon that affects the transcriptibility of the genome is histone modifications, which control the accessibility of the chromatin to the transcriptional machinery. In this section, we identified Cbx7, which happens to be an essential component of the chromatin modifying machinery, as an epigenetically regulated gene in GBM. We observed from the methylation array carried out in our lab, that Cbx7 was one of the highly methylated genes. We also validated that Cbx7 is downregulated in GBM and the same observation was further corroborated from other data sets. The hypermethylated state of Cbx7 was confirmed by DNA bisulphite sequencing and the expression levels of Cbx7 also got alleviated after 5-Aza-2′-deoxycytidine treatment, which is a DNA methylation inhibitor. This indicated that the down regulation of Cbx7 could be attributed to the methylation of its promoter region. In order to figure out the role of Cbx7 in GBM, we carried out transcriptome analysis of Cbx7 overexpressing cells compared to vector control condition by RNA sequencing. Gene ontology analysis revealed a significant enrichment of pathways involved in cell cycle, migration and invasion like processes. In fact, the exogenous overexpression of Cbx7 leads to cell death, reduced colony formation, retarded migration and invasion of cells. In order to explain the above phenotypes brought about by the exogenous expression of Cbx7, we further examined the RNA sequencing data and observed that many of the top most downregulated genes in Cbx7 overexpression state belonged to the Hippo signaling pathway. The effectors of the Hippo pathway, YAP and TAZ which essentially antagonize the pathway activity, are well known for their role in proliferation, migration and invasion in cancer. So we carried out a Gene Set Enrichment Analysis (GSEA) and found that there was a significant negative enrichment of YAP/TAZ targets in the Cbx7 regulated gene set. We validated some of these targets that were downregulated by Cbx7 overexpression. One of the most downregulated genes that we validated was Connective Tissue Growth Factor (CTGF), which also happens to be a bonafide target of YAP/TAZ. Independent downregulation of CTGF also resulted in reduced migration, thereby phenocopying the effects as were produced by Cbx7 overexpression. Moreover, we also observed that SAPK/JNK was the only kinase whose activity was abolished upon Cbx7 overexpression. Since CTGF is known to activate SAPK/JNK, we assessed the SAPK/JNK activity upon CTGF silencing. We found that levels of phospho-SAPK/JNK were significantly reduced in CTGF silenced condition. In addition to that, the inhibition of the SAPK/JNK by synthetic inhibitor also hampered the migration ability of the cells. We were also able to rescue the loss of migratory potential of glioma cells by the exogenous overexpression of CTGF in Cbx7 stable background. A similar rescue was also achieved by the overexpression of a constitutively active form of SAPK/JNK. This indicates that Cbx7 activates Hippo pathway to inhibit YAP/TAZ dependent transcription, resulting in the downregulation of CTGF, thereby inhibiting CTGF mediated activation of SAPK and thus resulting in the inhibition of glioma cell migration. PART 2: ROLE OF MIRNAS IN GLIOMA DEVELOPMENT AND PROGRESSION miRNAs are a class of small non-coding RNAs that are not translated into functional proteins but still contribute to numerous cellular processes, thereby adding yet another realm of regulation and control. miRNAs bring about gene regulation at the post-transcriptional level, either by degrading the mRNA or by translational repression and in this manner fine tune the expression of protein coding genes. miRNAs are often located in the most fragile sites of the genome which exposes them to grave genetic alterations, thus providing a circumstantial evidence of their etiological role in tumorigenesis. In a malignant state, miRNAs have been found to play pivotal roles in cellular transformation by altering various cellular phenotypes. Owing to their participation in diverse cellular functions, miRNAs have gained a strong foothold in gene regulation. Though a lot has been deciphered about the functional aspect of miRNAs, not much is known about the precise mechanisms which lead to their misregulation and therefore demands in-depth study. The expression of miRNAs can be modulated by a variety of genetic and epigenetic mechanisms. Section I: Role of miR-326 – a PI3 kinase regulated miRNA, in gliomagenesis The TCGA group in the year 2008 identified three major pathways which go disarray in GBM. These include the pro-tumorigenic receptor tyrosine kinase (RTK) pathway, and the p53 and the pRB tumour-suppressive pathways. The RTK signalling includes the PI3 kinase pathway, which is pivotal in gliomagenesis and many other cancers. This directed us to elucidate the set of miRNAs which are controlled by the aberrant functioning of the PI3 kinase pathway. We used synthetic inhibitor LY294002 to abrogate the PI3 kinase signalling and examined the miRNA profile in two glioma cell lines U87 and U251, which have an activated PI3 kinase pathway. Indeed the abrogation of the PI3 kinase pathway resulted in the modulation of a wide array of miRNAs. We validated miR-326 as one of the miRNAs that was upregulated upon PI3 kinase pathway abrogation. Furthermore, we observed that miR-326 was a down regulated miRNA in GBM and different glioma cell lines, as well as in many other publicly available data sets. We also observed that miR-326 is an intragenic miRNA and its host gene Arrestin β1 (ARRB1) also exhibited similar upregulation upon PI3K pathway inhibition. Over-expression of miR-326 resulted in various anti-tumorigenic affects like reduced proliferation, reduced migration and colony suppression. In order to find the targets of miR-326, we analysed the transcriptome by RNA sequencing upon pre-miR-326 transfection. We shortlisted and validated some of the genes which were getting regulated through miRNA over-expression and also explain the functional role of miR-326. Section II: Role of miR-4484 – a copy number deleted miRNA, in gliomagenesis In the TCGA study mentioned above, it was also unfurled that there are many genes in the RTK, p53 and pRB signalling pathways which are made dysfunctional through gene deletions and amplifications. We envisaged whether it is only the protein coding genes which are subjected to such regulations or the non-coding genes like miRNAs as well. In this pursuit, we identified miR-4484 as one of the miRNAs located in the deleted region of uroporphyrinogen III synthase (UROS) gene in the chromosome 11 of the GBM genome. As conceived, miR-4484 was observed to be a downregulated miRNA in association with its host gene UROS. We further elucidated that the downregulation was due to the co-deletion of a locus harbouring both the protein coding gene and the miRNA. In addition, upon over-expression of miR-4484, we observed reduced migration and colony formation, indicating its role as a tumour–suppressor. For seeking the targets of miR-4484, we extracted RNA from miR-4484 over-expression condition and subjected it to RNA sequencing. We shortlisted and validated some of the genes which were getting regulated through miRNA over-expression and possibly explain the functional role of miR-4484.

Eukaryotic genomes are typically organized as chromatin, the complex of DNA and proteins that forms chromosomes within the cell\\\''s nucleus. Chromatin has pivotal roles for a multitude of functions, most of which are carried out by a complex system of covalent chemical modifications of histone proteins. The propagation of patterns of these histone post-translational modifications across cell divisions is particularly important for maintenance of the cell state in general and the transcriptional program in particular. The discovery of epigenetic inheritance phenomena - mitotically and/or meiotically heritable changes in gene function resulting from changes in a chromosome without alterations in the DNA sequence - was remarkable because it disproved the assumption that information is passed to daughter cells exclusively through DNA. However, DNA replication constitutes a dramatic disruption of the chromatin state that effectively amounts to partial erasure of stored information. To preserve its epigenetic state the cell reconstructs (at least part of) the histone post-translational modifications by means of processes that are still very poorly understood. A plausible hypothesis is that the different combinations of reader and writer domains in histone-modifying enzymes implement local rewriting rules that are capable of \\\"recomputing\\\" the desired parental patterns of histone post-translational modifications on the basis of the partial information contained in that half of the nucleosomes that predate replication. It is becoming increasingly clear that both information processing and computation are omnipresent and of fundamental importance in many fields of the natural sciences and the cell in particular. The latter is exemplified by the increasingly popular research areas that focus on computing with DNA and membranes. Recent work suggests that during evolution, chromatin has been converted into a powerful cellular memory device capable of storing and processing large amounts of information. Eukaryotic chromatin may therefore also act as a cellular computational device capable of performing actual computations in a biological context. A recent theoretical study indeed demonstrated that even relatively simple models of chromatin computation are computationally universal and hence conceptually more powerful than gene regulatory networks. In the first part of this thesis, I establish a deeper understanding of the computational capacities and limits of chromatin, which have remained largely unexplored. I analyze selected biological building blocks of the chromatin computer and compare it to system components of general purpose computers, particularly focusing on memory and the logical and arithmetical operations. I argue that it has a massively parallel architecture, a set of read-write rules that operate non-deterministically on chromatin, the capability of self-modification, and more generally striking analogies to amorphous computing. I therefore propose a cellular automata-like 1-D string as its computational paradigm on which sets of local rewriting rules are applied asynchronously with time-dependent probabilities. Its mode of operation is therefore conceptually similar to well-known concepts from the complex systems theory. Furthermore, the chromatin computer provides volatile memory with a massive information content that can be exploited by the cell. I estimate that its memory size lies in the realms of several hundred megabytes of writable information per cell, a value that I compare with DNA itself and cis-regulatory modules. I furthermore show that it has the potential to not only perform computations in a biological context but also in a strict informatics sense. At least theoretically it may therefore be used to calculate any computable function or algorithm more generally. Chromatin is therefore another representative of the growing number of non-standard computing examples. As an example for a biological challenge that may be solved by the \\\"chromatin computer\\\", I formulate epigenetic inheritance as a computational problem and develop a flexible stochastic simulation system for the study of recomputation-based epigenetic inheritance of individual histone post-translational modifications. The implementation uses Gillespie\\\''s stochastic simulation algorithm for exactly simulating the time evolution of the chemical master equation of the underlying stochastic process. Furthermore, it is efficient enough to use an evolutionary algorithm to find a system of enzymes that can stably maintain a particular chromatin state across multiple cell divisions. I find that it is easy to evolve such a system of enzymes even without explicit boundary elements separating differentially modified chromatin domains. However, the success of this task depends on several previously unanticipated factors such as the length of the initial state, the specific pattern that should be maintained, the time between replications, and various chemical parameters. All these factors also influence the accumulation of errors in the wake of cell divisions. Chromatin-regulatory processes and epigenetic (inheritance) mechanisms constitute an intricate and sensitive system, and any misregulation may contribute significantly to various diseases such as Alzheimer\\\''s disease. Intriguingly, the role of epigenetics and chromatin-based processes as well as non-coding RNAs in the etiology of Alzheimer\\\''s disease is increasingly being recognized. In the second part of this thesis, I explicitly and systematically address the two hypotheses that (i) a dysregulated chromatin computer plays important roles in Alzheimer\\\''s disease and (ii) Alzheimer\\\''s disease may be considered as an evolutionarily young disease. In summary, I found support for both hypotheses although for hypothesis 1, it is very difficult to establish causalities due to the complexity of the disease. However, I identify numerous chromatin-associated, differentially expressed loci for histone proteins, chromatin-modifying enzymes or integral parts thereof, non-coding RNAs with guiding functions for chromatin-modifying complexes, and proteins that directly or indirectly influence epigenetic stability (e.g., by altering cell cycle regulation and therefore potentially also the stability of epigenetic states). %Notably, we generally observed enrichment of probes located in non-coding regions, particularly antisense to known annotations (e.g., introns). For the identification of differentially expressed loci in Alzheimer\\\''s disease, I use a custom expression microarray that was constructed with a novel bioinformatics pipeline. Despite the emergence of more advanced high-throughput methods such as RNA-seq, microarrays still offer some advantages and will remain a useful and accurate tool for transcriptome profiling and expression studies. However, it is non-trivial to establish an appropriate probe design strategy for custom expression microarrays because alternative splicing and transcription from non-coding regions are much more pervasive than previously appreciated. To obtain an accurate and complete expression atlas of genomic loci of interest in the post-ENCODE era, this additional transcriptional complexity must be considered during microarray design and requires well-considered probe design strategies that are often neglected. This encompasses, for example, adequate preparation of a set of target sequences and accurate estimation of probe specificity. With the help of this pipeline, two custom-tailored microarrays have been constructed that include a comprehensive collection of non-coding RNAs. Additionally, a user-friendly web server has been set up that makes the developed pipeline publicly available for other researchers. / Eukaryotische Genome sind typischerweise in Form von Chromatin organisiert, dem Komplex aus DNA und Proteinen, aus dem die Chromosomen im Zellkern bestehen. Chromatin hat lebenswichtige Funktionen in einer Vielzahl von Prozessen, von denen die meisten durch ein komplexes System von kovalenten Modifikationen an Histon-Proteinen ablaufen. Muster dieser Modifikationen sind wichtige Informationsträger, deren Weitergabe über die Zellteilung hinaus an beide Tochterzellen besonders wichtig für die Aufrechterhaltung des Zellzustandes im Allgemeinen und des Transkriptionsprogrammes im Speziellen ist. Die Entdeckung von epigenetischen Vererbungsphänomenen - mitotisch und/oder meiotisch vererbbare Veränderungen von Genfunktionen, hervorgerufen durch Veränderungen an Chromosomen, die nicht auf Modifikationen der DNA-Sequenz zurückzuführen sind - war bemerkenswert, weil es die Hypothese widerlegt hat, dass Informationen an Tochterzellen ausschließlich durch DNA übertragen werden. Die Replikation der DNA erzeugt eine dramatische Störung des Chromatinzustandes, welche letztendlich ein partielles Löschen der gespeicherten Informationen zur Folge hat. Um den epigenetischen Zustand zu erhalten, muss die Zelle Teile der parentalen Muster der Histonmodifikationen durch Prozesse rekonstruieren, die noch immer sehr wenig verstanden sind. Eine plausible Hypothese postuliert, dass die verschiedenen Kombinationen der Lese- und Schreibdomänen innerhalb von Histon-modifizierenden Enzymen lokale Umschreibregeln implementieren, die letztendlich das parentale Modifikationsmuster der Histone neu errechnen. Dies geschieht auf Basis der partiellen Informationen, die in der Hälfte der vererbten Histone gespeichert sind. Es wird zunehmend klarer, dass sowohl Informationsverarbeitung als auch computerähnliche Berechnungen omnipräsent und in vielen Bereichen der Naturwissenschaften von fundamentaler Bedeutung sind, insbesondere in der Zelle. Dies wird exemplarisch durch die zunehmend populärer werdenden Forschungsbereiche belegt, die sich auf computerähnliche Berechnungen mithilfe von DNA und Membranen konzentrieren. Jüngste Forschungen suggerieren, dass sich Chromatin während der Evolution in eine mächtige zelluläre Speichereinheit entwickelt hat und in der Lage ist, eine große Menge an Informationen zu speichern und zu prozessieren. Eukaryotisches Chromatin könnte also als ein zellulärer Computer agieren, der in der Lage ist, computerähnliche Berechnungen in einem biologischen Kontext auszuführen. Eine theoretische Studie hat kürzlich demonstriert, dass bereits relativ simple Modelle eines Chromatincomputers berechnungsuniversell und damit mächtiger als reine genregulatorische Netzwerke sind. Im ersten Teil meiner Dissertation stelle ich ein tieferes Verständnis des Leistungsvermögens und der Beschränkungen des Chromatincomputers her, welche bisher größtenteils unerforscht waren. Ich analysiere ausgewählte Grundbestandteile des Chromatincomputers und vergleiche sie mit den Komponenten eines klassischen Computers, mit besonderem Fokus auf Speicher sowie logische und arithmetische Operationen. Ich argumentiere, dass Chromatin eine massiv parallele Architektur, eine Menge von Lese-Schreib-Regeln, die nicht-deterministisch auf Chromatin operieren, die Fähigkeit zur Selbstmodifikation, und allgemeine verblüffende Ähnlichkeiten mit amorphen Berechnungsmodellen besitzt. Ich schlage deswegen eine Zellularautomaten-ähnliche eindimensionale Kette als Berechnungsparadigma vor, auf dem lokale Lese-Schreib-Regeln auf asynchrone Weise mit zeitabhängigen Wahrscheinlichkeiten ausgeführt werden. Seine Wirkungsweise ist demzufolge konzeptionell ähnlich zu den wohlbekannten Theorien von komplexen Systemen. Zudem hat der Chromatincomputer volatilen Speicher mit einem massiven Informationsgehalt, der von der Zelle benutzt werden kann. Ich schätze ab, dass die Speicherkapazität im Bereich von mehreren Hundert Megabytes von schreibbarer Information pro Zelle liegt, was ich zudem mit DNA und cis-regulatorischen Modulen vergleiche. Ich zeige weiterhin, dass ein Chromatincomputer nicht nur Berechnungen in einem biologischen Kontext ausführen kann, sondern auch in einem strikt informatischen Sinn. Zumindest theoretisch kann er deswegen für jede berechenbare Funktion benutzt werden. Chromatin ist demzufolge ein weiteres Beispiel für die steigende Anzahl von unkonventionellen Berechnungsmodellen. Als Beispiel für eine biologische Herausforderung, die vom Chromatincomputer gelöst werden kann, formuliere ich die epigenetische Vererbung als rechnergestütztes Problem. Ich entwickle ein flexibles Simulationssystem zur Untersuchung der epigenetische Vererbung von individuellen Histonmodifikationen, welches auf der Neuberechnung der partiell verlorengegangenen Informationen der Histonmodifikationen beruht. Die Implementierung benutzt Gillespies stochastischen Simulationsalgorithmus, um die chemische Mastergleichung der zugrundeliegenden stochastischen Prozesse über die Zeit auf exakte Art und Weise zu modellieren. Der Algorithmus ist zudem effizient genug, um in einen evolutionären Algorithmus eingebettet zu werden. Diese Kombination erlaubt es ein System von Enzymen zu finden, dass einen bestimmten Chromatinstatus über mehrere Zellteilungen hinweg stabil vererben kann. Dabei habe ich festgestellt, dass es relativ einfach ist, ein solches System von Enzymen zu evolvieren, auch ohne explizite Einbindung von Randelementen zur Separierung differentiell modifizierter Chromatindomänen. Dennoch ängt der Erfolg dieser Aufgabe von mehreren bisher unbeachteten Faktoren ab, wie zum Beispiel der Länge der Domäne, dem bestimmten zu vererbenden Muster, der Zeit zwischen Replikationen sowie verschiedenen chemischen Parametern. Alle diese Faktoren beeinflussen die Anhäufung von Fehlern als Folge von Zellteilungen. Chromatin-regulatorische Prozesse und epigenetische Vererbungsmechanismen stellen ein komplexes und sensitives System dar und jede Fehlregulation kann bedeutend zu verschiedenen Krankheiten, wie zum Beispiel der Alzheimerschen Krankheit, beitragen. In der Ätiologie der Alzheimerschen Krankheit wird die Bedeutung von epigenetischen und Chromatin-basierten Prozessen sowie nicht-kodierenden RNAs zunehmend erkannt. Im zweiten Teil der Dissertation adressiere ich explizit und auf systematische Art und Weise die zwei Hypothesen, dass (i) ein fehlregulierter Chromatincomputer eine wichtige Rolle in der Alzheimerschen Krankheit spielt und (ii) die Alzheimersche Krankheit eine evolutionär junge Krankheit darstellt. Zusammenfassend finde ich Belege für beide Hypothesen, obwohl es für erstere schwierig ist, aufgrund der Komplexität der Krankheit Kausalitäten zu etablieren. Dennoch identifiziere ich zahlreiche differentiell exprimierte, Chromatin-assoziierte Bereiche, wie zum Beispiel Histone, Chromatin-modifizierende Enzyme oder deren integrale Bestandteile, nicht-kodierende RNAs mit Führungsfunktionen für Chromatin-modifizierende Komplexe oder Proteine, die direkt oder indirekt epigenetische Stabilität durch veränderte Zellzyklus-Regulation beeinflussen. Zur Identifikation von differentiell exprimierten Bereichen in der Alzheimerschen Krankheit benutze ich einen maßgeschneiderten Expressions-Microarray, der mit Hilfe einer neuartigen Bioinformatik-Pipeline erstellt wurde. Trotz des Aufkommens von weiter fortgeschrittenen Hochdurchsatzmethoden, wie zum Beispiel RNA-seq, haben Microarrays immer noch einige Vorteile und werden ein nützliches und akkurates Werkzeug für Expressionsstudien und Transkriptom-Profiling bleiben. Es ist jedoch nicht trivial eine geeignete Strategie für das Sondendesign von maßgeschneiderten Expressions-Microarrays zu finden, weil alternatives Spleißen und Transkription von nicht-kodierenden Bereichen viel verbreiteter sind als ursprünglich angenommen. Um ein akkurates und vollständiges Bild der Expression von genomischen Bereichen in der Zeit nach dem ENCODE-Projekt zu bekommen, muss diese zusätzliche transkriptionelle Komplexität schon während des Designs eines Microarrays berücksichtigt werden und erfordert daher wohlüberlegte und oft ignorierte Strategien für das Sondendesign. Dies umfasst zum Beispiel eine adäquate Vorbereitung der Zielsequenzen und eine genaue Abschätzung der Sondenspezifität. Mit Hilfe der Pipeline wurden zwei maßgeschneiderte Expressions-Microarrays produziert, die beide eine umfangreiche Sammlung von nicht-kodierenden RNAs beinhalten. Zusätzlich wurde ein nutzerfreundlicher Webserver programmiert, der die entwickelte Pipeline für jeden öffentlich zur Verfügung stellt.

Heterochromatin is a silenced chromatin region essential for maintaining genomic stability in eukaryotes and for driving developmental processes in higher organisms. A hallmark of heterochromatin is the presence of specialized architectural proteins that alter chromatin structure to inhibit transcription and recombination. Although it is generally assumed that heterochromatin is highly condensed, surprisingly little is known about the structure of heterochromatin or its dynamics in solution. In budding yeast, heterochromatin assembly at telomeres and the HM silent mating type loci requires the Sir proteins: Sir3, believed to be the major structural component of SIR heterochromatin, and the Sir2/4 complex, responsible for SIR recruitment to silencing regions and deacetylation of lysine 16 of the histone H4 tail, a mark associated with active chromatin. A combination of sedimentation velocity, atomic force microscopy, and nucleosomal array capture was used to characterize the stoichiometry and conformation of SIR nucleosomal arrays. The results indicate that Sir3 interacts with nucleosomal arrays with a stoichiometry of two Sir3 monomers per nucleosome, and that Sir2/4 may additionally bind at a ratio of one per nucleosome. Despite Sir3’s ability to repress transcription in vivo and homologous recombination in vitro in the absence of Sir2/4, Sir3 fibers were found to be significantly less compact than canonical magnesium-induced 30 nanometer fibers. However, heterochromatin fibers composed of all three Sir proteins did adopt a more condensed, globular structure. These results suggest that heterochromatic silencing is mediated both by the creation of more stable nucleosomes and by the steric exclusion of external factors.

Eukaryotic DNA is incorporated into the nucleoprotein structure of chromatin. This structure is essential for the proper storage, maintenance, regulation, and function of the genomes’ constituent genes and genomic sequences. Importantly, cells generate discrete types of chromatin that impart distinct properties on genomic loci; euchromatin is an open and active compartment of the genome, and heterochromatin is a restricted and inactive compartment. Heterochromatin serves many purposes in vivo, from heritably silencing key gene loci during embryonic development, to preventing aberrant DNA repeat recombination. Despite this generally repressive role, the DNA contained within heterochromatin must still be repaired and replicated, creating a need for regulated dynamic access into silent heterochromatin. In this work, we discover and characterize activities that the ATP-dependent chromatin remodeling enzyme SWI/SNF uses to disrupt repressive heterochromatin structure. First, we find two specific physical interactions between the SWI/SNF core subunit Swi2p and the heterochromatin structural protein Sir3p. We find that disrupting these physical interactions results in a SWI/SNF complex that can hydrolyze ATP and slide nucleosomes like normal, but is defective in its ability to evict Sir3p off of heterochromatin. In vivo, we find that this Sir3p eviction activity is required for proper DNA replication, and for establishment of silent chromatin, but not for SWI/SNF’s traditional roles in transcription. These data establish new roles for ATP-dependent chromatin remodeling in regulating heterochromatin. Second, we discover that SWI/SNF can disrupt heterochromatin structures that contain all three Sir proteins: Sir2p, Sir3p and Sir4p. This new disruption activity requires nucleosomal contacts that are essential for silent chromatin formation in vivo. We find that SWI/SNF evicts all three heterochromatin proteins off of chromatin. Surprisingly, we also find that the presence of Sir2p and Sir4p on chromatin stimulates SWI/SNF to evict histone proteins H2A and H2B from nucleosomes. Apart from discovering a new potential mechanism of heterochromatin dynamics, these data also establish a new paradigm of chromatin remodeling enzyme regulation by nonhistone proteins present on the substrate.