Replisome Proximal Protein Associations and Dynamic Proteomic Changes at Stalled Replication Forks.

DNA replication is a fundamental cellular process that ensures the transfer of genetic information during cell division. Genome duplication takes place in S phase and requires a dynamic and highly coordinated recruitment of multiple proteins at replication forks. Various genotoxic stressors lead to fork instability and collapse, hence the need for DNA repair pathways. By identifying the multitude of protein interactions implicated in those events, we can better grasp the complex and dynamic molecular mechanisms that facilitate DNA replication and repair. Proximity-dependent biotin identification was used to identify associations with 17 proteins within four core replication components, namely the CDC45/MCM2-7/GINS helicase that unwinds DNA, the DNA polymerases, replication protein A subunits, and histone chaperones needed to disassemble and reassemble chromatin. We further investigated the impact of genotoxic stress on these interactions. This analysis revealed a vast proximity association network with 108 nuclear proteins further modulated in the presence of hydroxyurea; 45 being enriched and 63 depleted. Interestingly, hydroxyurea treatment also caused a redistribution of associations with 11 interactors, meaning that the replisome is dynamically reorganized when stressed. The analysis identified several poorly characterized proteins, thereby uncovering new putative players in the cellular response to DNA replication arrest. It also provides a new comprehensive proteomic framework to understand how cells respond to obstacles during DNA replication.

Deciphering of Adult Glioma Vulnerabilities through Expression Pattern Analysis of GABA, Glutamate and Calcium Neurotransmitter Genes.

Adult infiltrating gliomas are highly aggressive tumors of the central nervous system with a dismal prognosis despite intensive multimodal therapy (chemotherapy and/or radiotherapy). In this study, we studied the expression, methylation and interacting miRNA profiles of GABA-, glutamate- and calcium-related genes in 661 adult infiltrating gliomas available through the TCGA database. Neurotransmitter-based unsupervised clustering identified three established glioma molecular subgroups that parallel major World Health Organization glioma subclasses (IDH-wildtype astrocytomas, IDH-mutant astrocytomas, IDH-mutant oligodendroglioma). In addition, this analysis also defined a novel, neurotransmitter-related glioma subgroup (NT-1), mostly comprised of IDH-mutated gliomas and characterized by the overexpression of neurotransmitter-related genes. Lower expression of neurotransmission-related genes was correlated with increased aggressivity in hypomethylated IDH-wildtype tumors. There were also significant differences in the composition of the tumor inflammatory microenvironment between neurotransmission-based tumor categories, with lower estimated pools of M2-phenotype macrophages in NT-1 gliomas. This multi-omics analysis of the neurotransmission expression landscape of TCGA gliomas-which highlights the existence of neurotransmission-based glioma categories with different expression, epigenetic and inflammatory profiles-supports the existence of operational neurotransmitter signaling pathways in adult gliomas. These findings could shed new light on potential vulnerabilities to exploit in future glioma-targeting drug therapies.

miR profile in pagetic osteoclasts: from large-scale sequencing to gene expression study.

Paget's disease of bone (PDB) is characterized by excessive and disorganized bone remodeling, in which bone-resorbing osteoclasts play a key role. We investigated microRNA (miR) expression in osteoclasts derived from the blood of 40 PDB patients and 30 healthy controls. By deep sequencing, a preliminary analysis identified differentially expressed miRs in a discovery cohort of 9 PDB patients and 9 age and sex-matched healthy controls. Six mature miRs, miR-29b1-3p, miR-15b-5p, miR-181a-5p, let-7i-3p, miR-500b-5p, and miR-1246, were found to be significantly decreased in pagetic overactive osteoclasts. The differential expression of the miRs was confirmed by the analysis of a larger independent cohort using qPCR. In an integrative network biology analysis of the miR candidates, we identified strong validated interactions between the miRs and some pathways, primarily apoptosis, and major osteoclast signaling pathways including PI3K/Akt, IFNγ, or TGFß, as well as c-Fos, a transcription factor, and MMP-9, a metalloprotease. In addition, other genes like CCND2, CCND1, WEE1, SAMHD1, and AXIN2 were revealed in this network of interactions. Our results enhance the understanding of osteoclast biology in PDB; our work may also provide fresh perspectives on the research or therapeutic development of other bone diseases. KEY MESSAGES: miR profile in overactive osteoclasts from patients with Paget's disease of bone. Six mature miRs were significantly decreased in pagetic osteoclasts vs controls. miRs of interest: let7i-3p, miR-15b-5p, -29b1-3p, -181a-5p, -500b-5p, and -1246. Target genes and enriched pathways highlight the importance of apoptotic pathways.

Osteoclast signaling-targeting miR-146a-3p and miR-155-5p are downregulated in Paget's disease of bone.

MicroRNA (miRs) are small, non-coding RNA that post-transcriptionally regulate DNA expression. We hypothesized that specific miR profiles may be a feature of overactive osteoclasts in Paget's disease of bone (PDB), a disorder characterized by an increased and disorganized bone remodeling that typically begins with excessive bone resorption. We compared the expression profile of 13 miRs in human osteoclasts differentiated in vitro from peripheral blood mononuclear cells (PBMCs) of patients with PDB (n = 10) or age- and sex- matched healthy subjects (n = 10). We selected 13 miRs for testing, on the basis of their previously reported roles either in human osteoclast differentiation, in bone diseases, or in osteoclast important signaling pathways. From those expression results, 3 miRNAs were further selected for in-vitro studies aiming at modulating miR expression in human cord blood monocyte derived osteoclasts: 2 miRs (miR-146a-3p and miR-155-5p) whose expression was significantly reduced in pagetic osteoclasts, as well as miRNA-133a-3p, stable in PDB relative to controls, but with known regulatory importance within osteoclasts. We demonstrated a positive (miR-133a-3p) or negative (miR-155-5p, miR-146a-3p) impact of those miRs on the formation of osteoclasts and/or their bone resorption capacity in this human model. Signaling pathways were significantly affected, including p38 MAP-kinase (miR-133a-3p), RANKL-induced TRAF6/NFκB signaling (miR-146a-3p), and MITF expression (miR-155-5p). Osteoclast miRNA profiles might have an important value to yield significant new insights into the osteoclast phenotype in PDB and in other bone diseases with hyperactive osteoclasts.