eprintid: 31436
rev_number: 12
eprint_status: archive
userid: 6578
dir: disk0/00/03/14/36
datestamp: 2022-03-23 13:56:16
lastmod: 2022-03-24 12:48:27
status_changed: 2022-03-23 13:56:16
type: doctoralThesis
metadata_visibility: show
creators_name: Doppler, Sarah
title: The histone fold protein CHRAC-14 controls CENP-A loading and 
gene expression in Drosophila melanogaster
subjects: 570
divisions: 140001
adv_faculty: af-14
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.
date: 2022
id_scheme: DOI
id_number: 10.11588/heidok.00031436
ppn_swb: 1796570427
own_urn: urn:nbn:de:bsz:16-heidok-314363
date_accepted: 2022-02-24
advisor: HASH(0x5634a2add9b0)
language: eng
bibsort: DOPPLERSARTHEHISTONE2022
full_text_status: public
place_of_pub: Heidelberg
citation:   Doppler, Sarah  (2022) The histone fold protein CHRAC-14 controls CENP-A loading and gene expression in Drosophila melanogaster.  [Dissertation]     
document_url: https://archiv.ub.uni-heidelberg.de/volltextserver/31436/1/Dissertation_SDoppler_2022.pdf