The role of chromatin organization and structure in neuronal differentiation
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1.
2.
Matthias Karl Weng The role of chromatin organization and structure in neuronal differentiation
Publications and presentations
Publications integrated in this thesis Chapter 3
Weng MK, Zimmer B, Pöltl D, Broeg MP, Ivanova V, Gaspar JA, Sachinidis A, Wüllner U, Waldmann T and Leist M. Extensive transcriptional regulation of chromatin modifiers during human neurodevelopment. PLoS One 2012;
7(5):e36708. Epub 2012 May 9.
Chapter 4
Matthias K. Weng, Karthick Nataraj, Diana Scholz, Violeta Ivanova, Agapios Sachinidis, Jan G. Hengstler, Tanja Waldmann and Marcel Leist, Lineage- specific regulation of epigenetic modifier genes in human liver and brain. PLoS One 2014; in press PLoS One 2014
Chapter 5
Matthias K. Weng, Timo Trefzer, Tim-Oliver Buchholz, Christian Dietz, Michael Berthold, Lisa Hölting, Ferdinand Kappes, Veronika Lodenmeyer, Marcel Leist and Tanja Waldmann. Unique type of restrictive chromatin in postmitotic neurons is linked to stress-induced apoptosis in neurodegenerative disease. manuscript in preparation
Publications not integrated in this thesis
Pöltl D, Scholz D, Genewsky A, Weng M, Waldmann T, Schildknecht S and Leist M. Rapid, complete and large-scale generation of post-mitotic neurons from the human LUHMES cell line. J Neurochem 2011;119(5):957-971.
Balmer NV, Weng MK, Zimmer B, Ivanova VN, Chambers SM, et al. (2012) Epigenetic changes and disturbed neural development in a human embryonic stem cell-based model relating to the fetal valproate syndrome. Hum Mol Genet 21: 4104-4114
Balmer NV, Klima S, Rempel E, Ivanova VN, Kolde R, Weng MK, Meganathan K, Henri M, Sachinidis A, Berthold MR, Hengstler JG, Rahnenführer J, Waldmann T and Leist M, (2014) From transient transcriptome responses to disturbed neurodevelopment: divergent response patterns and epigenetic modifications triggered by the same drug. Arch Toxicol. Under revision
Oral presentations
“Bivalent state and lineage decision in neuronal differentiation”. Konstanz Research School Chemical Biology (KoRSCB) Retreat 2012, Gülstein, Deutschland.
Poster presentations
Matthias Weng, Tanja Waldmann, Marcel Leist. ”Chromatin states during neural and neuronal differentiation of human embryonic stem cells”. FMI 40th Anniversary Symposium, 2010, Basel, Schweiz.
Matthias Weng, Tanja Waldmann, Marcel Leist. ”Epigenetic modifier gene expression in different neuronal cell types”. 3rd SFB TR5 Symposium, 2010, München, Deutschland.
Matthias Weng, Nina Balmer, Tanja Waldmann, Marcel Leist. “Chromatin Immunoprecipitation: a tool to monitor epigenetic changes in toxicological test systems”. ToxNet-BW Symposium, 2011, Konstanz, Deutschland
Matthias Weng, Nina Balmer, Tanja Waldmann, Marcel Leist. ”Chromatin Immunoprecipitation: a tool to monitor epigenetic changes in toxicological test systems”. Insel Symposium, 2012, Konstanz, Deutschland.
Matthias Weng, Eva Gwosch, Tanja Waldmann, Marcel Leist. “The role of chromatin structure and organization on neural differentiation and stem cell maintenance”. Chromatin: from structure to epigenetics, 2012, Straßbourg, Frankreich.
Matthias Weng, Tanja Waldmann, Marcel Leist. ”Heterochromatin formation in postmitotic neurons: a model for neurodegeneration?”.
Epigenetics and Stem Cells, 2012, Cambridge, England.
Table of Contents
1 Summary ... 14
Zusammenfassung ... 15
2 General introduction ... 17
2.1 Epigenetics ... 17
2.2 Neurodevelopment... 18
2.3 Chromatin ………19
2.4 DNA methylation ... 19
2.5 Histone modifications and histone variants ... 21
2.5.1 Histone modification ... 21
2.5.2 Histone variants ... 25
2.6 Chromatin remodeling complexes (CRC) ... 26
2.7 Mammalian cell cycle ... 27
2.7.1 Cell cycle regulation and checkpoints ... 28
2.7.2 Cell death ... 30
2.7.3 Cell cycle, apoptosis and disease ... 30
2.8 Aims of the thesis ... 32
3 Extensive transcriptional regulation of chromatin modifiers during human neurodevelopment ... 34
3.1 Abstract ………35
3.2 Introduction ... 36
3.3 Materials and Methods ... 37
3.3.1 Cultivation of the hESC line H9 ... 37
3.3.2 Differentiation of H9 to NEP ... 37
3.3.3 Differentiation of H9 to NCP ... 37
3.3.4 Human brain samples ... 38
3.3.5 Immunostaining ... 38
3.3.6 Western blot analysis ... 38
3.3.7 Chromatin immuno precipitation... 38
3.3.8 Reverse transcription and quantitative qPCR ... 39
3.3.9 Normalization of qPCR data for cell type comparisons ... 39
3.3.10 Affymetrix gene chip analysis ... 40
3.3.11 Bioinformatics and data analysis ... 40
3.4 Results ………41
3.4.1 Chromatin changes during the differentiation of hESC to neuroectodermal progenitor cells ... 41
3.4.2 Compilation of a set of genes involved in chromatin modification ... 44
3.4.3 Pronounced changes of the epigenetic regulator transcript profile during human neuroepithelial (NEP) differentiation ... 45
3.4.4 Differentiation of hESC to neural crest precursor cells (NCP) ... 47
3.4.5 Changes of the epigenetic regulator transcript profile during differentiation of human pluripotent stem cells to neural crest ... 49
3.4.6 Relative abundance of epigenetic regulator transcript levels in cortical neurons relative to stem cells and neural precursors ... 49
3.5 Discussion ... 53
3.6 Acknowledgement ... 56
3.7 Supplementary figures ... 56
4 Lineage-specific regulation of epigenetic modifier genes in human liver and
brain ... 57
4.1 Abstract ………58
4.2 Introduction ... 59
4.3 Materials and Methods ... 60
4.3.1 Cultivation and differentiation of LUHMES cells ... 60
4.3.2 Fresh human hepatocyte samples ... 61
4.3.3 Differentiation of H9 cells to hepatocyte-like islets ... 61
4.3.4 Human brain samples ... 62
4.3.5 Immunostaining of neural cells ... 62
4.3.6 Reverse transcription and quantitative RT-PCR ... 62
4.3.7 Normalization of qPCR data for cell type comparisons ... 63
4.3.8 Bioinformatics and data analysis ... 63
4.4 Results and Discussion ... 64
4.4.1 Distinct profiles of epigenetic modifier expression in liver and brain ... 64
4.4.2 Comparison of the EMG profile of two hepatocyte populations ... 66
4.4.3 Similarities of EMG regulation in different neuronal cells ... 68
4.4.4 Cluster analysis of EMG expression across different cell types ... 70
4.4.5 Subgroup analysis of differentially expressed EMGs in neurons and hepatocytes ... 72
4.4.6 Overview of differentially expressed EMGs ... 76
4.5 Acknowledgments ... 78
4.6 Supplementary figures ... 78
5 Unique type of restrictive chromatin in postmitotic neurons is linked to stress-induced apoptosis in neurodegenerative disease ... 79
5.1 Abstract ………80
5.2 Introduction ... 80
5.3 Materials and Methods ... 81
5.3.1 Cultivation and differentiation of LUHMES cells ... 81
5.3.2 Cultivation and differentiation of NHLF cells... 82
5.3.3 Cultivation and differentiation of peripheral neurons ... 82
5.3.4 Microarray analysis ... 83
5.3.5 Immunofluorescence staining ... 83
5.3.6 Image analysis ... 83
5.3.7 Reverse transcription and quantitative RT-PCR ... 84
5.3.8 Chromatin immunoprecipitation (ChIP) ... 85
5.3.9 DNase digestion ... 85
5.3.10 Western Blot ... 86
5.4 Results ………87
5.4.1 LUHMES cells differentiate into postmitotic cells with a neuronal phenotype... 87
5.4.2 Gene expression correlates with chromatin environment and structure in LUHMES cells ... 88
5.4.3 Repressive PTMs relocate to nuclear periphery upon neuronal differentiation ... 90
5.4.4 Nuclear relocation of heterochromatin-associated histone modification is unique to postmitotic cells ... 92
5.4.5 The timing of nuclear relocation of H3K27me coincides with cell cycle exit ... 92
5.4.6 Stimulation of Cell cycle re-entry by FGF ... 95
5.4.7 Cells that are treated with FGF late during differentiation keep H3K27me at nuclear periphery and show signs of replication stress ... 97
5.5 Discussion ... 99
5.5.1 Gene repression via H3K27me3 and the spatial factor ... 99
5.5.2 Chromatin structure in postmitotic neurons might function as replication barrier ... 100
5.6 Supplementary figures ... 102
6 General discussion ... 110
6.1 Chromatin regulation in neuronal systems ... 110
6.2 Epigenetics in normal and diseased postmitotic neurons ... 112
6.3 LUHMES cells as model for neurodegenerative disease ... 115
6.4 Summary and Outlook ... 116
7 Bibliography ... 118
8 Appendix ... 134
8.1 Record of contribution ... 134
8.2 Abbreviations ... 135
8.3 Supplementary figures chapter 4... 136
Danksagung ... 148
1 Summary
Chromatin structure is more than just a simple packaging scaffold for DNA. It organizes and coordinates the precise spatio-temporal transcription processes a cell needs for a properly orchestrated development. Epigenetics is the study of this type of regulation. It describes the processes that lead to chromatin decondensation or compaction and involves a multitude of different protein complexes and enzyme families. They carry out various functions like DNA-methylation, histone modification or energy-dependent chromatin remodeling and work together to ensure correct transcription of the DNA template. A dynamic transcription is most important during development. Here, the cell needs to execute lineage restriction while staying responsive to outside cues in order to differentiate properly. Neuronal development is of particular interest because it produces long-lived cell types and early mistakes in regulation can lead to severe disease phenotypes also later in life. However, once differentiation is completed and the neuronal cells have reached a mature postmitotic state they need to maintain a certain transcriptional balance over a long period of time.
Recently, there have been a lot of studies linking epigenetic transcriptional regulation in neurons to processes that are also seen in neurodegenerative diseases. This thesis described the characterization of epigenetic modifiers in neurons and their possible implication in neurodegenerative disease development.
First, we provided a comparative transcriptional profiling of an epigenetic modifier gene set in five neuronal and three non-neuronal cell types. With this sensitive qPCR- based approach we were able to find a cell-type-specific regulation of subunits in remodelers like the SWI/SNF-complex. We also observed a neuron-specific expression of modifiers such as PRMT8, CHD5 and HDAC9. We continued by describing a form of repressive chromatin that is localized at the nuclear periphery of neurons and characteristic for postmitotic cells. This area is defined by a differentiation-dependent relocalization of the heterochromatin marks H3K27me3 and H3K9me3. For this part of the thesis we used the well-established LUHMES model cell line that produces mature postmitottic neurons within 6 days. Upon differentiation LUHMES cells stop to proliferate, exit the cell cycle and develop a functional neuronal network. LUHMES cells are widely used as model system to study neurodegeneration in dopaminergic neurons. In an attempt to combine the well-studied neurodegenerative aspects of this system with epigenetic research we restimulated cell cycle after differentiation. While this led to an upregulation of cell cycle markers, a condition also seen in early stages of neurodegeneration, the peripheral localization of H3K27me3 did not change. In addition, the restimulation of proliferation indicated activation but not progression of cell cycle; again a condition that is also found in neurodegenerative disease models.
Simultaneously, we could observe the expression of replication stress markers in those cells. We hypothesized that the death of cell-cycle-stimulated neurons is due to the restrictive nature of the “heterochromatin barrier” we observed. This structure appears block DNA-synthesis, cause replicative stress and eventually lead to apoptosis.
In summary, we developed a qPCR-based array for the characterization of epigenetic modifier genes and we were able to link H3K27me3-relocalization in postmitotic neurons to a replication-stress-phenotype that is also seen in many neurodegenerative diseases. This work also confirms the usefulness of the LUHMES system as tool to study mechanistic and biochemical details of neurodegeneration.
Zusammenfassung
Chromatinstruktur ist viel mehr als nur ein bloßes Verpackungsgerüst für DNA. Sie organisiert und koordiniert die genauen zeitlichen und räumlichen Transkriptionsprozesse, die eine Zelle für eine ordentliche Entwicklung benötigt. Die Epigenetik ist die Lehre solcher Regulationsprozesse. Sie beschreibt die Schritte, die zur Dekondensation oder Kompression von Chromatin führen, und sie umfasst eine Vielzahl an verschiedenen Proteinkomplexen und Enzymfamilien. Diese führen viele unterschiedliche Funktionen aus wie zum Beispiel DNA-Methylierung, Histonmodifikation und energieabhängige Chromatin-Remodellierung und sie sorgen gemeinsam für die korrekte Transkription der DNA-Matrize. Eine dynamische Transkription ist am wichtigsten während der Embryonalentwicklung. Um in dieser Phase ordentlichzu differenzieren, muss die Zelle zum einen in der Lage sein eine zelllinien-spezifische Restriktion durchzuführen und zum anderen muss sie aber auch auf externe Signale reagieren können. Die neuronale Entwicklung ist hier von besonderem Interesse, da sie langlebige Zellen hervorbringt und Fehler in der Regulation zu schweren Krankheitsbildern, auch im späteren Leben, führen können. Ist der Differenzierungsvorgang jedoch einmal abgeschlossen und die neuronalen Zellen haben einen reifen postmitotischen Zustand erreicht, müssen sie eine gewisses Gleichgewicht der Transkriptionsprozesse über einen sehr langen Zeitraum aufrecht erhalten. In jüngster Zeit wurden viele Studien veröffentlicht, die epigenetische Transkriptionsregulation mit Prozessen verbinden, die auch in neurodegenerativen Erkrankungen auftreten. In der hier vorgestellten Arbeit wurde die Charakterisierung epigenetischer Regulatorproteine in Neuronen beschrieben und deren mögliche Rolle in neurodegenerativen Erkrankungen untersucht.
Zunächst erstellten wir ein vergleichendes Transkriptionsprofil epigenetischer Regulatorproteine in fünf neuronalen und drei nicht-neuronalen Zelltypen. Mit Hilfe dieses sensitiven, auf qPCR-Daten basierenden Ansatzes waren wir in der Lage einen zelltypspezifischen Austausch von Untereinheiten in Remodellierungsproteinen wie dem SWI/SNF-Komplex nachzuweisen. Wir konnten außerdem eine neuronenspezifische Expression von epigenetischen Regulatoren wie PRMT8, CHD5 und HDAC9 beobachten. Desweiteren konnten wir eine Form von restriktivem Chromatin beschreiben, die an der nuleären Peripherie von Neuronen lokalisiert ist und postmitotische Zellen charakterisiert. Dieses Areal ist definiert durch eine differenzierungs-abhängige Umlagerung der Heterochromatinmarker H3K27me3 und H3K9me3. Für diesem Teil der Arbeit verwendeten wir die robuste LUHMES Zelllinie, die in der Lage ist innerhalb von 6 Tagen reife, postmitotische Neuronen zu produzieren. Während des Differenzierungsprozesses stellen LUHMES Zellen die Proliferation ein, treten aus dem Zellzyklus aus und bilden ein funktionelles neuronales Netzwerk. LUHMES Zellen sind als Modellsystem zur Untersuchung neurodegenerativer Erkrankungn in dopaminergen Neuronen sehr verbreitet. In einem Versuch die gut untersuchten neurodegenerativen Aspekte dieses Zellsystems mit epigenetischer Forschung zu verbinden, haben wir nach abgeschlossener Differenzierung erneut Zellzyklusprozesse stimuliert. Obwohl dies tatsächlich zu einem Anstieg in der Expression von Zellzyklusmarkern führte, ein Vorgang der gleichzeitig in frühen Stadien der Neurodegeneration beobachtet wird, änderte sich nichts an der periphären Lokalisation von H3K27me3. Zusätzlich deuteten stimulierte Zellen zwar auf eine Aktivierung jedoch nicht auf das Fortschreiten des Zellzykluses hin. Ein
Zustand der ebenfalls in Neurodegenerationsmodellen vorgefunden wird. Zeitgleich konnten wir die Expression von Replikationsstressmarkern in diesen Zellen beobachten. Wir stellten die Hypothese auf, dass das Absterben von zellzyklus- stimulierten Neuronen durch die restriktiven Eigenschaften der periphären
„Heterochromatinbarriere“, die wir beschreiben konnten, ausgelöst wird. Diese Struktur scheint DNA-Synthese zu blockieren, replikativen Stress hervorzurufen und schließlich Apoptose zu verursachen.
Zusammenfassend konnten wir ein qPCR-basiertes Verfahren zur Charakterisierung von epigenetischen Regulatoren entwickeln und wir waren in der Lage die Umlagerung von H3K27me3 in postmitotischen Neuronen mit einem Phänotyp zu verknüpfen, der häufig in neurodegenerativen Erkrankungen beobachtet wird. Diese Arbeit bestätigt außerdem die Nützlichkeit des LUHMES Zellsystems als Werkzeug für detailierte mechanistische und biochemische Studien in der Neurodegenerationsforschung.
2 General introduction
2.1 Epigenetics
Information is nothing without context. Although all somatic cells carry the same genomic DNA template, their expression profiles and therefore the phenotypical output can be very different in individual cells and tissues. The question that arises from this is exemplified in the developing embryo. How can a complex organism arise from one and the same pool of information? One of the first to address this question was Konrad H. Waddington. Even before it was clear that DNA is the carrier of genetic information and that its content is the same in every somatic cell (1), Waddington used the term epigenesis to describe the developmental processes that lead from a genotype to a certain phenotype (2). This relatively broad definition of the term emphasizes the fact that development is an act of creation (“genesis”) that depends on and evolves from (“epi-“) a preceding status quo. To simplify this view he used the concept of an epigenetic landscape (3) (Fig.1).
It illustrates the history of a developing somatic cell as a sphere rolling down a landscape of hills and valleys. Every point of diversion symbolizes a lineage decision until the cell reaches the differentiated adult state. He called the process that forms this landscape epigenesis and postulated that a phenotype is simply the combination of genetic expression and tissue interactions. Or to use the language of the metaphor; the deepness (immutability) of a chosen path is directly determined by characteristics in the terrain (combinatorial effects). It is also possible that the sphere (differentiating cell) is pushed over a ridge that is too shallow by internal and external stimuli to end up in a completely different developmental pathway. What is so intriguing about this model is the fact that it makes the two above described concepts of development, the coordination of lineage decisions and disturbances of normal differentiation through other factors, very easily explicable (Fig. 1).
As the knowledge of the relations between genotype and phenotype grew over the years, the term epigenetics changed into a more precise definition. The first epigenetic Figure 1: Illustration of Waddington`s epigenetic landscape (from „The strategy of the Genes“, 1957)
phenomenon that also matches the current description of epigenetics was the discovery of the so-called position effect variegation (4) in Drosophila. It showed that somatic change can have genetic repercussions. Further experiments on genetic assimilation could prove that a somatic change can precede a heritable phenotype (5). This lead to a redefinition of epigenetics as the study of “mitotic and meiotic heritable changes in gene function that are not based on DNA sequence” (6). Recently this definition was been extended into an model of triggers („epigenators“), initiators and maintainers of epigenetic processes in an attempt to incorporate the fast growing knowledge in this field (7). It seems ironic that today we use the description of “epigenetic” states (modern definition) to explain the concepts of epigenesis (developmental genetics) that was introduced more than half a century ago (8).
2.2 Neurodevelopment
Some claim that the brain is the most complex thing in the known universe and that studying its function is crucial in understanding both of them; the human mind and the universe (9). The functional units of the human brain are the cells of the nervous system; neurons, astrocytes and oligodendrocytes. Neurons are able to receive, transform and propagate electrochemical signals via their axons and synapses. The study of neuronal functions (Neuroscience) is more than one and a half centuries old (Remak, 1850). Since then, constant progress through new techniques and the use of model systems (10) has shed some light on the enourmous complexity and connectivity the conglomerate of neurons is able to create in the brain. But still, so far it seems we only have explored the coastline of the continent that is the human mind.
Neurogenesis, the creation of neurons, starts from a layer of neuroepithelial cells (neural plate) on the dorsal surface of the early embryo and marks the beginning of neural development. Every cell of the developing neural system goes through the same three basic stages during differentiation; induction, specification and maturation (11).
In a first step at the end of gastrulation the neural plate folds into the neural tube that gives rise to all the cells of the central nervous system (CNS) (12). At the top of the neural tube lies the neural crest, a region that produces several waves of migrating neural crest cells (13). Those cells will form the cells of the peripheral neural system (PNS) amongst over 100 other cell types. The huge diversity of neuronal cell types is achieved through spatial and temporal pattering (14). These two pattering processes define the properties of early neural stem cell (NSC) populations (15). Multipotent NSCs can differentiate into glial and neuronal cell lineages (16). Today most neural cell types, from precursors to postmitotic neurons can be generated in vitro (17). This ability has made neural differentiation one the most studied developmental processes (18). Even though there is still a need to generate purer and more robust systems for analysis, NSC-based differentiation systems represent promising tools for basic developmental as well as medical research. They will help us to discover and hopefully eliminate the reasons for developmental dispositions and neurodegenerative diseases like Alzheimer`s or Parkinson`s disease (17).
As it is seen in neural development, all multicellular organisms must regulate cellular identity and function to ensure survival and cellular plasticity. Cell identity is determined at different stages of development timely dependent on the specific cell
type and regulated by waves of gene expression (19). Gene expression, in turn, is organized and orchestrated by chromatin.
2.3 Chromatin
The nuclear DNA is more than an inert template for transcription. Cell division and changing gene expression during a cells life cycle require dynamic regulation and compaction of this macromolecular structure. This is achieved by a nucleoprotein complex called chromatin. It is organized in a repeating array of nucleosomes, the basic packaging unit of chromatin (20), and it controls DNA accessibility and structure (21).
The nucleosome itself consists of a histone octamer (22); one histone H3-4 tetramer and two histone H2A-H2B dimers (Fig. 2). Around each nucleosome approximately 146bp of DNA are wrapped like a piece of string on a spindle. This relatively open and accessible form of chromatin is classically called “euchromatin”. In order to produce a more compact chromatin structure, the linker histone H1 is placed between the regularly spaced nucleosomes to enable the folding of the nucleofilament into a solenoid structure (Fig. 2). High-mobility group proteins (HMG) and other DNA- binding factors (23) help to compress the nucleosomal array further, resulting in the inaccessible and densely packed “heterochromatin”. Finally, a well-organized higher- order chromatin structure is established inside the nucleus with different nuclear domains in varying degrees of compaction (Fig. 2) (24).
Even though terms like eu- and heterochromatin are historically used to distinguish between transcriptionally active and inactive parts of the genome, they are unsuited to describe the increasing complexity of different chromatin environments that can be found in the nucleus. Recently, there has been an effort to characterize genomic regions according to their composition (25). The proteins and complexes that alter chromatin structure and define the “epigenetic state” of a gene could help to categorize and distinguish different functional gene clusters. This includes for example the assessment of histone modification states at certain genes in order to find the previously postulated
“histone code” (26) that defines the transcriptional status.
The “epigenetic state” that regulates the transcription of the DNA template is determined mainly through 5 interrelated mechanisms. Those include DNA- methylation (27), posttranslational modification (PTM) of histones (28), chromatin remodeling (29), histone variants and non-histone structural components like HP-1 or non-coding RNAs (ncRNAs) (30,31). We will take a closer look at some of them in the following paragraphs.
2.4 DNA methylation
The idea that covalent modification on DNA could represent a heritable and functional feature of genome organization first came up through the process of X-chromosome inactivation (32) and has become one of the best-studied mechanisms of epigenetic gene regulation until today. After its deposition through DNA methyltransferase enzymes (DNMTs) (33) the 5-methylcytosine mark (5mC) can be semi-conservatively propagated through cell division (34). DNA methylation is associated with transcriptionally repressed chromatin (35) and mainly occurs in a CpG dinucleotide
context. Although 70-80% of the mammalian CpGs are methylated, larger clusters of them (also known as CpG islands) remain unmethylated in most cases. CpG islands are often found in the regulatory promoter regions of genes and their irregular hypermethylation is linked to cancer development (silencing of oncogene suppressors) (36). At the same time hypomethylation can have equally detrimental effects on genome stability since DNA methylation suppresses repetitive and retroviral sequences and plays an important role in genomic imprinting (37,38). Therefore a proper regulation of the methylation state is crucial during embryogenesis. The reorganization of DNA-methylation is particularly important right after fertilization before the maternal and paternal genomes are merged (39). At this stage paternal but not maternal methylation patterns are erased which leads to the so-called maternal imprinting effect.
But demethylation events were not only observed during early development but throughout differentiation and even in postmitotic cells (40,41). This underlines the importance of reversible DNA methylation in different biological settings.
To ensure the dynamic of this process several methylation and demethylation mechanisms exist (42). There are three different DNA methyltransferases known to date. DNMT1 is needed to maintain the methylation status of genes (43). It duplicates the 5mC mark onto the newly synthesized daughter strand after each replication.
Figure 2: schematic showing the general steps of chromatin compaction;
from nucleosomal
structure [bottom] to higher-order chromatin regions in the nucleus [top]. (adapted from Gotoh et al., 2010)
DNMT3A and DNMT3B, on the other hand, both are able to de novo methylate DNA and they help to set up a methylation pattern early in development (44). Compared to other covalent epigenetic marks like histone modifications, DNA methylation is relatively stable. In contrast to establishing the 5mC mark, the removal of the modification has been controversially discussed (45); and until recently the only known means of demethylation was a passive loss through successive cell divisions. The discovery of the ten-eleven translocation (TET) enzyme family brought a new understanding to those processes. TET proteins can initialize demethylation by oxidizing 5mC into 5-Hydroxymethylcytosine (5hmC) (46). Further oxidation steps allow either a passive dilution because 5hmC can no longer be propagated or active replacement through base excision repair (47).
In summary, DNA methylation is described by a dynamic methylation, oxidation and repair cycle model and it is one of the key epigenetic mechanisms to propagate and control non-sequence-based information throughout development.
2.5 Histone modifications and histone variants
As mentioned above the histone octamer of the nucleosome is the smallest packaging unit of chromatin. But apart from providing a structure for compaction histone proteins also possess additional features. The possibility of covalent modification at their protruding N- and C-terminal tails and the incorporation of different histone variants simultaneously convert them into transmitters and receivers of information. They represent an “indexing system” that brings variation into the chromatin structure and ensures a dynamic transcriptional regulation.
2.5.1 Histone modification
An early hypothesis about the function of histone proteins as gene repressors was already made over 60 years ago (48). Even before any knowledge about nucleosomal packaging of DNA (20) or histone modifications and their mechanisms of recruitment was available. The first established histone modifications (HPTMs) were acetylation and methylation (49). Since then, more than 60 modified histone residues have been identified with at least 8 different types of possible covalent modification (28). Those include smaller modifications like acetylation (50), methylation (51) and phosphorylation (52); but also larger ones like ubiquitination (53), sumoylation (54) and ADP-ribosylation (55).
Some of them, like acetylation and phosphorylation, are able to introduce additional charges and therefore change the binding properties of histones to DNA. This can either lead to an expansion or a compaction of the chromatin fiber and directly increase or decline DNA accessibility (56). A more indirect mechanism of histone modifications to influence chromatin structure is the recruitment of binding partners. Those can they flag histones to recruit protein complexes that also change chromatin structure and the transcriptional status quo.
Figure 3: Posttranslational modifications at the N-terminal tails (left) and core domains (right) of the four nucleosomal histone variants. ph = phosphorylation (red); ac = acetylation (green); me = methylation (blue); ub = ubiquitination (purple); (adapted from Bhaumik et al., 2007)
Histone acetylation
Since the acetylation of lysines belongs to the first known HPTMs, the enzyme families responsible for establishment and removal of the acetylation mark are very well studied (57,58). Acetylation is generally associated with an activation of transcription since it reduces the affinity to nucleosomal DNA by neutralizing the net charge of the lysine residue (59). This makes the chromatin more accessible. In addition, repressive chromatin complexes often possess a deacetylase function (e.g. the NuRD-complex) (60,61). Characterized by their catalytic domains there are 3 main groups of histone acetyltransferases (HATs) (28,62); GNAT, MYST and CBP/p300. Although there are examples of site- (63) and tissue-specific (64) acetylation HATs normally only have a limited specificity and acetylation represents a generally ubiquitous mark. This is also shown by the lysine-acetyltransferase KAT2B (also known as PCAF), a multimeric protein complex of the GNAT-family, which has a ubiquitous function and is expressed in most tissues (65). To still ensure a targeted modification of certain chromatin regions HAT-specificity is conferred through cofactors that are able to respond in a more exclusive way (66).
In most cases the reversal of acetylation leads to a transcriptional repression. Histone deacetylase proteins (HDACs) can be divided into 4 different classes corresponding to their yeast homologues (67). The HDACs of class 1, 2 and 4 share a Zn-dependent mechanism to remove the acetylation from the histone; whereas class 3 HDACs use a NAD-dependent process (68). Similar to HATs, HDAC proteins do not show much specificity but use cofactors to target genomic sequences instead (68). Although, there are exceptions to that generalization (69). The occurrence of distinct expression patterns in different tissues lead to the assumption that many HDACs have cell type related functions (70). Because of their biological roles in cell growth, differentiation and apoptosis HDAC proteins are also important drug targets in many studies on cancer therapy (71,72). Inhibitors of HDACs are also used as neuroprotective drugs in several psychatric disorders and to treat epilepsy (73).
Histone methylation
Of all HPTMs methylation by histone lysine methyltransferases (HKMTs) is the most specific. But the possibility of arginine and lysine residues to be mono- or dimethylated (even trimethylated in case of lysines) adds extra complexity to this type of modification (74). Depending on the degree of methylation the functional response can change dramatically. Since the first HKMT was identified in 2000 (75), more and more members of this protein family have been found to have one common feature; the so- called SET-domain. HKMTs can differ in substrate as well as product specificity meaning that different methyltransferases not only have varying target sites but also catalyze different degrees of methylation. In order to remove mthylation marks, an equally large group of lysine demethylases (KDMs) exists. So like DNA-methylation and histone acetylation the methylation of histones is a dynamic and reversible process that can be regulated through a multitude of different enzymes.
The methylation of lysine 4 of H3 (H3K4me), much like histone acetylation, is generally associated with transcriptionally active chromatin (28). H3K4me recruits remodeling complexes and other modifying enzymes (e.g. HATs) to open chromatin structure or to prevent transcriptional repressors from binding (76). It is also a good example for the important impact the methylation status of a residue has on the binding properties of associated factors; in so far as the trimethylated form of H3K4 (H3K4me3) only peaks at gene promoter regions and is associated with the active form of RNA-polymerase II (77). H3K4me2 is found in the coding regions of transcribed
Figure 4: Protein domains that are able to recognize methylated and acetylated lysines or phosphorylated serines. (adapted from Kouzarides et al., 2007)
genes instead (78). In addition, factors involved in transcriptional activation only bind to H3K4me2/3 and not to the monomethylated form. One of those is the CHD1 protein, a chromatin remodeling factor that binds via a tandem chromodomain to H3K4me2/3 (Fig. 5) (76). Another selective “reader” of H3K4me2/3 is the NURF complex. It performs nucleosomal rearrangement in active genes and binds via the PHD finger domains of its BPTF subunit (Fig. 5) (76).
To date there are over ten different HKMTs known to be specific for H3K4. Half of them (SET1, MLL1/3/5 and ASH1L) belong to the family of trithorax group proteins (trxG). TrxG proteins were first discovered in Drosophila and represent a highly conserved class of Hox-gene transcription regulators (79) that are crucial during embryonic development (80). They are a functionally diverse group and represent a counterpart to the repressive polycomb group (PcG) protein family (see H3K27me).
Although all of those enzymes methylate one specific residue there have been few examples of redundancy. Instead the loss of a single H3K4me-HKMT can lead to embryonic lethality and disease (81). This might indicate different gene targets and separate subsequent cellular functions.
The methylation of lysine 27 of histone 3 (H3K27me) is a repressive mark that is closely connected to the PcG protein family (82). The PcG family has crucial role in all developmental processes by establishing and maintaining lineage restriction (83) from an early embryonic state (84) to final tissue differentiation (85). The two enzymes that catalyze the methylation of H3K27, EZH1 and 2, are part of the polycomb repressive complex 2 (PRC2). Whereas EZH2 is associated with proliferative tissue (86) and self-renewal (85), EZH1 is more abundant in non-proliferating tissues (86).
Although both share a percentage of overlapping target genes, they do seem to have distinct functions during cellular development. After the H3K27me-mark is set it recruits PRC1, the second complex of the PcG-family (87). PRC1 impairs transcriptional elongation by establishing another histone modification, H2AK119ub (88), and it mediates gene silencing through chromatin compaction (89). The dysregulation of these gene silencing processes plays a crucial role in malignant transformation and many other diseases (90).
Sometimes both H3K27me and H3K4me can be colocalized at the same genomic region. In this case the respective chromatin environment is kept in a so-called bivalent state (84). Bivalency is a proposed mechanism to keep genes that have an important function during later developmental steps “poised” for expression. The model allows fast expressional changes and safeguarding from dysregulation throughout differentiation, but still remains controversial (91). In summary, the PcG- and TrxG- protein families help to maintain pluripotency and cooperate in the important regulation of expression during development through individual and coordinated functions (92).
The third of the three most studied histone methylations for far is the methylation of lysine 9 on H3 (H3K9me). In contrast to H3K4me and H3K27me it is associated with constitutively repressed chromatin (93). Several H3K9me-specific HKMTs have been identified. The G9a protein catalyzes mono- and dimethylation of H3K9 (94) and has an important role in the stable gene silencing during lineage commitment (95). The trimethylation of H3K9 in euchromatic regions is established by SETDB1 (ESET) (96), whereas propagation of the mark in already repressed chromatin is supported by the SUV39H1 methyltransferase (75). Mechanistically, this process shows parallels to the function of the PcG repressive systems. After H3K9 is methylated the structural protein HP-1 binds to the modification and recruits further SUV39H1 molecules, which then methylate adjacent nucleosomes. HP-1 binding is also stabilized by additional factors like Acf1 or DEK (30,97). Apart from that, the bound HP-1 proteins build multimeres and further close the chromatin structure. This way a cycle of setting, spreading and maintaining a closed repressive chromatin structure is established by the three components: SUV39H1, H3K9me and HP-1.
Recently, specific KDMs have been reported for the three above mentioned modifications. KDM5C (H3K4me3 (98)), KDM6B (H3K27me (99)), KDM4A (H3K9me (100)) are able to remove those modifications from their respective sites and erase their epigenetic label. These discoveries show the flexibility and dynamic of the regulatory system that is histone modification.
2.5.2 Histone variants
Because of their important role in fundamental cellular processes histones are a highly conserved class of proteins. Nevertheless, histone variants represent a crucial element of transcriptional regulation beside canonical histones (101). Except histone H4, all histones also have non-canonical forms and exist in different variations (102). With at least 11 isoforms, the linker histone H1 is the most diverse of them. Although the canonical form of H1 promotes chromatin condensation and repression, H1 variants are thought to have multiple functions both in active and repressed genomic regions (103).
All histone variants influence histone-histone interactions and confer their unique characteristics to the chromatin structure. One example is altered remodeling complex binding (104). Histone variants can be divided into two groups; somatic histone variants and a large group of testis-specific histone variants (105). The latter is owed to a remarkable and unique mechanism of chromatin compaction in sperm cells. A
Figure 5: Protein complexes that bind to different methylated lysines. (adapted from Kouzarides et al., 2007)
dynamic histone variant exchange is essential also after fertilization since it helps to establish the de novo chromatin landscape (106,107).
A very well-studied histone variant is H3.3, a histone preferentially marked by HPTMs that are associated with active transcription (108). A knock-down of H3.3 in mouse cells resulted in overcondensation, mis-segregation and developmental arrest (109).
Simultaneously, H3.3 is required for proper H3K27me placement at developmental genes (105). Another variant, H2A.Z, also shows developmental defects. Here, an ablation of H2A.Z decreases H3K4me and H3K27me3 levels at the same time (110,111). As a result the balance between self-renewal and lineage-commitment is disrupted and a premature differentiation takes place. Those two examples emphasize the importance of histone variants in early development and stem cell pluripotency.
Apart from that, they also seem to play a role in late development and non-dividing cells. H3.3 accumulates in the mature rodent brain which might indicate a role in neuron plasticity (112). Similarly there is evidence for a differential expression of H2A and H2B variants in neurons compared to mitotic cells (113).
But histone variants also have non-development related functions like DNA-damage response or mitosis. DNA damage repair is less efficient in absence of H2A.X and cells show a reduced proliferation capacity (114). The H3-like centromeric protein A (CENP-A) is essential for mitosis and cell survival because it maintains the identity of the centromere through cell division; and it is overexpressed in many human cancers (115). Further, the importance of histone variants is confirmed by the fact that mutations in their genes are often disease relevant factors (116).
In summary, the functions of histone variants are diverse and they play a role in almost every chromatin-related form of regulation; from chromatin organization before and after fertilization over crucial roles in proper development to DNA-damage response and dysregulation in cancer and disease. However, they do not stand alone but rather fulfill their function in combination with HPTMs and other epigenetic mechanisms like chromatin remodeling.
2.6 Chromatin remodeling complexes (CRC)
Chromatin remodeling is the ATP-driven process of nucleosomal sliding, eviction and insertion (117) that regulates and maintains transcriptional states (118). It also translates the epigenetic information from histone modifications or histone variants into changes of higher order chromatin structure (29). Besides transcriptional regulation, remodeling complexes also coordinate the assembly and spacing of nucleosomes on newly synthesized DNA strands. Chromatin remodelers can be characterized by five basic functions: an affinity for nucleosomes, a histone modification binding domain, a conserved ATPase subunit, an ATPase regulating subunit and several domains that interact with other proteins and transcription factors (29). CRCs are large multiprotein complexes (119) divided into four subgroups according to their characteristic functional motifs (120,121).
First evidence of a protein complex altering transcription and chromatin structure at the same time was found in yeast (122). The protein in question turned out to be the catalytic ATPase subunit of SWI/SNF; a highly conserved remodeling complex that consists of up to 14 subunits (123). In humans SWI/SNF is represented by the BAF
complex (Brg1- or Brahma-associated factor) (121,124). It lacks a role in chromatin assembly which is present in all other subgroups (29). The ATPase domain consists of either one of two subunits with different functions in vivo (125). Whereas BRG1 is absolutely essential for self-renewal and embryogenesis (126) and more abundant in proliferative tissue (127), BRM shows a differential expression pattern and is restricted to non-proliferating tissues (128). However, BRG1 also seems to possess a function in postmitotic neurons (129). Apart from two different ATPases the BAF complex family is characterized by functional varying subunit compositions (130). During mouse stem cell differentiation to neural progenitor cells the subunits BRG1, BAF53a and BAFF155 are exchanged for BRM and BAF170 (131). Similarly, in postmitotic neurons the subunits BAF53b, BAF45b and BAF45c are upregulated instead. This subunit switch is essential for the function of the BAF complex to maintain different developmental stages; and it correlates with stage-specific gene expression (121).
The ISWI family of remodelers distinguishes itself by a characteristic SANT-SLIDE domain which is important for substrate recognition and the interaction with histones and nucleosomal DNA (132). It is located in the C-terminal half of two ATPase complexes (SNF2H and SNF2L) (133). SNF2H is found in a large number of complexes (134) and also associates with subunits of the NuRD-complex (see CHD- family). SNF2L was found in the human NURF complex, a remodeling factor involved in transcriptional activation of neurodevelopmental genes (135). A subunit of the NURF complex, BPTF, specifically binds to H3K4me3 at HOX genes (136) and is important for mammalian development.
All members of the CHD family contain a tandem chromodomain that is able to bind methylated histone tails (137). They can be further subdivided into CHD1 and NuRD remodelers. The CHD1 group promotes active transcription through nucleosome sliding and eviction (29). On the other hand, members of the NuRD complex (CHD3/4) are involved in transcriptional silencing. The NuRD complex does not bind to free DNA but targets chromatin and methylated DNA (138). The two intrinsic HDACs 1 and 2 deacetylate target sites and further restrict translational activation (60).
The last group of remodelers is the so-called INO80 family with a typically split ATPase domain. The large insertion between the two halves of the enzyme allows other proteins to bind. INO80 members are important for DNA repair and nucleosome exchange (29).
The above description of some of the protein families involved in epigenetic processes shows that chromatin structure is not static. Instead it is very dynamically regulated by a multitude of interdependent mechanisms (139) to ensure proper control of the higher order chromatin structure that is so important for development .
2.7 Mammalian cell cycle
In order to sustain a tissue its cells need to fulfil three fundamental functions. Those are proliferartion, differentiation and cell death. The cell cycle is the highly coordinated sequence of those events that happen during an in between one cell division. Cell cycle itself consists two general phases. The shorter M-phase (or mitosis) is when the duplication of the chromosomes and the actual cell division take place. The Interphase is almost 10 times longer than mitosis and defined through cell growth and various
metabolic activities (140). Both interphase and mitosis are further subdivided into several other stages (141). The G1-phase signifies the first “gap”-phase after cell division in which cell organelles and cytoplasm are substituted. To prevent damaged DNA from being further propagated the cell also initializes DNA-repair steps during this phase. Lastly, in order to prepare for the subsequent DNA-synthesis phase (or S- phase) the cell starts to produce mRNA for protein synthesis, energy-rich compounds like ATP to compensate for the high energy demand during this phase and DNA synthesis enzymes (DNA-polymerases, ligases etc.). During S-phase the cell duplicates its genome and after this process each chromosome consists once again of two sister chromatids. The last phase of the interphase is the so-called postsynthetic or premitotic G2-Phase. Here the cell is preparing for the upcoming cell division by retracting extracellular contacts and taking up liquid in order to gain more volume. After completing the three stages of the interphase the cell is now ready to divide once again and enters mitosis. Mitosis itself is very short and only takes around 20 minutes. It is subdivided into 5 highly orchestrated phases named pro-, prometa-, meta-, ana-, and telophase. Upon completion of mitosis the cell either continues with the next G1-phase or exits the cell cycle to enter the so-called G0-phase. The G0-phase is a resting phase in which cells do not progress further through the cell cycle but remain static. This state can either be permanent or reversible depending on the cell type and differentiation state. Cells in G0 that are able to re-enter cell cycle are called “quiescent”. Those cells (e.g. hepatocytes) are able to proliferate again upon certain stimuli even after weeks and months in G0. Other cells (e.g. neurons) are not able to re-enter cell cycle once they are fully differentiated and thus are called “postmitotic”.
2.7.1 Cell cycle regulation and checkpoints
The cell cycle is an immensely coordinated biological process. To deal with such a complicated task in an orderly fashion an intricate ensemble of cell cycle–associated proteins is used. Those include cyclins, cyclin-dependent kinases (CDKs), kinases and phosphatases which all work together either in synchrony or rapid succession to ensure cell cycle progression. Cyclins and their respective kinases form complexes to exert their function. Each cyclin is expressed in a specific period during cell cycle and initiates the transition to a new cell cycle phase (142). After that it is rapidly degraded again while other cyclins are upregulated. If certain mitosis inducing factors (mitogens) are present in G1-phase the D-cyclins (CCND1, 2, 3) form a complex together with their specific protein kinases CDK2, 4 and 6 which is called SPF (S-phase promoting factor). For the transition from late G1- to S-phase another complex (cyclin-E-CDK2- complex) is needed. Shortly before the start of DNA replication cyclin A (CCNA) is upregulated. CCNA also activates CDK2 and both build a stable complex until the end of G2-phase. During late S- and G2-phase cyclin B (CCNB) is upregulated and associates with CDK1 to form the MPF (mitosis promoting factor). However, the phosphorylation of CDK1 keeps this complex inactive until the end of G2-phase. Upon dephosphorylation through the protein phosphatase CDC25 the CDK1-CCNB-complex is translocated into the nucleus where mitosis is initialized. There, the complex stays active until CCNB is degraded during meta- to anaphase transition.
Figure 6: cell cycle-dependent activity of CDK-complexes (adapted from Müller-Esterl, 2004)
To ensure a functional cell cycle there is also a vast amount of negative cell cycle regulators. They can be roughly divided into two groups. The first group is comprised of the CDK inhibitory proteins (CIP). By inhibiting all cyclin-kinase-complexes, they stop G0/1- to S-phase transition. One example for a CIP-family member is the p27 protein. P27 controls the transition from G0- to G1-phase. It is expressed in postmitotic cells and prevents cell cycle re-entry. The second group of inhibitors is called Ink4 (inhibitor of kinase 4). They solely inhibit the CDK4/6-CCND-complexes and stall G1- phase progression. Another type of cell cycle regulation is provided by tumour suppressor genes like the retinoblastoma protein (pRb). pRb also controls the transition from G1- to S-phase and therefore prevents the replication of damaged DNA. In its unphosphorylated state pRb encapsulates the transcription factor E2F which in turn is essential for the induction of the S-phase. Only upon phosphorylation by CDK4/6- CCND-complexes during G1-phase does pRb release E2F. The transcription factor then binds to the promoters of genes that are important for DNA replication (like DNA- polymerase) and activates their transcription.
Cyclins, CDKs and cell cycle inhibitors provide the basic schedule for normal cell cycle regulation. However, certain external and internal factors are able to stop cell cycle progression at specifically implemented checkpoints. Those are necessary to control for incomplete or erroneous cell cycle phases. External factors that can lead to cell cycle arrest and G0-phase entry are for example cell culture density or insufficient supply of nutrients. Internal factors like critical cell mass, DNA damage and incomplete replication can also induce a proliferation stop or even apoptosis. Each of these factors triggers one or more checkpoints at specific stages during cell cycle. DNA damage events for example activate cell cycle checkpoints before (G1-phase), during (mid-S-phase) and after (G2-phase) replication. Another critical step is the so-called
“spindle checkpoint”. It monitors the alignment of the chromosomes on the equatorial plate and the connection of spindle fibres to the kinetochore during metaphase. In the subsequent anaphase, the two sister chromatids of the chromosome are separated; but only if all chromosomes are correctly aligned and the spindle fibres are connected to both centromeres via the kinetochore.
Those checkpoints are the “safeguards” against improper cell division and DNA replication which might lead to uncontrolled cell proliferation and cancer. But what if
they detect irregularities in the cell cycle program and repair mechanisms fail to correct the mistake? In this case the cell goes into a self-destruction mode also known as apoptosis.
2.7.2 Cell death
Apoptosis is a tightly and actively controlled process that is crucial for many physiological functions like limb development. This makes it fundamentally different from necrosis, an involuntary and chaotic cell death often caused by severe external damage of tissue (e.g. sunburn). The process of apoptosis itself can be grouped into three different phases; initiation phase, effector phase and execution phase. Although there are many different stimuli that are able to trigger apoptosis, there are only two well-studied signalling pathways that lead to the induction of the process; the intrinsic and the extrinsic pathway. The extrinsic pathway is executed by the so-called “death – receptors” of the tumour necrosis factor (TNF) superfamily. The natural ligands of TNF receptors are TNF itself and other cytokines. TNF-receptors do not possess an enzymatic activity. That is why they recruit cytoplasmic adaptor proteins or “death domains”. They form the DISC (death inducing signalling complex) which starts a proteolytic signalling cascade that leads to cell death. The intrinsic pathway can take place with or without the help of death receptors. Internal signals like DNA damage or oxidative stress lead to caspase activation (see below) which in turn activates proteins that influence mitochondrial permeability. The increased permeability of the outer mitochondrial membrane leads to the release of cytochrome c into the cytoplasm. This activates further caspases and finally initiates apoptosis. In both extrinsic and intrinsic pathway, caspases play a decisive role. Caspases are intracellular protease enzymes that cut their substrate proteins selectively at aspartate residues. To date there are 14 different caspases known in man, with 7 of them involved in apoptosis. Caspases are expressed as inactive proenzymes which are activated through auto-proteolysis or other proteins. Some caspases participate in the induction of apoptosis (initiator caspases).
Those include the caspases 2, 8, 9 and 10. Others operate downstream of an amplifying proteolytic signal cascade and are involved in the final apoptotic degradation process of the cell. They are called effector caspases and include the caspases 3, 6 and 7. Apart from caspases there is a whole plethora of apoptotic and antiapoptotic molecules that help to regulate the apoptotic sensitivity and the apoptotic program of a cell (143).
2.7.3 Cell cycle, apoptosis and disease
If apoptotic and/or cell cycle regulation goes wrong, it can lead to severe problems in cellular function. On the opposing ends of this large spectrum of cellular defects are cancer or degeneration diseases (144). Therefore, controlling the balance between degeneration and proliferation helps to maintain a functional equilibrium in the body.
As mentioned above postmitotic cells have exited the cell cycle and are no longer able to re-enter it upon stimulation. Therefore they should not be affected by cell cycle deregulation and subsequent proliferative signals. It was shown however that cell cycle induction in postmitotic neurons leads to cell death (145). This finding is already more than 20 years old and since then researchers found evidence for cell cycle-related events in Alzheimer`s disease (AD), Parkinson`s disease (PD), amyotrophic lateral
sclerosis (ALS) and stroke ataxia-telangiectasia (AT) (146-148). Based on this, some hypothesized that the cell cycle induction seen in many neurodegenerative diseases is due to a deregulation of apoptosis and/or cell cycle; a process often seen in cancer phenotypes of other tissues (149). What strengthens this theory is that to date no cancer of real postmitotic origin has been reported. Re-entrance into cell cycle is associated with neuronal cell death in a number of mouse models (150,151) and in vitro studies (152,153). Apart from models that are not disease-related, several transgenic AD mouse models also show abnormal cell cycle processes including expression of cell cycle proteins and DNA replication (154). Recently, it was shown that by blocking cell cycle, cell death could be averted in cultures of neurodegenerative disease models (155).
But despite the availability of models that simulate a cell cycle-related neurodegeneration, it is still unclear what ultimately causes cell death in the real disease pathology. Some propose cell death as consequence of genomic instability (aneuploidy) caused by DNA replication (156). This would explain the high incidence of AD pathology in Down syndrome patients (157). Another possible explanation could be provided by the observation that cell cycle deregulation in postmitotic cells has a synergistic effect together with other cellular insults. Neurons treated with UV to induce DNA damage also need irregular cyclin activity to introduce cell death (158).
This observation led to the so-called double-hit hypothesis. In short, an irregularity like heightened oxidative stress levels or elevated cell cycle markers can persist for years without the cell going into apoptosis. Only after the second insult of the two takes place does apoptosis occur. This accumulative effect might explain the late onset of those neurodegenerative disease types.
2.8 Aims of the thesis
Neuronal development is one of the most complex processes in human ontology. The same is true for neurodegeneration. Both are very difficult to study because they represent slowly progressing events that depend on more or less precise spatio- temporal cues. Epigenetic regulation is crucial in neurodevelopment and neurodegeneration as it links chromatin structure and the transcriptional phenotype.
The overall aim of this thesis was to assess neuron-specific mechanisms in the chromatin structure and organization of neurons. To this end, we deployed several neuronal cell types and differentiation systems in order to:
1. find differences in the transcriptional profile of epigenetic modifier genes between our various cell systems
2. describe the postmitotic neuronal phenotype on an epigenetic level by using immunofluorecence stainings and ChIP analysis for histone modifications and chromatin structure proteins
3. create and characterize a new model system for cell cycle-related neurodegeneration
4. asses the role of epigenetic regulation in this model (in particular the function of transciptionally restrictive chromatin and its associated factors).
3 Extensive transcriptional regulation of chromatin modifiers during human neurodevelopment
Matthias K. Weng1,2, Bastian Zimmer1, Dominik Pöltl1,2, Marc P. Broeg3, Violeta Ivanova4,2,5, John A. Gaspar6, Agapios Sachinidis6, Ullrich Wüllner7, Tanja Waldmann1,
8, *and Marcel Leist1, *
1 Doerenkamp-Zbinden Department of in vitro Toxicology and Biomedicine, University of Konstanz, Konstanz, Germany.
2 Konstanz Research School Chemical Biology, University of Konstanz, Konstanz, Germany.
3 Department of Computer Graphics and Media Design, University of Konstanz, Konstanz, Germany.
4 Department of Bioinformatics and Information Mining, University of Konstanz, Konstanz, Germany.
5 Graduate school for Computer and Information Science, University of Konstanz, Konstanz, Germany.
6 Institute of Neurophysiology, University of Cologne, Cologne, Germany.
7 Department of Neurology, Bonn University Hospital, Bonn, Germany.
8 corresponding author: tanja.waldmann@uni-konstanz.de
*
these authors contributed equallyPLoS One 2012; 7(5):e36708. Epub 2012 May 9.
3.1 Abstract
Epigenetic changes, including histone modifications or chromatin remodeling are regulated by a large number of human genes. We developed a strategy to study the coordinate regulation of such genes, and to compare different cell populations or tissues. A set of 150 genes, comprising different classes of epigenetic modifiers was compiled. This new tool was used initially to characterize changes during the differentiation of human embryonic stem cells (hESC) to central nervous system neuroectoderm progenitors (NEP). qPCR analysis showed that more than 60% of the examined transcripts were regulated, and > 10% of them had a > 5-fold increased expression. For comparison, we differentiated hESC to neural crest progenitors (NCP), a distinct peripheral nervous system progenitor population. Some epigenetic modifiers were regulated into the same direction in NEP and NCP, but also distinct differences were observed. For instance, the remodeling ATPase SMARCA2 was up-regulated > 30- fold in NCP, while it remained unchanged in NEP; up-regulation of the ATP-dependent chromatin remodeler CHD7 was increased in NEP, while it was down-regulated in NCP. To compare the neural precursor profiles with those of mature neurons, we analyzed the epigenetic modifiers in human cortical tissue. This resulted in the identification of 30 regulations shared between all cell types, such as the histone methyltransferase SETD7. We also identified new markers for post-mitotic neurons, like the arginine methyl transferase PRMT8 and the methyl transferase EZH1. Our findings suggest a hitherto unexpected extent of regulation, and a cell type-dependent specificity of epigenetic modifiers in neurodifferentiation.
