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The eukaryotic Plasmodium falciparum parasite employs multiple levels of gene regulation to alter its morphology throughout its complex life cycle to survive different environments it encounters in mosquitoes and humans. Thus, epigenetic mechanisms that control gene regulation are integral to the parasite’s survival. Histone lysine acetylation is the post-translational modification of histones, generally associated with eukaryotic transcriptional activation. This epigenetic modification is recognised by bromodomain, and bromodomain-containing proteins (BDPs) can recruit transcription factors to promote gene expression. In the P. falciparum genome, eight bromodomain-containing proteins were identified but only two have been functionally characterised. This thesis detailed the works done on the uncharacterised P. falciparum bromodomain protein 2 (PfBDP2). I explored cellular and epigenomic localisations of PfBDP2 in the asexual schizont-stage P. falciparum parasites via biochemical assays and native chromatin immunoprecipitation, which revealed that PfBDP2 is a nuclear protein expressed throughout the asexual cycle and is enriched within the 5' intergenic region of invasion genes in schizonts. PfBDP2 is also enriched within heterochromatin, particularly across the promoters of silent multigene families and across var gene introns. This points to PfBDP2 being a chromatin protein with dual epigenetic roles, associated with both gene expression and silencing. This thesis also reports a growth delay upon the conditional disruption of PfBDP2 expression in vitro, indicating its importance for normal growth during the asexual intraerythrocytic cycle. The inability to recover stable PfBDP2 knockouts following knockout induction suggests that PfBDP2 may be essential to blood-stage growth and could be druggable by novel antimalarials. Finally, this thesis also detailed the epigenomic localisation of two unique Apicomplexan histone variants, PfH2A.Z and PfH2B.Z, and their acetyl

H2A.B is an evolutionarily distant histone H2A variant that accumulates on DNA repair sites, DNA replication sites, and actively transcribing regions in genomes. In cells, H2A.B exchanges rapidly in chromatin, but the mechanism has remained enigmatic. In the present study, we found that the H2A.B-H2B dimer incorporated within the nucleosome exchanges with the canonical H2A-H2B dimer without assistance from additional factors, such as histone chaperones and nucleosome remodelers. High-speed atomic force microscopy revealed that the H2A.B nucleosome, but not the canonical H2A nucleosome, transiently forms an intermediate “open conformation”, in which two H2A.B-H2B dimers may be detached from the H3-H4 tetramer and bind to the DNA regions near the entry/exit sites. Mutational analyses revealed that the H2A.B C-terminal region is responsible for the adoption of the open conformation and the H2A.B-H2B exchange in the nucleosome. These findings provide mechanistic insights into the histone exchange of the H2A.B nucleosome.

Canonical histones (H2A, H2B, H3, and H4) are present in all eukaryotes where they package genomic DNA and participate in numerous cellular processes, such as transcription regulation and DNA repair. In addition to the canonical histones, there are many histone variants, which have different amino acid sequences, possess tissue-specific expression profiles, and function distinctly from the canonical counterparts. A number of histone variants, including both core histones (H2A/H2B/H3/H4) and linker histones (H1/H5), have been identified to date. Htz1 (H2A.Z) and CENP-A (CenH3) are present from yeasts to mammals, and H3.3 is present from Tetrahymena to humans. In addition to the prevalent variants, others like H3.4 (H3t), H2A.Bbd, and TH2B, as well as several H1 variants, are found to be specific to mammals. Among them, H2BFWT, H3.5, H3.X, H3.Y, and H4G are unique to primates (or Hominidae). In this review, we focus on localization and function of primate- or hominidae-specific histone variants.

Canonical histones (H2A, H2B, H3, and H4) are present in all eukaryotes where they package genomic DNA and participate in numerous cellular processes, such as transcription regulation and DNA repair. In addition to the canonical histones, there are many histone variants, which have different amino acid sequences, possess tissue-specific expression profiles, and function distinctly from the canonical counterparts. A number of histone variants, including both core histones (H2A/H2B/H3/H4) and linker histones (H1/H5), have been identified to date. Htz1 (H2A.Z) and CENP-A (CenH3) are present from yeasts to mammals, and H3.3 is present from Tetrahymena to humans. In addition to the prevalent variants, others like H3.4 (H3t), H2A.Bbd, and TH2B, as well as several H1 variants, are found to be specific to mammals. Among them, H2BFWT, H3.5, H3.X, H3.Y, and H4G are unique to primates (or Hominidae). In this review, we focus on localization and function of primate- or hominidae-specific histone variants.

Arabidopsis thaliana 45S ribosomal genes (rDNA) are located in tandem arrays called nucleolus organizing regions on the termini of chromosomes 2 and 4 (NOR2 and NOR4) and encode rRNA, a crucial structural element of the ribosome. The current model of rDNA organization suggests that inactive rRNA genes accumulate in the condensed chromocenters in the nucleus and at the nucleolar periphery, while the nucleolus delineates active genes. We challenge the perspective that all intranucleolar rDNA is active by showing that a subset of nucleolar rDNA assembles into condensed foci marked by H3.1 and H3.3 histones that also contain the repressive H3K9me2 histone mark. By using plant lines containing a low number of rDNA copies, we further found that the condensed foci relate to the folding of rDNA, which appears to be a common mechanism of rDNA regulation inside the nucleolus. The H3K9me2 histone mark found in condensed foci represents a typical modification of bulk inactive rDNA, as we show by genome-wide approaches, similar to the H2A.W histone variant. The euchromatin histone marks H3K27me3 and H3K4me3, in contrast, do not colocalize with nucleolar foci and their overall levels in the nucleolus are very low. We further demonstrate that the rDNA promoter is an important regulatory region of the rDNA, where the distribution of histone variants and histone modifications are modulated in response to rDNA activity.

Testis-specific regulators of chromatin function are commonly ectopically expressed in human cancers, but their roles are poorly understood. Examination of 81 primary Hodgkin lymphoma (HL) samples showed that the ectopic expression of the eutherian testis-specific histone variant H2A.B is an inherent feature of HL. In experiments using two HL cell lines derived from different subtypes of HL, H2A.B knockdown inhibited cell proliferation. H2A.B was enriched in both nucleoli of these HL cell lines and primary HL samples. We found that H2A.B enhanced ribosomal DNA (rDNA) transcription, was enriched at the rDNA promoter and transcribed regions, and interacted with RNA Pol I. Depletion of H2A.B caused the loss of RNA Pol I from rDNA chromatin. Remarkably, H2A.B was also required for high levels of ribosomal protein gene expression being located at the transcriptional start site and within the gene body. H2A.B knockdown reduced gene body chromatin accessibility of active RNA Pol II genes concurrent with a decrease in transcription. Taken together, our data show that in HL H2A.B has acquired a new function, the ability to increase ribosome biogenesis.

Canonical histones (H2A, H2B, H3, and H4) are present in all eukaryotes where they package genomic DNA and participate in numerous cellular processes, such as transcription regulation and DNA repair. In addition to the canonical histones, there are many histone variants, which have different amino acid sequences, possess tissue-specific expression profiles, and function distinctly from the canonical counterparts. A number of histone variants, including both core histones (H2A/H2B/H3/H4) and linker histones (H1/H5), have been identified to date. Htz1 (H2A.Z) and CENP-A (CenH3) are present from yeasts to mammals, and H3.3 is present from Tetrahymena to humans. In addition to the prevalent variants, others like H3.4 (H3t), H2A.Bbd, and TH2B, as well as several H1 variants, are found to be specific to mammals. Among them, H2BFWT, H3.5, H3.X, H3.Y, and H4G are unique to primates (or Hominidae). In this review, we focus on localization and function of primate- or hominidae-specific histone variants.

NAD metabolism is essential for all forms of life. Compartmental regulation of NAD consumption, especially between the nucleus and the mitochondria, is required for energy homeostasis. However, how compartmental regulation evolved remains unclear. In the present study, we investigated the evolution of the macrodomain-containing histone variant macroH2A1.1, an integral chromatin component that limits nuclear NAD consumption by inhibiting poly(ADP-ribose) polymerase 1 in vertebrate cells. We found that macroH2A originated in premetazoan protists. The crystal structure of the macroH2A macrodomain from the protist Capsaspora owczarzaki allowed us to identify highly conserved principles of ligand binding and pinpoint key residue substitutions, selected for during the evolution of the vertebrate stem lineage. Metabolic characterization of the Capsaspora lifecycle suggested that the metabolic function of macroH2A was associated with nonproliferative stages. Taken together, we provide insight into the evolution of a chromatin element involved in compartmental NAD regulation, relevant for understanding its metabolism and potential therapeutic applications.

Organization of the genetic information into chromatin plays an important role in the regulation of all DNA templatebased reactions. The incorporation of different variant versions of the core histones H3, H2A, and H2B, or the linker histone H1 results in nucleosomes with unique properties. Histone variants can differ by only a few amino acids or larger protein domains and their incorporation may directly affect nucleosome stability and higher order chromatin organization or indirectly influence chromatin function through histone variant-specific binding partners. Histone variants employ dedicated histone deposition machinery for their timely and locus-specific incorporation into chromatin. Plants have evolved specific histone variants with unique expression patterns and features. In this review, we discuss our current knowledge on histone variants in Arabidopsis, their mode of deposition, variant-specific posttranslational modifications, and genome-wide distribution, as well as their role in defining different chromatin states.

BackgroundGene bodies of vertebrates and flowering plants are occupied by the histone variant H3.3 and DNA methylation. The origin and significance of these profiles remain largely unknown. DNA methylation and H3.3 enrichment profiles over gene bodies are correlated and both have a similar dependence on gene transcription levels. This suggests a mechanistic link between H3.3 and gene body methylation.ResultsWe engineered an H3.3 knockdown in Arabidopsis thaliana and observed transcription reduction that predominantly affects genes responsive to environmental cues. When H3.3 levels are reduced, gene bodies show a loss of DNA methylation correlated with transcription levels. To study the origin of changes in DNA methylation profiles when H3.3 levels are reduced, we examined genome-wide distributions of several histone H3 marks, H2A.Z, and linker histone H1. We report that in the absence of H3.3, H1 distribution increases in gene bodies in a transcription-dependent manner.ConclusionsWe propose that H3.3 prevents recruitment of H1, inhibiting H1's promotion of chromatin folding that restricts access to DNA methyltransferases responsible for gene body methylation. Thus, gene body methylation is likely shaped by H3.3 dynamics in conjunction with transcriptional activity.

BackgroundGene bodies of vertebrates and flowering plants are occupied by the histone variant H3.3 and DNA methylation. The origin and significance of these profiles remain largely unknown. DNA methylation and H3.3 enrichment profiles over gene bodies are correlated and both have a similar dependence on gene transcription levels. This suggests a mechanistic link between H3.3 and gene body methylation.ResultsWe engineered an H3.3 knockdown in Arabidopsis thaliana and observed transcription reduction that predominantly affects genes responsive to environmental cues. When H3.3 levels are reduced, gene bodies show a loss of DNA methylation correlated with transcription levels. To study the origin of changes in DNA methylation profiles when H3.3 levels are reduced, we examined genome-wide distributions of several histone H3 marks, H2A.Z, and linker histone H1. We report that in the absence of H3.3, H1 distribution increases in gene bodies in a transcription-dependent manner.ConclusionsWe propose that H3.3 prevents recruitment of H1, inhibiting H1's promotion of chromatin folding that restricts access to DNA methyltransferases responsible for gene body methylation. Thus, gene body methylation is likely shaped by H3.3 dynamics in conjunction with transcriptional activity.

CENP-A is a histone H3 variant key to epigenetic specification of mammalian centromeres. Using transient overexpression of CENP-A mutants, two recent reports in Developmental Cell proposed essential centromere functions for post-translational modifications of human CENP-A. Phosphorylation at Ser68 was proposed to have an essential role in CENP-A deposition at centromeres. Blockage of ubiquitination at Lys124 was proposed to abrogate localization of CENP-A to the centromere. Following gene inactivation and replacement in human cells, we demonstrate that CENP-A mutants that cannot be phosphorylated at Ser68 or ubiquitinated at Lys124 assemble efficiently at centromeres during G1, mediate early events in centromere establishment at an ectopic chromosomal locus, and maintain centromere function indefinitely. Thus, neither Ser68 nor Lys124 post-translational modification is essential for long-term centromere identity, propagation, cell-cycle-dependent deposition, maintenance, function, or mediation of early steps in centromere establishment.

Spermatogenesis involves some of the most dramatic chromatin remodelings observed during any cell differentiation process. It involves the intricate interplay of a variety of somatic and variant histones that, in the late stages, are replaced by transition proteins (TNPs) and subsequently by protamines. Alterations of these chromosomal proteins and their posttranslational modifications can have epigenetic consequences, leading to infertility. This chapter focuses primarily on the disturbance of spermatogenesis resulting from alterations of somatic (H3.3, H2A.X, and H2A.Z) and spermatogenic-specific (TH2B, TH2A, H2BFW, H2A.Bbd, H1t, and HILS1) histone variants, and their implications for infertility. A brief overview of the role of TNPs (TNP1/TNP2) and protamines (P1/P2) is also provided.

Spermatogenesis involves some of the most dramatic chromatin remodelings observed during any cell differentiation process. It involves the intricate interplay of a variety of somatic and variant histones that, in the late stages, are replaced by transition proteins (TNPs) and subsequently by protamines. Alterations of these chromosomal proteins and their posttranslational modifications can have epigenetic consequences, leading to infertility. This chapter focuses primarily on the disturbance of spermatogenesis resulting from alterations of somatic (H3.3, H2A.X, and H2A.Z) and spermatogenic-specific (TH2B, TH2A, H2BFW, H2A.Bbd, H1t, and HILS1) histone variants, and their implications for infertility. A brief overview of the role of TNPs (TNP1/TNP2) and protamines (P1/P2) is also provided.

Spermatogenesis involves some of the most dramatic chromatin remodelings observed during any cell differentiation process. It involves the intricate interplay of a variety of somatic and variant histones that, in the late stages, are replaced by transition proteins (TNPs) and subsequently by protamines. Alterations of these chromosomal proteins and their posttranslational modifications can have epigenetic consequences, leading to infertility. This chapter focuses primarily on the disturbance of spermatogenesis resulting from alterations of somatic (H3.3, H2A.X, and H2A.Z) and spermatogenic-specific (TH2B, TH2A, H2BFW, H2A.Bbd, H1t, and HILS1) histone variants, and their implications for infertility. A brief overview of the role of TNPs (TNP1/TNP2) and protamines (P1/P2) is also provided.

The packaging of genomic information and the regulation of gene expression are both fundamentally important to eukaryotic life. Meters of human DNA must fit inside the micron-diameter nucleus while still rapidly becoming available for templated processes such as transcription, replication, and repair. Therefore, the DNA-protein complex known as chromatin must dynamically transition between more compact, closed states and more accessible, open ones. To fully understand chromatin structure and dynamics, it is necessary to employ a multifaceted approach, integrating different general philosophies and scientific techniques that include experiment and computation. Since the DNA in chromatin is organized into arrays of nucleosomes, we take a bottom-up approach in this dissertation, striving first to understand the structure and dynamics of an individual nucleosome and subdomains thereof. Atomistic computational methods have provided useful tools to study DNA and protein dynamics at the nanosecond, and recently microsecond, timescale. In this dissertation, we present recent developments in the understanding of the nucleosome though atomistic molecular dynamics (MD) simulations. By applying different all-atom MD computational techniques, we demonstrate that replacing the canonical H3 histone with the centromere-specific variant CENP-A translates to greater structural flexibility in the nucleosome, that replacing H3 with CENP-A increases the plasticity of an individual histone dimer, and that the effects of acetylation on the H4 histone tail are cumulative and specific to lysine 16 mono-acetylation.

The protozoan parasite Plasmodium falciparum is the main agent responsible for the severe and potentially fatal form of human malaria, which is predominantly prevalent in tropical and sub-tropical countries. Malaria is transmitted through the bite of an infected female Anopheles mosquito and the parasites eventually invade and multiply asexually in red blood cells. Malaria pathogenesis is associated with the expression of clonally variant proteins on the surface of the infected erythrocyte which mediate cytoadhesion of the infected red blood cell to the endothelium of blood vessels. Thereby the infected erythrocytes sequester in the vasculature and avoid clearance by the spleen. Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) is the main variant antigen expressed on the surface of the infected erythrocyte that is responsible for cytoadhesion and immune evasion. PfEMP1 is encoded by the var multi-gene family and var genes are expressed in a mutually exclusive manner. To escape the human immune system, the parasite switches expression of the single expressed var gene. These pathogenic processes of invasion and cytoadhesion are tightly regulated by epigenetic mechanisms. Epigenetics refers to heritable changes in gene activity that are caused by changes in chromatin structure rather than changes in the DNA sequence. Nucleosomes are the basic repeat elements that make up chromatin and are formed by DNA wrapping around a histone octamer. Chromatin can assume two principle states; the open and transcriptionally competent euchromatin or the tightly packed and silent heterochromatin. The majority of the parasite’s genome exists in a euchromatic state. Genes in the euchromatin compartment are not necessarily active; those that are not are maintained in a readily inducible state and can be transcribed later in the parasite’s life cycle. Some of the gene families involved in pathogenic processes such as invasion and cytoadhesion are largely restricted to the subt

Structural variability within histone families, such as H2A, can be achieved through 2 primary mechanisms: the expression of histone variants and the incorporation of chemical modifications. The histone H2A family contains several variants in addition to the canonical H2A forms. In this review, recent developments in the study of the heteromorphous variants H2A.X, H2A.Z, and macroH2A will be discussed. Particular focus will be given to the post-translational modifications (PTMs) of these variants, including phosphorylation, ubiquitination, acetylation, and methylation. The combination of the newly identified N- and C-terminal tail PTMs expands the multiplicity of roles that the individual H2A variants can perform. It is of additional interest that analogous sites within these different histone variants can be similarly modified. Whether this is a redundant function or a finely tuned one, designed to meet specific needs, remains to be elucidated.

Structural variability within histone families, such as H2A, can be achieved through 2 primary mechanisms: the expression of histone variants and the incorporation of chemical modifications. The histone H2A family contains several variants in addition to the canonical H2A forms. In this review, recent developments in the study of the heteromorphous variants H2A.X, H2A.Z, and macroH2A will be discussed. Particular focus will be given to the post-translational modifications (PTMs) of these variants, including phosphorylation, ubiquitination, acetylation, and methylation. The combination of the newly identified N- and C-terminal tail PTMs expands the multiplicity of roles that the individual H2A variants can perform. It is of additional interest that analogous sites within these different histone variants can be similarly modified. Whether this is a redundant function or a finely tuned one, designed to meet specific needs, remains to be elucidated.

Structural variability within histone families, such as H2A, can be achieved through 2 primary mechanisms: the expression of histone variants and the incorporation of chemical modifications. The histone H2A family contains several variants in addition to the canonical H2A forms. In this review, recent developments in the study of the heteromorphous variants H2A.X, H2A.Z, and macroH2A will be discussed. Particular focus will be given to the post-translational modifications (PTMs) of these variants, including phosphorylation, ubiquitination, acetylation, and methylation. The combination of the newly identified N- and C-terminal tail PTMs expands the multiplicity of roles that the individual H2A variants can perform. It is of additional interest that analogous sites within these different histone variants can be similarly modified. Whether this is a redundant function or a finely tuned one, designed to meet specific needs, remains to be elucidated.

The fundamental building blocks of chromatin are the nucleosomes. Each such unit is composed of about 200 bp of DNA, the well-conserved core histones (H2A, H2B, H3 and H4) and a linker histone (H1). The DNA is wound around two dimers of H2A–H2B and a tetramer comprising two molecules each of H3 and H4, and there is approximately one linker histone molecule positioned on the exterior of the DNA–protein octamer complex. The nucleosome directs the various structural transitions in chromatin that are needed for proper transcriptional regulation during differentiation and development of the organism in question. The gene activity can be regulated by different histone variants, DNA–protein interactions, and protein–protein interactions, all of which are influenced by the enormous amounts of post-translational modifications that occur in the histone tails. The research underlying this thesis focused on different aspects of post-translational modifications during aging, differentiation, and progression of the cell cycle, and also on expression of linker histone variants and linker histone-chromatin interactions in a variety of cells and tissues. The present results are the first to show that H4 can be trimethylated at lysine 20 in mammalian cells. The trimethylated H4K20 was found in rat kidney and liver at levels that rose with increasing age of the nimals, and it was also detected in trace amounts in human cell lines. Furthermore, in differentiating MEL cells, trimethylated H4K20 was localized to heterochromatin, and levels of trimethylated H4K20 increased during the course of cell differentiation and were correlated with the increasing compaction of the chromatin. The chromatin of terminally differentiated chicken and frog erythrocytes is highly condensed, and the linker histone variants it contains vary between the two species. Cytofluorometric analyses revealed that the linker histones in the chicken erythrocytes exhibited higher affinity for chromatin than did those i

The fundamental building blocks of chromatin are the nucleosomes. Each such unit is composed of about 200 bp of DNA, the well-conserved core histones (H2A, H2B, H3 and H4) and a linker histone (H1). The DNA is wound around two dimers of H2A–H2B and a tetramer comprising two molecules each of H3 and H4, and there is approximately one linker histone molecule positioned on the exterior of the DNA–protein octamer complex. The nucleosome directs the various structural transitions in chromatin that are needed for proper transcriptional regulation during differentiation and development of the organism in question. The gene activity can be regulated by different histone variants, DNA–protein interactions, and protein–protein interactions, all of which are influenced by the enormous amounts of post-translational modifications that occur in the histone tails. The research underlying this thesis focused on different aspects of post-translational modifications during aging, differentiation, and progression of the cell cycle, and also on expression of linker histone variants and linker histone-chromatin interactions in a variety of cells and tissues. The present results are the first to show that H4 can be trimethylated at lysine 20 in mammalian cells. The trimethylated H4K20 was found in rat kidney and liver at levels that rose with increasing age of the nimals, and it was also detected in trace amounts in human cell lines. Furthermore, in differentiating MEL cells, trimethylated H4K20 was localized to heterochromatin, and levels of trimethylated H4K20 increased during the course of cell differentiation and were correlated with the increasing compaction of the chromatin. The chromatin of terminally differentiated chicken and frog erythrocytes is highly condensed, and the linker histone variants it contains vary between the two species. Cytofluorometric analyses revealed that the linker histones in the chicken erythrocytes exhibited higher affinity for chromatin than did those i

The fundamental building blocks of chromatin are the nucleosomes. Each such unit is composed of about 200 bp of DNA, the well-conserved core histones (H2A, H2B, H3 and H4) and a linker histone (H1). The DNA is wound around two dimers of H2A–H2B and a tetramer comprising two molecules each of H3 and H4, and there is approximately one linker histone molecule positioned on the exterior of the DNA–protein octamer complex. The nucleosome directs the various structural transitions in chromatin that are needed for proper transcriptional regulation during differentiation and development of the organism in question. The gene activity can be regulated by different histone variants, DNA–protein interactions, and protein–protein interactions, all of which are influenced by the enormous amounts of post-translational modifications that occur in the histone tails. The research underlying this thesis focused on different aspects of post-translational modifications during aging, differentiation, and progression of the cell cycle, and also on expression of linker histone variants and linker histone-chromatin interactions in a variety of cells and tissues. The present results are the first to show that H4 can be trimethylated at lysine 20 in mammalian cells. The trimethylated H4K20 was found in rat kidney and liver at levels that rose with increasing age of the nimals, and it was also detected in trace amounts in human cell lines. Furthermore, in differentiating MEL cells, trimethylated H4K20 was localized to heterochromatin, and levels of trimethylated H4K20 increased during the course of cell differentiation and were correlated with the increasing compaction of the chromatin. The chromatin of terminally differentiated chicken and frog erythrocytes is highly condensed, and the linker histone variants it contains vary between the two species. Cytofluorometric analyses revealed that the linker histones in the chicken erythrocytes exhibited higher affinity for chromatin than did those i

The fundamental building blocks of chromatin are the nucleosomes. Each such unit is composed of about 200 bp of DNA, the well-conserved core histones (H2A, H2B, H3 and H4) and a linker histone (H1). The DNA is wound around two dimers of H2A–H2B and a tetramer comprising two molecules each of H3 and H4, and there is approximately one linker histone molecule positioned on the exterior of the DNA–protein octamer complex. The nucleosome directs the various structural transitions in chromatin that are needed for proper transcriptional regulation during differentiation and development of the organism in question. The gene activity can be regulated by different histone variants, DNA–protein interactions, and protein–protein interactions, all of which are influenced by the enormous amounts of post-translational modifications that occur in the histone tails. The research underlying this thesis focused on different aspects of post-translational modifications during aging, differentiation, and progression of the cell cycle, and also on expression of linker histone variants and linker histone-chromatin interactions in a variety of cells and tissues. The present results are the first to show that H4 can be trimethylated at lysine 20 in mammalian cells. The trimethylated H4K20 was found in rat kidney and liver at levels that rose with increasing age of the nimals, and it was also detected in trace amounts in human cell lines. Furthermore, in differentiating MEL cells, trimethylated H4K20 was localized to heterochromatin, and levels of trimethylated H4K20 increased during the course of cell differentiation and were correlated with the increasing compaction of the chromatin. The chromatin of terminally differentiated chicken and frog erythrocytes is highly condensed, and the linker histone variants it contains vary between the two species. Cytofluorometric analyses revealed that the linker histones in the chicken erythrocytes exhibited higher affinity for chromatin than did those i

The fundamental building blocks of chromatin are the nucleosomes. Each such unit is composed of about 200 bp of DNA, the well-conserved core histones (H2A, H2B, H3 and H4) and a linker histone (H1). The DNA is wound around two dimers of H2A–H2B and a tetramer comprising two molecules each of H3 and H4, and there is approximately one linker histone molecule positioned on the exterior of the DNA–protein octamer complex. The nucleosome directs the various structural transitions in chromatin that are needed for proper transcriptional regulation during differentiation and development of the organism in question. The gene activity can be regulated by different histone variants, DNA–protein interactions, and protein–protein interactions, all of which are influenced by the enormous amounts of post-translational modifications that occur in the histone tails. The research underlying this thesis focused on different aspects of post-translational modifications during aging, differentiation, and progression of the cell cycle, and also on expression of linker histone variants and linker histone-chromatin interactions in a variety of cells and tissues. The present results are the first to show that H4 can be trimethylated at lysine 20 in mammalian cells. The trimethylated H4K20 was found in rat kidney and liver at levels that rose with increasing age of the nimals, and it was also detected in trace amounts in human cell lines. Furthermore, in differentiating MEL cells, trimethylated H4K20 was localized to heterochromatin, and levels of trimethylated H4K20 increased during the course of cell differentiation and were correlated with the increasing compaction of the chromatin. The chromatin of terminally differentiated chicken and frog erythrocytes is highly condensed, and the linker histone variants it contains vary between the two species. Cytofluorometric analyses revealed that the linker histones in the chicken erythrocytes exhibited higher affinity for chromatin than did those i

Zusammenfassung Zentromere sind spezifische Abschnitte auf den Chromosomen, welche für den Zusammenbau der Kinetochore während der Mitose unerlässlich sind und die gleichmäßige Aufteilung der Schwesterchromatiden auf die Tochterzellen ermöglichen. Die Überexpression der zentromerspezifischen Histon-H3-Variante CENP-A führt zu einer Fehllokalisierung entlang der Chromosomenarme, was zu einer Fehlregulation der Transkription, zu Segregationsdefekten und folglich zu genomischer Instabilität führt und so zur Tumorentstehung beiträgt. Daher sind Mechanismen notwenig, welche den Einbau von CENP-A auf die Zentromer-Region beschränken. Während einige Aspekte der CENP-A Integration ausführlich untersucht wurden, gibt es noch viele offene Fragen. In dieser Arbeit wurde der Einbau von CENP-A und dessen Verbleib im zentromeren Chromatin des Modellorganismus Drosophila melanogaster untersucht. Im ersten Teil dieser Studie wurden verschiedene Komplexe von CENP-A mit anderen Proteinen identifiziert und charakterisiert. Insbesondere haben wir mit der Histon-Acetyltransferase HAT1 einen bisher unbekannten CENP-A-Komplex identifiziert. Neben der subzellulären Verteilung dieses Komplexes konnten wir zeigen, dass HAT1 für eine effiziente CENP-A Integration in das Chromatin erforderlich ist. Basierend auf diesen Erkenntnissen wurde im zweiten Teil der Arbeit der Beitrag verschiedener posttranslationaler Modifikationen zur Funktion von CENP-A untersucht. Dabei ergab sich, dass die Phosphorylierung von CENP-A an S20 sowohl am neusynthetisierten als auch am Chromatin-gebundenen Protein stattfindet, und dass sie von der Protein Kinase CKII katalysiert wird. Unter Verwendung einer Kombination von biochemischen und in vivo-Markierungsansätzen haben wir gezeigt, dass S20-Phosphorylierung den Abbau von CENP-A in einer von der E3-Ligase SCFPpa und dem Proteasom abhängigen Weise reguliert. Zusätzlich konnten wir beweisen, dass S20-Phosphorylierung die schnelle Entfernung von CENP-A von extra-zent, Centromeres are specialized regions on chromosomes which are required for kinetochore assembly during mitosis to facilitate the faithful segregation of sister chromatids. Overexpression of the centromere-specific histone H3 variant CENP-A results in its mislocalization along chromosome arms leading to transcriptional misregulation, segregation defects and consequently to genomic instability promoting tumorigenesis. Thus, mechanisms must exist to restrict assembly of CENP-A into centromeric chromatin. While some aspects of CENP-A loading have been studied in considerable detail, there are still many open questions. In this work, we have investigated the processes that govern CENP-A loading into and maintenance at centromeric chromatin in the model organism Drosophila melanogaster. In the first part of this study, we have characterized different CENP-A preloading complexes. In particular, we have identified a so far unknown complex of CENP-A with the histone acetyltransferase HAT1. We determined the subcellular distribution of this complex and showed that HAT1 is required for efficient CENP-A assembly into chromatin. Based on these findings, we characterized the posttranslational modification pattern of CENP-A in different cellular fractions and within the different pre-loading complexes. We found that phosphorylation at S20 of CENP-A occurs in soluble as well as chromatin-bound fractions of CENP-A, and we identified CKII as the kinase responsible for this modification. Using a combination of biochemical and in vivo labeling approaches we demonstrate that S20 phosphorylation regulates the degradation of CENP-A in a manner that is dependent on the E3 ligase SCFPpa and the proteasome. We show that S20 phosphorylation promotes the fast clearance of CENP-A from extracentromeric sites, while it does not affect stability of centromeric CENP-A suggesting additional roles for this modification at the centromere. Next we addressed the role of phosphorylation of S77 in, Anming Huang, MAg, Kumulative Dissertation aus drei Artikeln, Zusammenfassung in deutscher Sprache, Dissertation Medical University of Innsbruck 2020

© 2016, International Society for Cellular Oncology.Background: Cancer development is a complex process involving both genetic and epigenetic changes. Genetic changes in oncogenes and tumor-suppressor genes are generally considered as primary causes, since these genes may directly regulate cellular growth. In addition, it has been found that changes in epigenetic factors, through mutation or altered gene expression, may contribute to cancer development. In the nucleus of eukaryotic cells DNA and histone proteins form a structure called chromatin which consists of nucleosomes that, like beads on a string, are aligned along the DNA strand. Modifications in chromatin structure are essential for cell type-specific activation or repression of gene transcription, as well as other processes such as DNA repair, DNA replication and chromosome segregation. Alterations in epigenetic factors involved in chromatin dynamics may accelerate cell cycle progression and, ultimately, result in malignant transformation. Abnormal expression of remodeler and modifier enzymes, as well as histone variants, may confer to cancer cells the ability to reprogram their genomes and to yield, maintain or exacerbate malignant hallmarks. At the end, genetic and epigenetic alterations that are encountered in cancer cells may culminate in chromatin changes that may, by altering the quantity and quality of gene expression, promote cancer development. Methods: During the last decade a vast number of studies has uncovered epigenetic abnormalities that are associated with the (anomalous) packaging and remodeling of chromatin in cancer genomes. In this review I will focus on recently published work dealing with alterations in the primary structure of chromatin resulting from imprecise arrangements of nucleosomes along DNA, and its functional implications for cancer development. Conclusions: The primary chromatin structure is regulated by a variety of epigenetic mechanisms that may be deregulated through gen

© 2016, International Society for Cellular Oncology.Background: Cancer development is a complex process involving both genetic and epigenetic changes. Genetic changes in oncogenes and tumor-suppressor genes are generally considered as primary causes, since these genes may directly regulate cellular growth. In addition, it has been found that changes in epigenetic factors, through mutation or altered gene expression, may contribute to cancer development. In the nucleus of eukaryotic cells DNA and histone proteins form a structure called chromatin which consists of nucleosomes that, like beads on a string, are aligned along the DNA strand. Modifications in chromatin structure are essential for cell type-specific activation or repression of gene transcription, as well as other processes such as DNA repair, DNA replication and chromosome segregation. Alterations in epigenetic factors involved in chromatin dynamics may accelerate cell cycle progression and, ultimately, result in malignant transformation. Abnormal expression of remodeler and modifier enzymes, as well as histone variants, may confer to cancer cells the ability to reprogram their genomes and to yield, maintain or exacerbate malignant hallmarks. At the end, genetic and epigenetic alterations that are encountered in cancer cells may culminate in chromatin changes that may, by altering the quantity and quality of gene expression, promote cancer development. Methods: During the last decade a vast number of studies has uncovered epigenetic abnormalities that are associated with the (anomalous) packaging and remodeling of chromatin in cancer genomes. In this review I will focus on recently published work dealing with alterations in the primary structure of chromatin resulting from imprecise arrangements of nucleosomes along DNA, and its functional implications for cancer development. Conclusions: The primary chromatin structure is regulated by a variety of epigenetic mechanisms that may be deregulated through gen