DNA double-strand breaks (DSBs) are harmful DNA lesions, which elicit catastrophic consequences for genome stability if not properly repaired. DSBs can be repaired by either non-homologous end joining (NHEJ) or homologous recombination (HR). The choice between these two pathways depends on which proteins bind to the DSB ends and how their action is regulated. NHEJ initiates with the binding of the Ku complex to the DNA ends, while HR is initiated by the nucleolytic degradation of the 5′-ended DNA strands, which requires several DNA nucleases/helicases and generates single-stranded DNA overhangs. DSB repair occurs within a precisely organized chromatin environment, where the DNA is wrapped around histone octamers to form the nucleosomes. Nucleosomes impose a barrier to the DNA end processing and repair machinery. Chromatin organization around a DSB is modified to allow proper DSB repair either by the removal of entire nucleosomes, thanks to the action of chromatin remodeling factors, or by post-translational modifications of histones, thus increasing chromatin flexibility and the accessibility of repair enzymes to the DNA. Here, we review histone post-translational modifications occurring around a DSB in the yeast Saccharomyces cerevisiae and their role in DSB repair, with particular attention to DSB repair pathway choice.

E2-conjugating enzymes (E2s) play a central role in the enzymatic cascade that leads to the attachment of ubiquitin to a substrate. This process, termed ubiquitylation, is required to maintain cellular homeostasis and affects almost all cellular process. By interacting with multiple E3 ligases, E2s dictate the ubiquitylation landscape within the cell. Since its discovery, ubiquitylation has been regarded as a posttranslational modification that specifically targets lysine side chains (canonical ubiquitylation). We used Matrix-Assisted Laser Desorption/Ionization-Time Of Flight Mass Spectrometry to identify and characterize a family of E2s that are instead able to conjugate ubiquitin to serine and/or threonine. We used structural modeling and prediction tools to identify the key activity determinants that these E2s use to interact with ubiquitin as well as their substrates. Our results unveil the missing E2s necessary for noncanonical ubiquitylation, underscoring the adaptability and versatility of ubiquitin modifications.

KDM5B histone demethylase is overexpressed in many cancers and plays an ambivalent role in oncogenesis, depending on the specific context. This ambivalence could be explained by the expression of KDM5B protein isoforms with diverse functional roles, which could be present at different levels in various cancer cell lines. We show here that one of these isoforms, namely KDM5B-NTT, accumulates in breast cancer cell lines due to remarkable protein stability relative to the canonical PLU-1 isoform, which shows a much faster turnover. This isoform is the truncated and catalytically inactive product of an mRNA with a transcription start site downstream of the PLU-1 isoform, and the consequent usage of an alternative ATG for translation initiation. It also differs from the PLU-1 transcript in the inclusion of an additional exon (exon-6), previously attributed to other putative isoforms. Overexpression of this isoform in MCF7 cells leads to an increase in bulk H3K4 methylation and induces derepression of a gene cluster, including the tumor suppressor Cav1 and several genes involved in the interferon-alpha and -gamma response. We discuss the relevance of this finding considering the hypothesis that KDM5B may possess regulatory roles independent of its catalytic activity.

Histone lysine demethylases (KDMs) play an essential role in biological processes such as transcription regulation, RNA maturation, transposable element control, and genome damage sensing and repair. In most cases, their action requires catalytic activities, but non-catalytic functions have also been shown in some KDMs. Indeed, some strictly KDM-related proteins and some KDM isoforms do not act as histone demethylase but show other enzymatic activities or relevant non-enzymatic functions in different cell types. Moreover, many studies have reported on functions potentially supported by catalytically dead mutant KDMs. This is probably due to the versatility of the catalytical core, which can adapt to assume different molecular functions, and to the complex multi-domain structure of these proteins which encompasses functional modules for targeting histone modifications, promoting protein–protein interactions, or recognizing nucleic acid structural motifs. This rich modularity and the availability of multiple isoforms in the various classes produced variants with enzymatic functions aside from histone demethylation or variants with non-catalytical functions during the evolution. In this review we will catalog the proteins with null or questionable demethylase activity and predicted or validated inactive isoforms, summarizing what is known about their alternative functions. We will then go through some experimental evidence for the non-catalytical functions of active KDMs.

We analyzed the morphology and the transcriptomic changes of human neural stem progenitor cells (hNSPCs) grown on laminin in adherent culture conditions and subjected to simulated microgravity for different times in a random positioning machine apparatus. Low-cell-density cultures exposed to simulated microgravity for 24 h showed cell aggregate formation and significant modulation of several genes involved in focal adhesion, cytoskeleton regulation, and cell cycle control. These effects were much more limited in hNSPCs cultured at high density in the same conditions. We also found that some of the genes modulated upon exposure to simulated microgravity showed similar changes in hNSPCs grown without laminin in non-adherent culture conditions under normal gravity. These results suggest that reduced gravity counteracts the interactions of cells with the extracellular matrix, inducing morphological and transcriptional changes that can be observed in low-density cultures.

Deciphering patient-specific mechanisms of cancer cell reprogramming remains a crucial challenge in systems oncology, as it is key to improving patient diagnosis and treatment. For this reason, comprehensive and patient-specific multi-omic characterization of tumor specimens has become increasingly common in clinical practice. Here, we developed PatientProfiler , a computational workflow that integrates proteogenomic data with curated causal interaction networks to generate mechanistic models of signal transduction for individual patients. PatientProfiler allows multi-omic data analysis and standardization, generation of patient-specific mechanistic models of signal transduction, and extraction of network-based prognostic biomarkers. We successfully benchmarked the tool on proteogenomic and clinical data derived from 122 biopsies of treatment-naïve breast cancer, available through the CPTAC portal. We identified patient-specific mechanistic models that recapitulate oncogenic signaling pathways. In-depth topological exploration of these networks revealed seven subgroups of patients, associated with unique transcriptomic signatures and distinct prognostic values. We identified well-known Basal-like 1 and 2 subtypes, while also highlighting distinct mechanistic drivers such as the MYC–CDK4/6 axis or NF-kappaB-mediated inflammatory programs. Beyond breast cancer, PatientProfiler offers a generalizable framework to transform cohort-level multi-omic data into interpretable mechanistic models, making it applicable across diverse cancer types and other complex diseases.