A Site-Specific Click Chemistry Approach to Di-ubiquitylate H1 Variants Reveals Position-dependent Stimulation of the DNA Repair Protein RNF168.

Ubiquitylation of histone H2A at lysines 13 and 15 by the E3 ligase RNF168 plays a key role in orchestrating DNA double-strand break (DSB) repair, which is often deregulated in cancer. RNF168 activity is triggered by DSB signaling cascades, reportedly through K63-linked poly-ubiquitylation of linker histone H1. However, direct experimental evidence of this mechanism has been elusive, primarily due to the lack of methods to specifically poly-ubiquitylate H1. Here, we developed a versatile click-chemistry approach to covalently link multiple proteins in a site-specific, controlled, and stepwise manner. Applying this method, we synthesized H1 constructs bearing triazole-linked di-ubiquitin on four DNA repair-associated ubiquitylation hotspots (H1KxUb2, at K17, 46, 64 and 96). Integrated into nucleosome arrays, the H1KxUb2 variants stimulated H2A ubiquitylation by RNF168 in a position-dependent manner, with H1K17Ub2 showing the strongest RNF168 activation effect. Moreover, we show that di-ubiquitin binding is the driving force underlying RNF168 recruitment, introducing H1K17Ub2 into living U-2 OS cells. Together, our results support the hypothesis of poly-ubiquitylated H1 guiding RNF168 recruitment to DSB sites. Moreover, we demonstrate how the streamlined synthesis of H1KxUb2 variants enables mechanistic studies into RNF168 regulation, with potential implications for its inhibition in susceptible cancers.

Innovative Techniques and Strategies for a Reliable High-Throughput Genotoxicity Assessment.

Damage to DNA is a central mechanism to the initiation of carcinogenesis. As a consequence, precise DNA damage detection is essential for an effective risk assessment of xenobiotics and constitutes a powerful tool for human biomonitoring and early stage cancer risk assessment. Here we highlight four innovative approaches for determining genotoxicity in a reliable and in the future high-throughput manner. In this context, we discuss and evaluate recent improvements to well-established methods and present promising new techniques.

Exploring Flow Cytometry-Based Micronucleus Scoring for Reliable Nanomaterial Genotoxicity Assessment.

The increased use of engineered nanomaterials (ENM) such as SiO2 and TiO2 in industrial products, especially in food, raises concerns with regard to their effect on human health. In particular, ENM-induced genotoxicity is crucial to investigate, since DNA damage can cause induction or promotion of carcinogenesis. However, current in vitro and in vivo nanogenotoxicological data are highly contradictory, which impedes interpretation and extrapolation. Hence, robust, reliable, and ideally scalable in vitro methods for nanogenotoxicity assessment are of great interest. This work aimed at evaluating the suitability of flow cytometry-based micronuclei scoring for reliable nanogenotoxicological assessment in human intestinal cells. Therefore, we have evaluated the genotoxicity of differently sized SiO2 and TiO2 from different sources (food-relevant, commercially available, and laboratory-synthesized) using the well-established alkaline single cell gel electrophoresis (Comet assay) and the micronucleus (MN) assay employing a flow cytometric readout. Our study demonstrates that physiologically relevant doses of several types of SiO2 and TiO2 did not cause genotoxicity, as assessed by the Comet assay, and the MN flow cytometry assay under the particular experimental conditions described. To improve data reliability, we identified ENM-induced interferences with flow cytometric scoring employing a set of interference controls, which is generally applicable for any nanomaterial and any cell line. In conclusion, flow cytometry-based MN scoring appears to be a promising methodology in nanogenotoxicity testing since data acquisition and analysis are significantly faster, highly scalable in terms of throughput, and less operator-dependent compared to the traditional microscopic evaluation. In particular, ENM-induced false-positive or false-negative results, which have not been addressed sufficiently in the literature, can be detected easily, thus enhancing data reliability.

Decoding Chromatin Ubiquitylation: A Chemical Biology Perspective.

Since Strahl and Allis proposed the "language of covalent histone modifications", a host of experimental studies have shed light on the different facets of chromatin regulation by epigenetic mechanisms. Initially proposed as a concept for controlling gene transcription, the regulation of deposition and removal of histone post-translational modifications (PTMs), such as acetylation, methylation, and phosphorylation, have been implicated in many chromatin regulation pathways. However, large PTMs such as ubiquitylation challenge research on many levels due to their chemical complexity. In recent years, chemical tools have been developed to generate chromatin in defined ubiquitylation states in vitro. Chemical biology approaches are now used to link specific histone ubiquitylation marks with downstream chromatin regulation events on the molecular level. Here, we want to highlight how chemical biology approaches have empowered the mechanistic study of chromatin ubiquitylation in the context of gene regulation and DNA repair with attention to future challenges.

Examining the role of phosphorylation of p19INK4d in its stability and ubiquitination using chemical protein synthesis.

p19INK4d plays an important role in the regulation of the cell cycle by inhibiting the function of cyclin-dependent kinases 4/6 that is responsible for the phosphorylation and deactivation of the retinoblastoma protein (pRb) tumour suppressor. Recently, it was reported that phosphorylation of p19INK4d at Ser76 and Ser66 causes structural changes, which lead to its ubiquitination and degradation. Yet the exact contribution of each phosphorylation site remains unclear. To shed light on the role of these sites, we developed the chemical synthesis of unmodified, mono- and doubly phosphorylated p19INK4d using state of the art methods for chemical protein synthesis. The synthesized proteins were characterized by circular dichroism and biochemical methods to examine the effect of phosphorylation on the thermal stability and ubiquitination, respectively. Our results provide clear determination of p19INK4d stability upon phosphorylation at different sites and reveal that phosphorylation of both Ser residues might be necessary for promoting ubiquitination of p19INK4d.