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Post-translational modifications on histones are well known to regulate chromatin structure and function, but much less information is available on modifications of the centromeric histone H3 variant and their effect at the kinetochore. Here, we report two modifications on the centromeric histone H3 variant CENP-A/Cse4 in the yeast Saccharomyces cerevisiae, methylation at arginine 143 (R143me) and lysine 131 (K131me), that affect centromere stability and kinetochore function. Both R143me and K131me lie in the core region of the centromeric nucleosome, near the entry/exit sites of the DNA from the nucleosome. Unexpectedly, mutation of Cse4-R143 (cse4-R143A) exacerbated the kinetochore defect of mutations in components of the NDC80 complex of the outer kinetochore (spc25-1) and the MIND complex (dsn1-7). The analysis of suppressor mutations of the spc25-1 cse4-R143A growth defect highlighted residues in Spc24, Ndc80, and Spc25 that localize to the tetramerization domain of the NDC80 complex and the Spc24-Spc25 stalk, suggesting that the mutations enhance interactions among NDC80 complex components and thus stabilize the complex. Furthermore, the Set2 histone methyltransferase inhibited kinetochore function in spc25-1 cse4-R143A cells, possibly by methylating Cse4-K131. Taken together, our data suggest that Cse4-R143 methylation and Cse4-K131 methylation affect the stability of the centromeric nucleosome, which is detrimental in the context of defective NDC80 tetramerization and can be compensated for by strengthening interactions among NDC80 complex components., Peer Reviewed

We have used chromosome engineering to replace native centromeric DNA with different test sequences at native centromeres in two different strains of the fission yeast Schizosaccharomyces pombe and have discovered that A + T rich DNA, whether synthetic or of bacterial origin, will function as a centromere in this species. Using genome size as a surrogate for the inverse of effective population size (N-e) we also show that the relative A + T content of centromeric DNA scales with N-e across 43 animal, fungal and yeast (Opisthokonta) species. This suggests that in most of these species the A + T content of the centromeric DNA is determined by a balance between selection and mutation. Combining the experimental results and the evolutionary analyses allows us to conclude that A + T rich DNA of almost any sequence will function as a centromere in most Opisthokonta species. The fact that many G/C to A/T substitutions are unlikely to be selected against may contribute to the rapid evolution of centromeric DNA. We also show that a neo-centromere sequence is not simply a weak version of native centromeric DNA and suggest that neo-centromeres require factors either for their propagation or establishment in addition to those required by native centromeres.

Although central to evolution, the causes of hybrid inviability that drive reproductive isolation are poorly understood. Embryonic lethality occurs when the eggs of the frog X. tropicalis are fertilized with either X. laevis or X. borealis sperm. We observed that distinct subsets of paternal chromosomes failed to assemble functional centromeres, causing their mis-segregation during embryonic cell divisions. Core centromere DNA sequence analysis revealed little conservation among the three species, indicating that epigenetic mechanisms that normally operate to maintain centromere integrity are disrupted on specific paternal chromosomes in hybrids. In vitro reactions combining X. tropicalis egg extract with either X. laevis or X. borealis sperm chromosomes revealed that paternally matched or overexpressed centromeric histone CENP-A and its chaperone HJURP could rescue centromere assembly on affected chromosomes in interphase nuclei. However, although the X. laevis chromosomes maintained centromeric CENP-A in metaphase, X. borealis chromosomes did not and also displayed ultra-thin regions containing ribosomal DNA. Both centromere assembly and morphology of X. borealis mitotic chromosomes could be rescued by inhibiting RNA polymerase I or preventing the collapse of stalled DNA replication forks. These results indicate that specific paternal centromeres are inactivated in hybrids due to the disruption of associated chromatin regions that interfere with CENP-A incorporation, at least in some cases due to conflicts between replication and transcription machineries. Thus, our findings highlight the dynamic nature of centromere maintenance and its susceptibility to disruption in vertebrate interspecies hybrids.

We have used chromosome engineering to replace native centromeric DNA with different test sequences at native centromeres in two different strains of the fission yeast Schizosaccharomyces pombe and have discovered that A + T rich DNA, whether synthetic or of bacterial origin, will function as a centromere in this species. Using genome size as a surrogate for the inverse of effective population size (N-e) we also show that the relative A + T content of centromeric DNA scales with N-e across 43 animal, fungal and yeast (Opisthokonta) species. This suggests that in most of these species the A + T content of the centromeric DNA is determined by a balance between selection and mutation. Combining the experimental results and the evolutionary analyses allows us to conclude that A + T rich DNA of almost any sequence will function as a centromere in most Opisthokonta species. The fact that many G/C to A/T substitutions are unlikely to be selected against may contribute to the rapid evolution of centromeric DNA. We also show that a neo-centromere sequence is not simply a weak version of native centromeric DNA and suggest that neo-centromeres require factors either for their propagation or establishment in addition to those required by native centromeres.

Centromeres are rapidly evolving across eukaryotes, despite performing a conserved function to ensure high-fidelity chromosome segregation. CENP-A chromatin is a hallmark of a functional centromere in most organisms. Due to its critical role in kinetochore architecture, the loss of CENP-A is tolerated in only a few organisms, many of which possess holocentric chromosomes. Here, we characterize the consequence of the loss of CENP-A in the fungal kingdom. Mucor circinelloides, an opportunistic human pathogen, lacks CENP-A along with the evolutionarily conserved CENP-C but assembles a monocentric chromosome with a localized kinetochore complex throughout the cell cycle. Mis12 and Dsn1, two conserved kinetochore proteins, were found to co-localize to a short region, one in each of nine large scaffolds, composed of an ∼200-bp AT-rich sequence followed by a centromere-specific conserved motif that echoes the structure of budding yeast point centromeres. Resembling fungal regional centromeres, these core centromere regions are embedded in large genomic expanses devoid of genes yet marked by Grem-LINE1s, a novel retrotransposable element silenced by the Dicer-dependent RNAi pathway. Our results suggest that these hybrid features of point and regional centromeres arose from the absence of CENP-A, thus defining novel mosaic centromeres in this early-diverging fungus.

Centromeres are rapidly evolving across eukaryotes, despite performing a conserved function to ensure high-fidelity chromosome segregation. CENP-A chromatin is a hallmark of a functional centromere in most organisms. Due to its critical role in kinetochore architecture, the loss of CENP-A is tolerated in only a few organisms, many of which possess holocentric chromosomes. Here, we characterize the consequence of the loss of CENP-A in the fungal kingdom. Mucor circinelloides, an opportunistic human pathogen, lacks CENP-A along with the evolutionarily conserved CENP-C but assembles a monocentric chromosome with a localized kinetochore complex throughout the cell cycle. Mis12 and Dsn1, two conserved kinetochore proteins, were found to co-localize to a short region, one in each of nine large scaffolds, composed of an ∼200-bp AT-rich sequence followed by a centromere-specific conserved motif that echoes the structure of budding yeast point centromeres. Resembling fungal regional centromeres, these core centromere regions are embedded in large genomic expanses devoid of genes yet marked by Grem-LINE1s, a novel retrotransposable element silenced by the Dicer-dependent RNAi pathway. Our results suggest that these hybrid features of point and regional centromeres arose from the absence of CENP-A, thus defining novel mosaic centromeres in this early-diverging fungus.

Centromeres are rapidly evolving across eukaryotes, despite performing a conserved function to ensure high-fidelity chromosome segregation. CENP-A chromatin is a hallmark of a functional centromere in most organisms. Due to its critical role in kinetochore architecture, the loss of CENP-A is tolerated in only a few organisms, many of which possess holocentric chromosomes. Here, we characterize the consequence of the loss of CENP-A in the fungal kingdom. Mucor circinelloides, an opportunistic human pathogen, lacks CENP-A along with the evolutionarily conserved CENP-C but assembles a monocentric chromosome with a localized kinetochore complex throughout the cell cycle. Mis12 and Dsn1, two conserved kinetochore proteins, were found to co-localize to a short region, one in each of nine large scaffolds, composed of an ∼200-bp AT-rich sequence followed by a centromere-specific conserved motif that echoes the structure of budding yeast point centromeres. Resembling fungal regional centromeres, these core centromere regions are embedded in large genomic expanses devoid of genes yet marked by Grem-LINE1s, a novel retrotransposable element silenced by the Dicer-dependent RNAi pathway. Our results suggest that these hybrid features of point and regional centromeres arose from the absence of CENP-A, thus defining novel mosaic centromeres in this early-diverging fungus.

At the most basic level, inheritance in living beings occurs by passing the genomic information such as the DNA sequences from the parent generation to the offspring generation. Hence, it is a fundamental goal for every generation to efficiently express the genomic information and safely pass it on to the next generation. In human and other eukaryotic species, this mission is mediated via chromatin, a macromolecule with intricate hierarchical structure. The fundamental unit of chromatin is called a nucleosome, a complex of histone proteins wrapped around with DNA. To carry out diverse biological functions such as transcription and DNA replication, the DNA-protein complex must dynamically transition between more compact, closed states and more accessible, open ones. To fully understand the chromatin structure and dynamics, it is essential to comprehend the basic structural unit of chromatin, nucleosome. In this dissertation, I present my doctoral research in the exploration of the nucleosome dynamics problem, focusing on the assembly process of histone proteins. From histone monomer to dimer, then to tetramer, octamer, and nucleosome, I used different computational modeling theories and techniques, together with different experimental collaborations, to investigate the overall thermodynamics and specific mechanistic details of nucleosome dynamics at different levels. My work has shed light on the fundamental principles governing the histone protein folding and histone complex assembly, in particular, highlighting similarities and differences between the canonical and variant CENP-A histones.

Chromosome instability (CIN) contributes to the development of many cancer. In this paper, we summarize our recent finding that a novel pathway by which FBW7 loss promotes Centromere Protein A (CENP-A) phosphorylation on Serine 18 through Cyclin E1/CDK2, therefore promoting CIN and tumorigenesis. Our finding demonstrates the importance of CENP-A post-translational modification on modulating centromere and mitotic functions in cancer.

Chromosome instability (CIN) contributes to the development of many cancer. In this paper, we summarize our recent finding that a novel pathway by which FBW7 loss promotes Centromere Protein A (CENP-A) phosphorylation on Serine 18 through Cyclin E1/CDK2, therefore promoting CIN and tumorigenesis. Our finding demonstrates the importance of CENP-A post-translational modification on modulating centromere and mitotic functions in cancer.

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.

The centromere plays a crucial role in genome inheritance — ensuring proper segregation of sister chromatids into each daughter cell. In cancer, especially in later stages of solid tumours, cells are often presented with extensive chromosomal abnormalities. However, the involvement of defective centromeres in the disease formation and progression has not been carefully studied. Hence, my PhD project aimed to investigate the role of defective centromeres in cancer progression using the NCI-60 panel of cancer cell lines. The spectrum of centromere-related abnormalities were first characterised with pan-centromeric FISH probes and then with the addition of CENP-A immunofluorescence. For HOP-92 and SN12C, cell lines showing high prevalence of functional dicentric chromosomes, expansion of single cell clones from the initial heterogeneous population was carried out to further study the involvement of dicentric chromosomes in cancer genomic instability. Neocentromere formation sites in cancer cell lines were investigated using cell lines with high prevalence of neocentric chromosomes. A neocentromere was found in T-47D which marked the first report of a neocentromere discovered in a long-established and widely used cell line, and also the first neocentromere to be reported in breast cancer. Separately, an antibody screen to identify antibodies recognising components of an active centromere in methanol-acetic acid stored cells was performed because thus far, antibodies that are widely used for functional centromere detection mainly worked on freshly harvested cells whereas most cytogenetic samples are stored long-term in methanol-acetic acid fixative. I found a commercially available rabbit monoclonal anti-CENP-C that worked on fixed samples and in addition, I combined three methods (FISH, immunofluorescence and mFISH) to obtain more information from the same metaphase spread. I then proceeded to test anti-CENP-C in my method, CENP-IF-cenFISH-mFISH, on dicentric- and neoce

Human centromeres are defined by chromatin containing the histone H3 variant CENP-A assembled onto repetitive alphoid DNA sequences. By inducing rapid, complete degradation of endogenous CENP-A, we now demonstrate that once the first steps of centromere assembly have been completed in G1/S, continued CENP-A binding is not required for maintaining kinetochore attachment to centromeres or for centromere function in the next mitosis. Degradation of CENP-A prior to kinetochore assembly is found to block deposition of CENP-C and CENP-N, but not CENP-T, thereby producing defective kinetochores and failure of chromosome segregation. Without the continuing presence of CENP-A, CENP-B binding to alphoid DNA sequences becomes essential to preserve anchoring of CENP-C and the kinetochore to each centromere. Thus, there is a reciprocal interdependency of CENP-A chromatin and the underlying repetitive centromere DNA sequences bound by CENP-B in the maintenance of human chromosome segregation.

cntrmr is a part f th chrmsm which is crucially imprtant fr sgrgatin f chrmsms during cll divisin and chsin f sistr chrmatids. Cntrmr idntity and functin is dtrmind by th presence f H3 histn variant CENP-A (Cid in Drsphila) at specific locations in the chromatin, which serves as a nucleation site for the assembly of a host of kinetochore proteins, many of which are yet to be characterized in Drsphila. T identify proteins that interact with Drosophila CENP-A and therefore might be involved in the generation of centromeric chromatin, we applied an affinity purification strategy for CENP-A-associated proteins. To this end, w gnratd cell lines with stably integrated transgenes allowing for inducible expression of CENP-A-GFP or CENP-A-StrepII-Flag fusion proteins. Mass spectrometry analysis after affinity purification using either anti-GFP or Strep-Tactin and anti-Flag affinity resins f nuclear extracts resulted in the identification of a number of potential partners f CENP-A which belong t different functional groups. Among the proteins that were identified with both affinity purification strategies were fly homologues of the previously identified interaction partners of mammalian CENP-A, Caf1 and the FACT complex subunit Dre4. In addition, CG2051 protein was detected. CG2051 corresponds to histn actyl transfras 1 (HAT1) whose orthologs in other species were shown to actylat newly synthesized histn H4 at lysine 5 (K5) and lysine 12 (K12) and which marks H3/H4 dimrs fr lading t chrmatin. Here we find that Drosophila CG2051 is a bona fide histone acetyltransferase and that CENP-A might also serve as a substrate for CG2051. Using a number of biochemical approaches ranging from mutual co-immunoprecipitation experiments to gel filtration and interaction studies of recombinant proteins, we characterized different CENP-A-containing preloading complexes. Furthermore, we showed that RNAi-mediated knock-down of HAT1 in S2 cells leads to a reduction of CENP-A incorporation into ch, Th cntrmr is a part f th chrmsm which is crucially imprtant fr sgrgatin f chrmsms during cll divisin and chsin f sistr chrmatids. Cntrmr idntity and functin is dtrmind by th presence f H3 histn variant CENP-A (Cid in Drsphila) at specific locations in the chromatin, which serves as a nucleation site for the assembly of a host of kinetochore proteins, many of which are yet to be characterized in Drsphila. T identify proteins that interact with Drosophila CENP-A and therefore might be involved in the generation of centromeric chromatin, we applied an affinity purification strategy for CENP-A-associated proteins. To this end, w gnratd cell lines with stably integrated transgenes allowing for inducible expression of CENP-A-GFP or CENP-A-StrepII-Flag fusion proteins. Mass spectrometry analysis after affinity purification using either anti-GFP or Strep-Tactin and anti-Flag affinity resins f nuclear extracts resulted in the identification of a number of potential partners f CENP-A which belong t different functional groups. Among the proteins that were identified with both affinity purification strategies were fly homologues of the previously identified interaction partners of mammalian CENP-A, Caf1 and the FACT complex subunit Dre4. In addition, CG2051 protein was detected. CG2051 corresponds to histn actyl transfras 1 (HAT1) whose orthologs in other species were shown to actylat newly synthesized histn H4 at lysine 5 (K5) and lysine 12 (K12) and which marks H3/H4 dimrs fr lading t chrmatin. Here we find that Drosophila CG2051 is a bona fide histone acetyltransferase and that CENP-A might also serve as a substrate for CG2051. Using a number of biochemical approaches ranging, Mark Boltengagen, Enth. u.a. 3 Veröff. d. Verf. aus den Jahren 2009 - 2013, Innsbruck, Med. Univ., Diss., 2015, OeBB, (VLID)336634

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 centromere, responsible for chromosome segregation during mitosis, is epigenetically defined by CENP-A containing chromatin. The amount of centromeric CENP-A has direct implications for both the architecture and epigenetic inheritance of centromeres. Using complementary strategies, we determined that typical human centromeres contain ∼400 molecules of CENP-A, which is controlled by a mass-action mechanism. This number, despite representing only ∼4% of all centromeric nucleosomes, forms a ∼50-fold enrichment to the overall genome. In addition, although pre-assembled CENP-A is randomly segregated during cell division, this amount of CENP-A is sufficient to prevent stochastic loss of centromere function and identity. Finally, we produced a statistical map of CENP-A occupancy at a human neocentromere and identified nucleosome positions that feature CENP-A in a majority of cells. In summary, we present a quantitative view of the centromere that provides a mechanistic framework for both robust epigenetic inheritance of centromeres and the paucity of neocentromere formation.DOI: http://dx.doi.org/10.7554/eLife.02137.001.

The centromere, responsible for chromosome segregation during mitosis, is epigenetically defined by CENP-A containing chromatin. The amount of centromeric CENP-A has direct implications for both the architecture and epigenetic inheritance of centromeres. Using complementary strategies, we determined that typical human centromeres contain ∼400 molecules of CENP-A, which is controlled by a mass-action mechanism. This number, despite representing only ∼4% of all centromeric nucleosomes, forms a ∼50-fold enrichment to the overall genome. In addition, although pre-assembled CENP-A is randomly segregated during cell division, this amount of CENP-A is sufficient to prevent stochastic loss of centromere function and identity. Finally, we produced a statistical map of CENP-A occupancy at a human neocentromere and identified nucleosome positions that feature CENP-A in a majority of cells. In summary, we present a quantitative view of the centromere that provides a mechanistic framework for both robust epigenetic inheritance of centromeres and the paucity of neocentromere formation.DOI: http://dx.doi.org/10.7554/eLife.02137.001.

The first centromeric protein identified in any species was CENP-A, a divergent member of the histone H3 family that was recognised by autoantibodies from patients with scleroderma-spectrum disease. It has recently been suggested to rename this protein CenH3. Here, we argue that the original name should be maintained both because it is the basis of a long established nomenclature for centromere proteins and because it avoids confusion due to the presence of canonical histone H3 at centromeres.

The first centromeric protein identified in any species was CENP-A, a divergent member of the histone H3 family that was recognised by autoantibodies from patients with scleroderma-spectrum disease. It has recently been suggested to rename this protein CenH3. Here, we argue that the original name should be maintained both because it is the basis of a long established nomenclature for centromere proteins and because it avoids confusion due to the presence of canonical histone H3 at centromeres.

CENP-A is an essential histone H3 variant found in all eukaryotes examined to date. To begin to determine how CENP-A is assembled into chromatin, we developed a binding assay using sperm chromatin in cell-free extract derived from Xenopus eggs. Our data suggest that the catalytic activities of an unidentified deoxycytidine deaminase and UNG2, a uracil DNA glycosylase, are involved in CENP-A assembly. In support of this model, inhibiting deoxycytidine deaminase with zebularine, or uracil DNA glycosylase with Ugi, uracil or UTP results in a lack of detectable CENP-A on sperm DNA. Conversely, inducing DNA damage increases the level of CENP-A detected on sperm chromatin. Our data suggest that base excision repair may be involved in assembly of this histone H3 variant.

CENP-A is an essential histone H3 variant found in all eukaryotes examined to date. To begin to determine how CENP-A is assembled into chromatin, we developed a binding assay using sperm chromatin in cell-free extract derived from Xenopus eggs. Our data suggest that the catalytic activities of an unidentified deoxycytidine deaminase and UNG2, a uracil DNA glycosylase, are involved in CENP-A assembly. In support of this model, inhibiting deoxycytidine deaminase with zebularine, or uracil DNA glycosylase with Ugi, uracil or UTP results in a lack of detectable CENP-A on sperm DNA. Conversely, inducing DNA damage increases the level of CENP-A detected on sperm chromatin. Our data suggest that base excision repair may be involved in assembly of this histone H3 variant.

The kinetochore is an essential structure for chromosome segregation. Although the kinetochore is usually formed on a centromere locus, it can be artificially formed at a non-centromere locus by protein tethering. An artificial kinetochore can be formed by tethering of CENP-C or CENP-I, members of the constitutive centromere-associated network (CCAN). However, how CENP-C or CENP-I recruit the centromere-specific histone CENP-A to form an artificial kinetochore remains unclear. In this study, we analyzed this issue using the tethering assay combined with an auxin-inducible degron (AID)-based knockout method in chicken DT40 cells. We found that tethering of CENP-C or CENP-I induced CENP-A incorporation at the non-centromeric locus in the absence of Knl2 (or MIS18BP1), a component of the Mis18 complex, and that Knl2 tethering recruited CENP-A in the absence of CENP-C. We also showed that CENP-C coimmunoprecipitated with HJURP, independently of Knl2. Considering these results, we propose that CENP-C recruits CENP-A by HJURP binding to form an artificial kinetochore. Our results suggest that CENP-C or CENP-I exert CENP-A recruitment activity, independently of Knl2, for artificial kinetochore formation in chicken DT40 cells. This gives us a new insight into mechanisms for CENP-A incorporation.

peer-reviewed, The histone H3 variant CENP-A epigenetically defines the centromere and is critical for chromosome segregation. Here we report an interaction between CENP-A and subunits of the mitochondrial ATP synthase complex in the germline of male Drosophila. Furthermore, we report that knockdown of CENP-A, as well as subunits ATPsyn-α, -βlike (a testis-specific paralogue of ATPsyn-β) and -γ disrupts sister centromere cohesion in meiotic prophase I. We find that this disruption is likely independent of reduced ATP levels. We identify that ATPsyn- α and -βlike localise to meiotic centromeres and that this localisation is dependent on the presence of CENP-A. We show that ATPsyn-α directly interacts with the N-terminus of CENP-A in vitro and that truncation of its N terminus perturbs sister centromere cohesion in prophase I. We propose that the CENP-A N-terminus recruits ATPsyn-α and -βlike to centromeres to promote sister centromere cohesion in a nuclear function that is independent of oxidative phosphorylation.

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

In many eukaryotes, kinetochores may form over a variety of unrelated centromeric sequences. This fact has led to the idea that an epigenetic process determines kinetochore location. To investigate the nature of this epigenetic process, I have established a system that allows the manipulation of the chromosomal centromeric DNA in a genetically tractable model organism, Schizosaccharomyces pombe. The system enables the definition of the relationship between centromeric DNA size, sequence, chromosome position and function. I have used this system to measure how binding of the conserved kinetochore protein Cnp1 (CENP-A homolog) varies as a function of the amount, sequence and position of the centromeric DNA. In humans, cytogenetic data suggests that the number of centromere-specific CENP-A nucleosomes at each centromere is approximately uniform regardless of the sequence and length of the centromeric DNA. These observations suggest that the number of these centromere-specific nucleosomes is tightly regulated. I set out to test whether such mechanism is evolutionarily conserved in the fission yeast S. pombe. I did this by manipulating the amount of the centromeric DNA at one centromere and then measuring the amount of Cnp1 bound to it. The results showed that in S. pombe, the amount of bound Cnp1 is proportional to the amount of centromeric DNA and thus the relationship between centromeric DNA size and CENP-A binding differs from humans. During these measurements, I observed that the size and sequence of the centromeric DNA do have a role in determining Cnp1 binding to centromeric DNA but, as described by others, it is not sufficient and sometimes not necessary for functional centromere formation. The requirement of the kinetochore protein Swi6 for neo-centromere formation but not for the maintenance of a pre-established centromere was also confirmed.

The kinetochore is an essential structure for chromosome segregation. Although the kinetochore is usually formed on a centromere locus, it can be artificially formed at a non-centromere locus by protein tethering. An artificial kinetochore can be formed by tethering of CENP-C or CENP-I, members of the constitutive centromere-associated network (CCAN). However, how CENP-C or CENP-I recruit the centromere-specific histone CENP-A to form an artificial kinetochore remains unclear. In this study, we analyzed this issue using the tethering assay combined with an auxin-inducible degron (AID)-based knockout method in chicken DT40 cells. We found that tethering of CENP-C or CENP-I induced CENP-A incorporation at the non-centromeric locus in the absence of Knl2 (or MIS18BP1), a component of the Mis18 complex, and that Knl2 tethering recruited CENP-A in the absence of CENP-C. We also showed that CENP-C coimmunoprecipitated with HJURP, independently of Knl2. Considering these results, we propose that CENP-C recruits CENP-A by HJURP binding to form an artificial kinetochore. Our results suggest that CENP-C or CENP-I exert CENP-A recruitment activity, independently of Knl2, for artificial kinetochore formation in chicken DT40 cells. This gives us a new insight into mechanisms for CENP-A incorporation.