[Lsr2: A Nucleoid Associated Protein (NAP) and a transcription factor in mycobacteria]. / Lsr2 : protéine associée au nucléoïde (NAP) et facteur transcriptionnel chez les mycobactéries.

Lsr2, a small protein mainly found in actinobacteria, plays a crucial role in the virulence and adaptation of mycobacteria to environmental conditions. As a member of the nucleoid-associated protein (NAPs) superfamily, Lsr2 influences DNA organization by facilitating the formation of chromosomal loops in vitro and, therefore, may be a major player in the three-dimensional folding of the genome. Additionally, Lsr2 also acts as a transcription factor, regulating the expression of numerous genes responsible for coordinating a myriad of cellular and molecular processes essential for the actinobacteria. Similar to the H-NS protein, its ortholog in enterobacteria, its role in transcriptional repression likely relies on oligomerization, rigidifying, and bridging of DNA, thereby disrupting RNA polymerase recruitment as well as the elongation of RNA transcripts.

Title: Lsr2 : protéine associée au nucléoïde (NAP) et facteur transcriptionnel chez les mycobactéries. Abstract: Lsr2, une petite protéine conservée chez les actinobactéries, joue un rôle crucial dans la virulence et l'adaptation des mycobactéries aux conditions environnementales. Membre de la superfamille des protéines associées au nucléoïde (NAP), Lsr2 influence l'organisation de l'ADN en facilitant la formation de boucle chromosomique in vitro, ce qui suggère qu'elle pourrait être un acteur majeur du repliement tridimensionnel du génome. Lsr2 agit également comme un facteur de transcription, régulant l'expression de nombreux gènes responsables de la coordination d'une multitude de processus cellulaires et moléculaires essentiels chez les actinobactéries. Tout comme la protéine H-NS, son orthologue chez les entérobactéries, son rôle de répresseur transcriptionnel repose probablement sur son oligomérisation conduisant à la rigidification de l'ADN et, dans certaines situations, sur le pontage de fragments génomiques distants. Ces mécanismes pourraient perturber le recrutement de l'ARN polymérase sur les promoteurs ainsi que l'élongation des transcrits.

Lsr2, a pleiotropic regulator at the core of the infectious strategy of Mycobacterium abscessus.

Mycobacterium abscessus is a non-tuberculous mycobacterium, causing lung infections in cystic fibrosis patients. During pulmonary infection, M. abscessus switches from smooth (Mabs-S) to rough (Mabs-R) morphotypes, the latter being hyper-virulent. Previously, we isolated the lsr2 gene as differentially expressed during S-to-R transition. lsr2 encodes a pleiotropic transcription factor that falls under the superfamily of nucleoid-associated proteins. Here, we used two functional genomic methods, RNA-seq and chromatin immunoprecipitation-sequencing (ChIP-seq), to elucidate the molecular role of Lsr2 in the pathobiology of M. abscessus. Transcriptomic analysis shows that Lsr2 differentially regulates gene expression across both morphotypes, most of which are involved in several key cellular processes of M. abscessus, including host adaptation and antibiotic resistance. These results were confirmed through quantitative real-time PCR, as well as by minimum inhibitory concentration tests and infection tests on macrophages in the presence of antibiotics. ChIP-seq analysis revealed that Lsr2 extensively binds the M. abscessus genome at AT-rich sequences and appears to form long domains that participate in the repression of its target genes. Unexpectedly, the genomic distribution of Lsr2 revealed no distinctions between Mabs-S and Mabs-R, implying more intricate mechanisms at play for achieving target selectivity.IMPORTANCELsr2 is a crucial transcription factor and chromosome organizer involved in intracellular growth and virulence in the smooth and rough morphotypes of Mycobacterium abscessus. Using RNA-seq and chromatin immunoprecipitation-sequencing (ChIP-seq), we investigated the molecular role of Lsr2 in gene expression regulation along with its distribution on M. abscessus genome. Our study demonstrates the pleiotropic regulatory role of Lsr2, regulating the expression of many genes coordinating essential cellular and molecular processes in both morphotypes. In addition, we have elucidated the role of Lsr2 in antibiotic resistance both in vitro and in vivo, where lsr2 mutant strains display heightened sensitivity to antibiotics. Through ChIP-seq, we reported the widespread distribution of Lsr2 on M. abscessus genome, revealing a direct repressive effect due to its extensive binding on promoters or coding sequences of its targets. This study unveils the significant regulatory role of Lsr2, intricately intertwined with its function in shaping the organization of the M. abscessus genome.

The environmentally-regulated interplay between local three-dimensional chromatin organisation and transcription of proVWX in E. coli.

Nucleoid associated proteins (NAPs) maintain the architecture of bacterial chromosomes and regulate gene expression. Thus, their role as transcription factors may involve three-dimensional chromosome re-organisation. While this model is supported by in vitro studies, direct in vivo evidence is lacking. Here, we use RT-qPCR and 3C-qPCR to study the transcriptional and architectural profiles of the H-NS (histone-like nucleoid structuring protein)-regulated, osmoresponsive proVWX operon of Escherichia coli at different osmolarities and provide in vivo evidence for transcription regulation by NAP-mediated chromosome re-modelling in bacteria. By consolidating our in vivo investigations with earlier in vitro and in silico studies that provide mechanistic details of how H-NS re-models DNA in response to osmolarity, we report that activation of proVWX in response to a hyperosmotic shock involves the destabilization of H-NS-mediated bridges anchored between the proVWX downstream and upstream regulatory elements (DRE and URE), and between the DRE and ygaY that lies immediately downstream of proVWX. The re-establishment of these bridges upon adaptation to hyperosmolarity represses the operon. Our results also reveal additional structural features associated with changes in proVWX transcript levels such as the decompaction of local chromatin upstream of the operon, highlighting that further complexity underlies the regulation of this model operon. H-NS and H-NS-like proteins are wide-spread amongst bacteria, suggesting that chromosome re-modelling may be a typical feature of transcriptional control in bacteria.

STAT1 deficiency underlies a proinflammatory imprint of naive CD4+ T cells in spondyloarthritis.

Introduction: In spondyloarthritis (SpA), an increased type 3 immune response, including T helper cells (Th) 17 excess, is observed in both human and SpA animal models, such as the HLA-B27/human ß2-microglobulin transgenic rat (B27-rat). Methods: To investigate this unexplained Th17-biased differentiation, we focused on understanding the immunobiology of B27-rat naive CD4+ T cells (Tn). Results: We observed that neutrally stimulated B27-rat Tn developed heightened Th17 profile even before disease onset, suggesting an intrinsic proinflammatory predisposition. In parallel with this observation, transcriptomic and epigenomic analyses showed that B27-rat Tn exhibited a decreased expression of Interferon/Th1- and increased expression of Th17-related genes. This molecular signature was predicted to be related to an imbalance of STAT1/STAT3 transcription factors activity. Stat1 mRNA and STAT1 protein expression were decreased before disease onset in Tn, even in their thymic precursors, whereas Stat3/STAT3 expression increased upon disease establishment. Confirming the relevance of these results, STAT1 mRNA expression was also decreased in Tn from SpA patients, as compared with healthy controls and rheumatoid arthritis patients. Finally, stimulation of B27-rat Tn with a selective STAT1 activator abolished this preferential IL-17A expression, suggesting that STAT1-altered activity in B27-rats allows Th17 differentiation. Discussion: Altogether, B27-rat Tn harbor a STAT1 deficiency preceding disease onset, which may occur during their thymic differentiation, secondarily associated with a persistent Th17 bias, which is imprinted at the epigenomic level. This early molecular phenomenon might lead to the persistent proinflammatory skew of CD4+ T cells in SpA patients, thus offering new clues to better understand and treat SpA.

HI-NESS: a family of genetically encoded DNA labels based on a bacterial nucleoid-associated protein.

The interplay between three-dimensional chromosome organisation and genomic processes such as replication and transcription necessitates in vivo studies of chromosome dynamics. Fluorescent organic dyes are often used for chromosome labelling in vivo. The mode of binding of these dyes to DNA cause its distortion, elongation, and partial unwinding. The structural changes induce DNA damage and interfere with the binding dynamics of chromatin-associated proteins, consequently perturbing gene expression, genome replication, and cell cycle progression. We have developed a minimally-perturbing, genetically encoded fluorescent DNA label consisting of a (photo-switchable) fluorescent protein fused to the DNA-binding domain of H-NS - a bacterial nucleoid-associated protein. We show that this DNA label, abbreviated as HI-NESS (H-NS-based indicator for nucleic acid stainings), is minimally-perturbing to genomic processes and labels chromosomes in eukaryotic cells in culture, and in zebrafish embryos with preferential binding to AT-rich chromatin.

Epigenetics of spondyloarthritis.

Spondyloarthritis (SpA) is a chronic inflammatory disorder resulting from a combination of genetic predisposition and environmental factors. Despite recent advances, a substantial fraction of its genetic basis remains poorly understood. Several mechanisms have been proposed to account for this unexplained heritability, including epigenetics which can play a role at the interface between genetic and environmental susceptibility factors. Epigenetics refers to changes in gene expression that are not encoded in the DNA sequence itself. Such mechanisms may include DNA methylation, histone modifications and non-coding RNAs. Disruption of one of these systems can lead to inappropriate gene expression, which in turn might favour the development of disease. Thanks to recent technological progress, there has been a growing interest in the field of epigenetics in complex diseases, including SpA. However, epigenetic studies face some methodological limitations that hamper interpretation of their results: small sample size, absence of biological replication, lack of adequate controls for potential confounders, studies not performed in the most relevant cell/tissues. In the future, integration of epigenetics with other "omics" data will probably be necessary to improve our understanding of SpA pathogenesis. These issues need to be addressed before considering the use of epigenetic marks in clinical routine, as biomarkers or as drug targets.

Lsr2 Is an Important Determinant of Intracellular Growth and Virulence in Mycobacterium abscessus.

Mycobacterium abscessus, a pathogen responsible for severe lung infections in cystic fibrosis patients, exhibits either smooth (S) or rough (R) morphotypes. The S-to-R transition correlates with inhibition of the synthesis and/or transport of glycopeptidolipids (GPLs) and is associated with an increase of pathogenicity in animal and human hosts. Lsr2 is a small nucleoid-associated protein highly conserved in mycobacteria, including M. abscessus, and is a functional homolog of the heat-stable nucleoid-structuring protein (H-NS). It is essential in Mycobacterium tuberculosis but not in the non-pathogenic model organism Mycobacterium smegmatis. It acts as a master transcriptional regulator of multiple genes involved in virulence and immunogenicity through binding to AT-rich genomic regions. Previous transcriptomic studies, confirmed here by quantitative PCR, showed increased expression of lsr2 (MAB_0545) in R morphotypes when compared to their S counterparts, suggesting a possible role of this protein in the virulence of the R form. This was addressed by generating lsr2 knock-out mutants in both S (Δlsr2-S) and R (Δlsr2-R) variants, demonstrating that this gene is dispensable for M. abscessus growth. We show that the wild-type S variant, Δlsr2-S and Δlsr2-R strains were more sensitive to H2O2 as compared to the wild-type R variant of M. abscessus. Importantly, virulence of the Lsr2 mutants was considerably diminished in cellular models (macrophage and amoeba) as well as in infected animals (mouse and zebrafish). Collectively, these results emphasize the importance of Lsr2 in M. abscessus virulence.

Determination of the 3D Genome Organization of Bacteria Using Hi-C.

The spatial organization of genomes is based on their hierarchical compartmentalization in topological domains. There is growing evidence that bacterial genomes are organized into insulated domains similar to the Topologically Associating Domains (TADs) detected in eukaryotic cells. Chromosome conformation capture (3C) technologies are used to analyze in vivo DNA proximity based on ligation of distal DNA segments crossed-linked by bridging proteins. By combining 3C and high-throughput sequencing, the Hi-C method reveals genome-wide interactions within topological domains and global genome structure as a whole. This chapter provides detailed guidelines for the preparation of Hi-C sequencing libraries for bacteria.

GEMMER: GEnome-wide tool for Multi-scale Modeling data Extraction and Representation for Saccharomyces cerevisiae.

Motivation: Multi-scale modeling of biological systems requires integration of various information about genes and proteins that are connected together in networks. Spatial, temporal and functional information is available; however, it is still a challenge to retrieve and explore this knowledge in an integrated, quick and user-friendly manner. Results: We present GEMMER (GEnome-wide tool for Multi-scale Modeling data Extraction and Representation), a web-based data-integration tool that facilitates high quality visualization of physical, regulatory and genetic interactions between proteins/genes in Saccharomyces cerevisiae. GEMMER creates network visualizations that integrate information on function, temporal expression, localization and abundance from various existing databases. GEMMER supports modeling efforts by effortlessly gathering this information and providing convenient export options for images and their underlying data. Availability and implementation: GEMMER is freely available at http://gemmer.barberislab.com. Source code, written in Python, JavaScript library D3js, PHP and JSON, is freely available at https://github.com/barberislab/GEMMER. Supplementary information: Supplementary data are available at Bioinformatics online.

The volumes and transcript counts of single cells reveal concentration homeostasis and capture biological noise.

Transcriptional stochasticity can be measured by counting the number of mRNA molecules per cell. Cell-to-cell variability is best captured in terms of concentration rather than molecule counts, because reaction rates depend on concentrations. We combined single-molecule mRNA counting with single-cell volume measurements to quantify the statistics of both transcript numbers and concentrations in human cells. We compared three cell clones that differ only in the genomic integration site of an identical constitutively expressed reporter gene. The transcript number per cell varied proportionally with cell volume in all three clones, indicating concentration homeostasis. We found that the cell-to-cell variability in the mRNA concentration is almost exclusively due to cell-to-cell variation in gene expression activity, whereas the cell-to-cell variation in mRNA number is larger, due to a significant contribution of cell volume variability. We concluded that the precise relationship between transcript number and cell volume sets the biological stochasticity of living cells. This study highlights the importance of the quantitative measurement of transcript concentrations in studies of cell-to-cell variability in biology.

Genomic looping: a key principle of chromatin organization.

The effective volume occupied by the genomes of all forms of life far exceeds that of the cells in which they are contained. Therefore, all organisms have developed mechanisms for compactly folding and functionally organizing their genetic material. Through recent advances in fluorescent microscopy and 3C-based technologies, we finally have a first glimpse into the complex mechanisms governing the 3-D folding of genomes. A key feature of genome organization in all domains of life is the formation of DNA loops. Here, we describe the main players in DNA organization with a focus on DNA-bridging proteins. Specifically, we discuss the properties of the bacterial DNA-bridging protein H-NS. Via two different modes of binding to DNA, this protein is a key driver of bacterial genome organization and provides a link between 3-D organization and transcription regulation. Importantly, H-NS function is modulated in response to environmental cues, which are translated into adapted gene expression patterns. We delve into the mechanisms underlying DNA looping and explore the complex and subtle modulation of these diverse, yet difficult-to-study, structures. DNA looping is universal and a conserved mechanism of genome organization throughout all domains of life.

A new regulatory principle for in vivo biochemistry: pleiotropic low affinity regulation by the adenine nucleotides--illustrated for the glycolytic enzymes of Saccharomyces cerevisiae.

Enzymology tends to focus on highly specific effects of substrates, allosteric modifiers, and products occurring at low concentrations, because these are most informative about the enzyme's catalytic mechanism. We hypothesized that at relatively high in vivo concentrations, important molecular monitors of the state of living cells, such as ATP, affect multiple enzymes of the former and that these interactions have gone unnoticed in enzymology. We test this hypothesis in terms of the effect that ATP, ADP, and AMP might have on the major free-energy delivering pathway of the yeast Saccharomyces cerevisiae. Assaying cell-free extracts, we collected a comprehensive set of quantitative kinetic data concerning the enzymes of the glycolytic and the ethanol fermentation pathways. We determined systematically the extent to which the enzyme activities depend on the concentrations of the adenine nucleotides. We found that the effects of the adenine nucleotides on enzymes catalysing reactions in which they are not directly involved as substrate or product, are substantial. This includes effects on the Michaelis-Menten constants, adding new perspective on these, 100 years after their introduction.

Drosophila retinal pigment cell death is regulated in a position-dependent manner by a cell memory gene.

The stereotyped organization of the Drosophila compound eye depends on the elimination by apoptosis of about 25% of the inter-ommatidial pigment cell precursors (IOCs) during metamorphosis. This program of cell death is under antagonistic effects of the Notch and the EGFR pathways. In addition, uncharacterized positional cues may underlie death versus survival choices among IOCs. Our results provide new genetic evidences that cell death is regulated in a position- dependent manner in the eye. We show that mutations in Trithorax-like (Trl) and lola-like/batman specifically block IOC death during eye morphogenesis. These genes share characteristics of both Polycomb-Group and trithorax-Group genes, in that they are required for chromatin-mediated repression and activation of Hox genes. However, Trl function in triggering IOC death is independent from a function in repressing Hox gene expression during eye development. Analysis of mosaic ommatidiae containing Trl mutant cells revealed that Trl function for IOC death is required in cone cells. Strikingly, cell death suppression in Trl mutants depends on the position of IOCs. Our results further support a model whereby death of IOCs on the oblique sides of ommatidiae requires Trl-dependent reduction of a survival signal, or an increase of a death signal, emanating from cone cells. Trl does not have the same effect on horizontal IOCs whose survival seems to involve additional topological constraints.

Imaging in situ protein-DNA interactions in the cell nucleus using FRET-FLIM.

Although the distribution of DNA-binding proteins inside the cell nucleus can be analyzed by immunolabeling or by tagging proteins with GFP, we cannot establish whether the protein is bound to DNA or not. Here, we describe a novel approach that allows imaging of the in situ interaction between a GFP-fusion protein and DNA in the cell nucleus, using fluorescence resonance energy transfer (FRET). We used fluorescence lifetime imaging microscopy (FLIM) as a reliable tool to detect protein in contact with DNA. The method was successfully applied to the DNA-binding proteins histone H2B and the glucocorticoid receptor and to the heterochromatin-associated proteins HP1alpha and HP1beta.