Preview

PDF, English Download (27MB) | Lizenz: Rights reserved - Free Access

Abstract

Centromeres are specialized chromatin domains present on each chromosome, which determine the location of kinetochore formation – the multiprotein platform for spindle microtubule attachment during mitosis. Since the underlying centromeric DNA sequence is not conserved, centromere inheritance and function rely on the conserved epigenetic marker CENP-A (also known as CID in Drosophila). CENP-A is highly enriched in centromeric chromatin and partially replaces canonical histone H3. Specific loading and stabilization of CENP-A at the centromere is ensured by a concert of different factors such as its dedicated chaperone called CAL1 in Drosophila. Whereas the primary DNA sequence is dispensable for centromere formation, CENP-A is both absolutely essential and sufficient to maintain and establish functional centromeres. When overabundant due to transcriptional upregulation for example in cancer cells, CENP-A escapes the endogenous loading pathway and hijacks other histone loading machineries, which leads to promiscuous non-centromeric CENP-A incorporation throughout the genome with detrimental effects for genome stability. Even though ectopic CENP-A accumulation existentially threatens cellular and organismal viability, CENP-A is distributed at basal levels throughout the genome under physiological conditions. The function and mechanistic details of genome-wide CENP-A loading remain elusive, but the field speculates, that low levels of ectopic CENP-A confer epigenetic plasticity and prime chromatin for neocentromere formation in the event of ancestral centromere loss. Previous work in our lab identified the histone fold protein CHRAC-14 as an important regulator of ectopic CENP-A incorporation and as a new DNA damage factor in Drosophila melanogaster. CHRAC-14 depletion leads to the increase of CENP-A levels, increased CENP-A ectopic loading – possibly at telomeric DNA repair sites – and to DNA repair defects accompanied by G2/M checkpoint failure. Since we lacked detailed mechanistic insights and clear data whether ectopic CENP-A localizes to and has a role at DNA lesions, I set out to further elucidate this pathway. Employing a portfolio of biochemical and molecular biological techniques such as CUT&Tag-Seq, RNA-Seq and immunoprecipitation combined with mass spectrometry analysis, I report in this thesis, that CHRAC-14 knockdown leads to the accumulation of CENP-A at centromeres and genome-wide. In addition, altered expression of genes associated with gene ontology categories such as ‘mitotic progression’ and ‘DNA damage response’ was observed. Furthermore, I identified candidate genes, which are mis-expressed whilst showing increased CENP-A binding. Moreover, I found, that both CHRAC-14 and CENP-A interact with Casein kinase 2 (CK2) and are phosphorylation substrates thereof. Interestingly, CK2 phosphorylation of CHRAC-14 seems to promote the turnover of a post translationally modified version of CENP-A, whereas CK2 phosphorylation of endogenous CENP-A itself seems to be essential for protein stability. Taken together, my study provides new insights into CHRAC-14 mediated CENP-A titration in chromatin and opens up a novel direction involving CK2 as a key regulator in this pathway.