Effects of histone 3.3K27M-mutation on CREB binding protein (CBP/p300)- and bromodomain and extra terminal domain (BET) protein- mediated gene regulation in diffuse intrinsic pontine gliomas (DIPG)

Kubiak, Klaudia

dc.contributor.advisor	Gärtner, Jutta Prof. Dr.
dc.contributor.author	Kubiak, Klaudia
dc.date.accessioned	2023-04-25T15:55:05Z
dc.date.available	2024-03-08T00:50:10Z
dc.date.issued	2023-04-25
dc.identifier.uri	http://resolver.sub.uni-goettingen.de/purl?ediss-11858/14641
dc.identifier.uri	http://dx.doi.org/10.53846/goediss-9838
dc.format.extent	XXX Seiten	de
dc.language.iso	eng	de
dc.rights.uri	http://creativecommons.org/licenses/by/4.0/
dc.subject.ddc	610
dc.title	Effects of histone 3.3K27M-mutation on CREB binding protein (CBP/p300)- and bromodomain and extra terminal domain (BET) protein- mediated gene regulation in diffuse intrinsic pontine gliomas (DIPG)	de
dc.type	doctoralThesis	de
dc.contributor.referee	Gärtner, Jutta Prof. Dr.
dc.date.examination	2023-03-09	de
dc.description.abstracteng	Diffuse intrinsic pontine gliomas (DIPG) are incurable pediatric high-grade tumors. The majority of all DIPG carries a mutation in one of the histone 3 genes leading to a substitution of lysine 27 to methionine (H3 K27M) characterized by malfunction of the histone methyltransferase enhancer of zeste homolog 2 (EZH2) that mediates H3K27 trimethylation (H3K27me3). Presence of H3.3K27M-mutation results in an epigenetic imbalance characterized by global loss of H3K27me3 and gain of H3K27 acetylation (H3K27ac) mediated by CREB binding protein (CBP/p300). Therefore it could be hypothesized that the histone acetyltransferase (HAT) CBP/p300 and readers recognizing H3K27ac like bromodomain and extra-terminal domain (BET) proteins might play a dominant role in H3.3K27M mutated DIPG cells. Thus, this project aims to identify the probable distinct genetic and resulting tumor-biological effects in DIPG cells caused by presence of H3.3K27M-mutation within the same cellular and genetic background, as well as to determine the H3.3K27M-dependent functions of H3-writers (CBP/p300) and readers (BET proteins) in DIPG cells with H3-wildtype and H3.3K27M-mutation. In order to study the impact of H3.3K27M-mutation in DIPG cells, isogenic cell lines with and without H3.3K27M-mutation were generated using CRISPR/Cas9 system and compared with each other to investigate H3.3K27M-dependent eigenome, transcriptome and consequent expression tumor-associated characteristics. Introduction of H3.3K27M-mutation resulted in loss of H3K27me3 and gain of H3K27ac, while removal of H3.3K27M-mutation led to reduction of H3K27ac and restored H3K27me3 level. Moreover, presence of H3.3K27M-mutation caused elevated cell proliferation, clonogenicity and migration/invasion ability of isoDIPG cells. Furthermore, H3.3K27M-mutation influenced the expression of several different histone marks as well as epigenetic key players in DIPG cells. To investigate and dissect the function of BET family members and CBP/p300 in isoDIPG cells, small molecules were used to inhibit specific functions of the writer/reader proteins. Subsequently, siRNA-mediated knockdown of the proteins of interest was performed to dissect specific functions of BRD2, 3 and 4 or CBP and p300. Indeed CBP and p300 as well as BET family members BRD2 and BRD4 had a more profound impact on reduction of tumor associated characteristics in isoDIPG-H3.3K27M compared to isoDIPG-H3WT cells. Interestingly, western blot analyses revealed that CBP catalyzed H3K27ac in isoDIPG-H3.3K27M cells, while p300 was responsible for H3K27ac isoDIPG-H3WT cells. Moreover, the study pointed out to a specific function of CBP in regulation of apoptosis, while p300 turned out to mediate MAPK cascades in isoDIPG-H3.3K27M cells. Within BET proteins family, BRD2 and BRD4 played a more profound role than BRD3 in isoDIPG-H3.3K27M cells, regulating biological processes including cell cycle and Wnt signaling. In summary, the present study reveals that presence of H3.3K27M-mutation in isoDIPG cells is a leading cause for the malignant phenotype observed in DIPG cells. Moreover, H3.3K27M-mutation modulates the function of H3K27-associated CBP writer and BRD2 and BRD4 readers in isoDIPG cells in vitro and in vivo, playing important roles in H3.3K27M-DIPG cells.	de
dc.contributor.coReferee	Dobbelstein, Matthias Prof. Dr.
dc.subject.ger	diffuse intrinsic pontine glioma	de
dc.subject.ger	H3K27M	de
dc.subject.ger	CBP/p300	de
dc.subject.ger	BETs	de
dc.subject.ger	EZH2	de
dc.subject.ger	H3K27me3	de
dc.subject.ger	H3K27ac	de
dc.subject.eng	diffuse intrinsic pontine glioma	de
dc.subject.eng	H3K27M	de
dc.subject.eng	CBP/p300	de
dc.subject.eng	BETs	de
dc.subject.eng	EZH2	de
dc.subject.eng	H3K27me3	de
dc.subject.eng	H3K27ac	de
dc.identifier.urn	urn:nbn:de:gbv:7-ediss-14641-7
dc.affiliation.institute	Medizinische Fakultät
dc.subject.gokfull	Molekularbiologie {Medizin} (PPN619875186)	de
dc.subject.gokfull	Pädiatrie / Neonatologie / Kinderchirurgie - Allgemein- und Gesamtdarstellungen (PPN619876093)	de
dc.subject.gokfull	Onkologie (PPN619875895)	de
dc.description.embargoed	2024-03-08	de
dc.identifier.ppn	1843673355
dc.notes.confirmationsent	Confirmation sent 2023-04-25T19:45:01	de

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