Biology subjects

Tihanyi, G.

Publications and source records attributed to Tihanyi, G..

3 recordsLinked to original sources

bioRxiv2021

Nonlinear control of transcription through enhancer-promoter interactions

Chromosome structure in mammals is thought to regulate transcription by modulating the three-dimensional interactions between enhancers and promoters, notably through CTCF-mediated interactions and topologically associating domains (TADs)1-4. However, how chromosome interactions are actually translated into transcriptional outputs remains unclear. To address this question we use a novel assay to position an enhancer at a large number of densely spaced chromosomal locations relative to a fixed promoter, and measure promoter output and interactions within a genomic region with minimal regulatory and structural complexity. Quantitative analysis of hundreds of cell lines reveal that the transcriptional effect of an enhancer depends on its contact probabilities with the promoter through a non-linear relationship. Mathematical modeling and validation against experimental data further provide evidence that nonlinearity arises from transient enhancer-promoter interactions being memorized into longer-lived promoter states in individual cells, thus uncoupling the temporal dynamics of interactions from those of transcription. This uncovers a potential mechanism for how enhancers control transcription across large genomic distances despite rarely meeting their target promoters, and for how TAD boundaries can block distal enhancers. We finally show that enhancer strength additionally determines not only absolute transcription levels, but also the sensitivity of a promoter to CTCF-mediated functional insulation. Our unbiased, systematic and quantitative measurements establish general principles for the context-dependent role of chromosome structure in long-range transcriptional regulation.

molecular biology↗

bioRxiv2020

Genome-wide alterations of uracil distribution patterns in human DNA upon chemotherapeutic treatments

Numerous anti-cancer drugs perturb thymidylate biosynthesis and lead to genomic uracil incorporation contributing to their antiproliferative effect. Still, it is not yet characterized if uracil incorporations have any positional preference. Here, we aimed to uncover genome-wide alterations in uracil pattern upon drug-treatment in human cancer cell-line HCT116. We developed a straightforward U-DNA sequencing method (U-DNA-Seq) that was combined with in situ super-resolution imaging. Using a novel robust analysis pipeline, we found broad regions with elevated probability of uracil occurrence both in treated and non-treated cells. Correlation with chromatin markers and other genomic features shows that non-treated cells possess uracil in the late replicating constitutive heterochromatic regions, while drug treatment induced a shift of incorporated uracil towards more active/functional segments. Data were corroborated by colocalization studies via dSTORM microscopy. This approach can also be applied to study the dynamic spatio-temporal nature of genomic uracil.

biochemistry↗

bioRxiv2018

CRISPR/Cas9-mediated knock-out of dUTPase in mice leads to early embryonic lethality

Sanitization of nucleotide pools is essential for genome maintenance. Among the enzymes significant in this mechanism, deoxyuridine 5'-triphosphate nucleotidohydrolase (dUTPase) performs cleavage of dUTP into dUMP and inorganic pyrophosphate. By this reaction the enzyme efficiently prevents uracil incorporation into DNA and provides dUMP, the substrate for de novo thymidylate biosynthesis. Despite its physiological significance, knock-out models of dUTPase have not yet been investigated in mammals, only in unicellular organisms, such as bacteria and yeast. Here we generate CRISPR/Cas9-mediated dUTPase knock-out in mice. We find that heterozygous dut +/-animals are viable while the decreased dUTPase level is clearly observable. We also show that the enzyme is essential for embryonic development. Based on the present results, early dut -/-embryos can still reach the blastocyst stage, however, they die shortly after implantation. Analysis of preimplantion embryos indicate perturbed growth of both inner cell mass (ICM) and trophectoderm (TE). We conclude that dUTPase is indispensable for post-implantation development in mice. The gene targeting model generated in the present study will allow further detailed studies in combination with additional gene knock-outs.

developmental biology↗