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Stem cells are defined by their capacity to self-renew and their potential to differentiate into multiple cell lineages. Pluripotent embryonic stem (ES) cells can renew indefinitely while keeping the potential to differentiate into any of the three germ layers (ectoderm, endoderm or mesoderm). For decades, ES cells are in the focus of research because of these unique features. When ES cells differentiate they form spheroid aggregates termed “embryoid bodies” (EBs). These EBs mimic post- implantation embryonic development and therefore facilitate the understanding of developmented mechanisms.
During ES cell differentiation, de-repression or repression of genes accompanies the changes in chromatin structure. In ES cells, several mechanisms are involved in the regulation of the chromatin architecture, including post-translational modifications of histones. Post-translational histone methylation marks became one of the best- investigated epigenetic modifications, and they are essential for maintaining pluripotency. Until the first histone demethylase KDM1A was discovered in 2004 histone modifications were considered to be irreversible. Since then, a great number of histone demethylases have been identified. Their activity is linked to gene regulation as well as to stem cell self-renewal and differentiation.
KDM6A and KDM6B are H3K27me3/2-specific histone demethylases, which are known to play a central role in the regulation of posterior development by regulating HOX gene expression. So far less is known about the molecular function of KDM6A or KDM6B in undifferentiated and differentiating ES cells. In order to completely abrogate KDM6A and KDM6B demethylase activity in undifferentiated and differentiating ES cells, a specific inhibitor (GSK-J4) was employed. Treatment with GSK-J4 had no effect on the viability or proliferation on ES cells. However, in the presence of GSK-J4 ES cell differentiation was completely abrogated with cells arrested in G1-phase and an increased rate of apoptosis. Global transcriptome analyses in early-differentiating ES cells revealed that only a limited set of genes were differentially regulated in response to GSK-J4 treatment with more genes up- regulated than down-regulated. Many of the up-regulated genes are linked to DNA damage response (DDR). In agreement with this, DNA damage was found in EBs incubated with GSK-J4. A co-localization of H3K27me3 or KDM6B with γH2AX foci, marking DNA breaks, could be excluded. However, differentiating Eed knockout (KO) ES cells, which are devoid of the H3K27me3 mark, showed an attenuated GSK-J4- induced DDR. Finally, hematopoietic differentiation in the presence of GSK-J4 resulted in a reduced colony-forming potential. This leads to the conclusion that differentiation in the presence of GSK-J4 is also restricted to hematopoietic differentiation.
In conclusion, my results show that the enzymatic activity of KDM6A and KDM6B is not essential for maintaining the pluripotent state of ES cells. In contrast, the enzymatic activity of both proteins is indispensable for ES cell and hematopoietic differentiation. Additionally KDM6A and KDM6B enzymatic inhibition in differentiating ES cells leads to increased DNA damage with an activated DDR. Therefore, KDM6A and KDM6B are associated with DNA damage and in DDR in differentiating ES cells.
The integrity of our genome is continuously endangered by DNA damaging factors. Several cellular mechanisms have evolved to recognize and remove different types of DNA lesions. Despite the wealth of information on the three-dimensional structure and the catalytic mechanism of DNA repair enzymes, the essential process of target site search and identification remains more elusive. How can a small number of repair proteins find and detect the rare sites of damage rapidly and efficiently over an excess of millions of undamaged bases?
To address this pivotal question in DNA repair, I focused on the central players from the two DNA damage excision repair pathways in my studies: nucleotide excision repair (NER) and base excision repair (BER). As examples for completely different approaches of damage search, recognition and verification, I compared the NER protein Xeroderma pigmentosum group D (XPD) with the BER proteins human thymine DNA glycosylase (hTDG) and human 8-oxoguanine glycosylase (hOgg1).
In particular, the single molecule approach of atomic force microscopy (AFM) imaging and complementary biochemical and biophysical techniques were applied. I established a simple, optimized preparation approach, which yields homogeneous and pure samples of long (several hundreds to thousands of base pairs) DNA substrates suitable for the AFM studies with DNA repair proteins. Via this sample preparation, a single target site of interest can be introduced into DNA at a known position, which allows separate analysis of specific protein-DNA complexes bound to the lesion site and nonspecific complexes bound to non-damaged DNA.
The first part of the thesis investigates the XPD protein involved in eukaryotic NER. In general, the NER mechanism removes helix-distorting lesions – carcinogenic UV light induced photoproducts, such as cyclobutane pyrimidine dimers (CPDs) as well as bulky DNA adducts. The 5’-3’ helicase XPD has been proposed to be one of the key players in DNA damage verification in eukaryotic NER, which is still a matter of hot debate. In the studies, I focused on XPD from the archaeal species Thermoplasma acidophilum (taXPD), which shares a relatively high sequence homology with the sequence of the human protein and may serve as a good model for its eukaryotic counterpart. Based on AFM experiments and accompanying DNA binding affinity measurements with the biosensor technology Biolayer Interferometry (BLI), a clear role of XPD in damage verification was deciphered. Specifically, the data suggested that the ATP-dependent 5’-3’ helicase activity of XPD was blocked by the presence of damage leading to stalled XPD-DNA damage verification complexes at the lesion sites.
Successful damage verification led to ATP-dependent conformational changes visible by a significant transition in DNA bend angles from ~ 50° to ~ 65° at the site of the bound protein. Remarkably, this DNA bend angle shift was observed both in the presence of ATP and ATPγs (non-hydrolyzable ATP analog) indicating that ATP-binding instead of ATP hydrolysis was sufficient to induce repair competent conformational changes of XPD. Most importantly, detailed protein binding position and DNA bend angle analyses revealed for the first time that XPD preferably recognizes a bulky fluorescein lesion on the translocated strand, whereas a CPD lesion is preferentially detected on the opposite, non-translocated strand. Despite the different recognition strategies for both types of damages, they share a common verification complex conformation, which may serve as a signal for the recruitment of further NER factors.
In the second part of the thesis, AFM imaging and a 2-Aminopurine fluorescence-based base-flipping assay were combined to investigate damage search and recognition by DNA glycosylases in BER. Exemplarily, I chose to study hTDG as a representative of the vast glycosylase family. hTDG excises thymine and uracil from mutagenic G:T and G:U mispairs contributing to cancer and genetic disease. The AFM data suggested that hTDG uses the intrinsic flexibility of G:T and G:U wobble pairs for initial damage sensing, while scanning DNA as a search complex (SC, slightly bent DNA). Remarkably, hTDG has been indicated to continuously switch between the search and interrogation conformation (IC, stronger bent DNA) during damage search. In the IC, target bases are interrogated by extrahelical base flipping, which is facilitated by protein-induced DNA bending and enhanced DNA flexibility at mismatches. AFM and fluorescence analyses revealed that the flipped base is stabilized via hTDG’s arginine finger. Correct target bases are perfectly stabilized within the enzyme’s catalytic pocket resulting in prolonged residence time and enhanced excision probability. To test for the generalizability of the proposed hTDG damage search model to BER glycosylases, identical studies were performed with a second glycosylase, hOgg1. The data on hOgg1, which removes structurally more stable 8-oxoguanine lesions, supported the hypothesis developed for lesion recognition by hTDG as a common strategy employed by BER glycosylases