Thesis supervisor: Gábor Szabó
Location of studies (in Hungarian): University of Debrecen Abbreviation of location of studies: DEÁOK
Description of the research topic:
Nucleosomal structure is repressive for transcription, hence eukaryotic gene regulation is primarily based on de-repression at the level of the chromatin. It is fine-tuned, among other factors, by histone posttranslational modifications (PTMs), and entails and depends on negative supercoling of the DNA. In line with this global picture, supercoil relaxation elicits a strong, PTM-dependent nucleosome destabilizing effect according to our published and unpublished data. Our previous work has also led us to recognize that DNA superhelicity has a strong and direct impact on the target size and binding affinity of two major classes of anticancer drugs: DNA intercalators and interstrand-crosslinking agents. We have formulated the concept that the intertwined effects of PTMs and regulation of supercoiling are integrated at the level of nucleosome stability what may be an important overall read-out of drug effects.
Current projects:
In a 3-decade long pursuit of the peculiar vulnerability of chromatin at loop-size intervals we have come to the working hypothesis that these phenomena are related to the presence of promoter-proximal endogeneous DNA breaks that may have a role in transcriptional regulation. We are investigating the mechanism of their generation, their possible functional role and their effect on higher-order chromatin structure, in S. cerevisiae and in human cells, including peripheral blood mononuclear cells.
Using our laser scanning cytometric assay of nucleosome stability (see Imre at al., Nucleosome stability measured in situ by automated quantitative imaging. Sci Rep. 2017 Oct 6;7(1):12734), we are investigating the molecular determinants of stability in the case of nucleosomes containing different PTMs or histone variants, with special interest in H2A.Z.
We strive to make use of the cell biological perspective obtained on chromatin structure in anticancer drug research investigating the determinants of accessibility of small molecules to their target within the chromatin, by studying the interaction of DNA intercalators with chromatin in intact cells, by assessing the potentials of modulating superhelicity by intercalators on the toxicity of DNA cross-linking agents and their effect on nuclear architecture as reflected by the large-scale nuclear, nucleolar and nucleocytoplasmic reorganizations and relocations detectable by confocal microscopy.
Our experimental systems include mammalian cells, mouse embryonic stem cells and S. cerevisiae, studied by (a) confocal microscopic analysis of the localisation patterns of free 3’OHs detected by in situ nick translation or terminal transferase reaction relative to those of transcription related R-loops (RNA/DNA-hybrids), topoisomerase enzymes and epigenetic marks (PTMs); (b) a laser scanning cytometric (LSC) assay of nucleosome stability, measured in a PTM and cell cycle dependent manner; (c) an LSC assay of superhelical density and size distribution of loops; (d) molecular combing of DNA; (e,f) various gelelectrophoretic techniques (denaturing urea-agarose, CHEF), including single-cell electrophoresis of large DNA molecules; (g) a reverse South-Western blotting technique to map strand breaks and R-loops; (h) a chip-on-beads assay to measure the co-occurrence of PTMs on a particular nucleosome and (i) standard genomic techniques (microarray,chip seq).
Number of students who can be accepted: 1
Deadline for application: 2020-09-30
2024. IV. 17. ODT ülés Az ODT következő ülésére 2024. június 14-én, pénteken 10.00 órakor kerül sor a Semmelweis Egyetem Szenátusi termében (Bp. Üllői út 26. I. emelet).
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