Control of Intestinal Stemness and Cell Lineage by Histone Variant H2A.Z Isoforms.

The histone variant H2A.Z plays important functions in the regulation of gene expression. In mammals, it is encoded by two genes, giving rise to two highly related isoforms named H2A.Z.1 and H2A.Z.2, which can have similar or antagonistic functions depending on the promoter. Knowledge of the physiopathological consequences of such functions emerges, but how the balance between these isoforms regulates tissue homeostasis is not fully understood. Here, we investigated the relative role of H2A.Z isoforms in intestinal epithelial homeostasis. Through genome-wide analysis of H2A.Z genomic localization in differentiating Caco-2 cells, we uncovered an enrichment of H2A.Z isoforms on the bodies of genes which are induced during enterocyte differentiation, stressing the potential importance of H2A.Z isoforms dynamics in this process. Through a combination of in vitro and in vivo experiments, we further demonstrated the two isoforms cooperate for stem and progenitor cells proliferation, as well as for secretory lineage differentiation. However, we found that they antagonistically regulate enterocyte differentiation, with H2A.Z.1 preventing terminal differentiation and H2A.Z.2 favoring it. Altogether, these data indicate that H2A.Z isoforms are critical regulators of intestine homeostasis and may provide a paradigm of how the balance between two isoforms of the same chromatin structural protein can control physiopathological processes.

TDP1 mutation causing SCAN1 neurodegenerative syndrome hampers the repair of transcriptional DNA double-strand breaks.

TDP1 removes transcription-blocking topoisomerase I cleavage complexes (TOP1ccs), and its inactivating H493R mutation causes the neurodegenerative syndrome SCAN1. However, the molecular mechanism underlying the SCAN1 phenotype is unclear. Here, we generate human SCAN1 cell models using CRISPR-Cas9 and show that they accumulate TOP1ccs along with changes in gene expression and genomic distribution of R-loops. SCAN1 cells also accumulate transcriptional DNA double-strand breaks (DSBs) specifically in the G1 cell population due to increased DSB formation and lack of repair, both resulting from abortive removal of transcription-blocking TOP1ccs. Deficient TDP1 activity causes increased DSB production, and the presence of mutated TDP1 protein hampers DSB repair by a TDP2-dependent backup pathway. This study provides powerful models to study TDP1 functions under physiological and pathological conditions and unravels that a gain of function of the mutated TDP1 protein, which prevents DSB repair, rather than a loss of TDP1 activity itself, could contribute to SCAN1 pathogenesis.

Deciphering Black Extrinsic Tooth Stain Composition in Children Using Metaproteomics.

The present study focuses on the use of a metaproteomic approach to analyze Black Extrinsic Tooth Stains, a specific type of pigmented extrinsic substance. Metaproteomics is a powerful emerging technology that successfully enabled human protein and bacterial identification of this specific dental biofilm using high-resolution tandem mass spectrometry. A total of 1600 bacterial proteins were identified in black stain (BS) samples and 2058 proteins in dental plaque (DP) samples, whereas 607 and 582 human proteins were identified in BS and DP samples, respectively. A large diversity of bacteria genera (142) in BS and DP was identified, showing a high prevalence of Rothia, Kingella, Neisseria, and Pseudopropionibacterium in black stain samples. In this work, the high diversity of the dental microbiota and its proteome is highlighted, including significant differences between black stain and dental plaque samples.

Increasing the power of interpretation for soil metaproteomics data.

BACKGROUND: Soil and sediment microorganisms are highly phylogenetically diverse but are currently largely under-represented in public molecular databases. Their functional characterization by means of metaproteomics is usually performed using metagenomic sequences acquired for the same sample. However, such hugely diverse metagenomic datasets are difficult to assemble; in parallel, theoretical proteomes from isolates available in generic databases are of high quality. Both these factors advocate for the use of theoretical proteomes in metaproteomics interpretation pipelines. Here, we examined a number of database construction strategies with a view to increasing the outputs of metaproteomics studies performed on soil samples. RESULTS: The number of peptide-spectrum matches was found to be of comparable magnitude when using public or sample-specific metagenomics-derived databases. However, numbers were significantly increased when a combination of both types of information was used in a two-step cascaded search. Our data also indicate that the functional annotation of the metaproteomics dataset can be maximized by using a combination of both types of databases. CONCLUSIONS: A two-step strategy combining sample-specific metagenome database and public databases such as the non-redundant NCBI database and a massive soil gene catalog allows maximizing the metaproteomic interpretation both in terms of ratio of assigned spectra and retrieval of function-derived information. Video abstract.

Estimating relative biomasses of organisms in microbiota using "phylopeptidomics".

BACKGROUND: There is an important need for the development of fast and robust methods to quantify the diversity and temporal dynamics of microbial communities in complex environmental samples. Because tandem mass spectrometry allows rapid inspection of protein content, metaproteomics is increasingly used for the phenotypic analysis of microbiota across many fields, including biotechnology, environmental ecology, and medicine. RESULTS: Here, we present a new method for identifying the biomass contribution of any given organism based on a signature describing the number of peptide sequences shared with all other organisms, calculated by mathematical modeling and phylogenetic relationships. This so-called "phylopeptidomics" principle allows for the calculation of the relative ratios of peptide-specified taxa by the linear combination of such signatures applied to an experimental metaproteomic dataset. We illustrate its efficiency using artificial mixtures of two closely related pathogens of clinical interest, and with more complex microbiota models. CONCLUSIONS: This approach paves the way to a new vision of taxonomic changes and accurate label-free quantitative metaproteomics for fine-tuned functional characterization. Video abstract.

Proteotyping Environmental Microorganisms by Phylopeptidomics: Case Study Screening Water from a Radioactive Material Storage Pool.

The microbial diversity encompassed by the environmental biosphere is largely unexplored, although it represents an extensive source of new knowledge and potentially of novel enzymatic catalysts for biotechnological applications. To determine the taxonomy of microorganisms, proteotyping by tandem mass spectrometry has proved its efficiency. Its latest extension, phylopeptidomics, adds a biomass quantitation perspective for mixtures of microorganisms. Here, we present an application of phylopeptidomics to rapidly and sensitively screen microorganisms sampled from an industrial environment, i.e., a pool where radioactive material is stored. The power of this methodology is demonstrated through the identification of both prokaryotes and eukaryotes, whether as pure isolates or present as mixtures or consortia. In this study, we established accurate taxonomical identification of environmental prokaryotes belonging to the Actinobacteria, Bacteroidetes, Firmicutes, and Proteobacteria phyla, as well as eukaryotes from the Ascomycota phylum. The results presented illustrate the potential of tandem mass spectrometry proteotyping, in particular phylopeptidomics, to screen for and rapidly identify microorganisms.

Combining proteogenomics and metaproteomics for deep taxonomic and functional characterization of microbiomes from a non-sequenced host.

Metaproteomics of gut microbiomes from animal hosts lacking a reference genome is challenging. Here we describe a strategy combining high-resolution metaproteomics and host RNA sequencing (RNA-seq) with generalist database searching to survey the digestive tract of Gammarus fossarum, a small crustacean used as a sentinel species in ecotoxicology. This approach provides a deep insight into the full range of biomasses and metabolic activities of the holobiont components, and differentiates between the intestine and hepatopancreatic caecum.