Browsing by Author "Gutekunst, Nicole"

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    Development of a BiAD sensor for locus-specific detection of cellular histone acetylation dynamics by fluorescence microscopy
    (2025) Köhler, Anja R.; Gutekunst, Nicole; Harsch, Annika; Bashtrykov, Pavel; Jeltsch, Albert
    Background: Dynamic changes in histone acetylation play crucial roles during cellular differentiation and disease development, but their detection in living cells is still a challenging task. Objectives: Here, we developed a Bimolecular Anchor Detector (BiAD) sensor for the detection of locus-specific changes in histone acetylation in living cells by fluorescence microscopy. Methods: We used the BRD9 bromodomain cloned as tandem double domain (2xBRD9-BD) as a reader of histone acetylation. It was integrated into a dual-color BiAD chassis that was previously described by us. Results: We identified the gene body of TTC34 as a potential target for our sensor, because it contains dense histone acetylation and 392 local sequence repeats. Using a binding-deficient mutant of 2xBRD9-BD as a negative control, we established a successful readout of histone acetylation at the TTC34 locus. A single-domain reader did not function, indicating the requirement for the double reader to enhance the affinity and specificity of the chromatin interaction via avidity effects. With this sensor, we could detect dynamic increases in histone acetylation at the TTC34 locus after the treatment of cells with the histone deacetylase inhibitor Trichostatin A for 6 h indicating the applicability of the sensor for single-cell epigenome studies. Conclusions: Our data demonstrate that active chromatin modifications can be detected by BiAD sensors using 2xBRD9-BD as a reader. This complements the toolkit of the available BiAD sensors and documents the modularity of BiAD sensors.
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    Non-canonical functions of UHRF1 maintain DNA methylation homeostasis in cancer cells
    (2024) Yamaguchi, Kosuke; Chen, Xiaoying; Rodgers, Brianna; Miura, Fumihito; Bashtrykov, Pavel; Bonhomme, Frédéric; Salinas-Luypaert, Catalina; Haxholli, Deis; Gutekunst, Nicole; Aygenli, Bihter Özdemir; Ferry, Laure; Kirsh, Olivier; Laisné, Marthe; Scelfo, Andrea; Ugur, Enes; Arimondo, Paola B.; Leonhardt, Heinrich; Kanemaki, Masato T.; Bartke, Till; Fachinetti, Daniele; Jeltsch, Albert; Ito, Takashi; Defossez, Pierre-Antoine
    DNA methylation is an essential epigenetic chromatin modification, and its maintenance in mammals requires the protein UHRF1. It is yet unclear if UHRF1 functions solely by stimulating DNA methylation maintenance by DNMT1, or if it has important additional functions. Using degron alleles, we show that UHRF1 depletion causes a much greater loss of DNA methylation than DNMT1 depletion. This is not caused by passive demethylation as UHRF1-depleted cells proliferate more slowly than DNMT1-depleted cells. Instead, bioinformatics, proteomics and genetics experiments establish that UHRF1, besides activating DNMT1, interacts with DNMT3A and DNMT3B and promotes their activity. In addition, we show that UHRF1 antagonizes active DNA demethylation by TET2. Therefore, UHRF1 has non-canonical roles that contribute importantly to DNA methylation homeostasis; these findings have practical implications for epigenetics in health and disease.