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Differential Crosslinking and Contractile Motors Drive Nuclear Chromatin Compaction.

During interphase, a typical cell nucleus features spatial compartmentalization of transcriptionally active euchromatin and repressed heterochromatin domains. In conventional nuclear organization, euchromatin predominantly occupies the nuclear interior, while heterochromatin, which is approximately 50% more dense than euchromatin, is positioned near the nuclear periphery. Peripheral chromatin organization can be further modulated by the nuclear lamina, which is itself a deformable structure. While a number of biophysical mechanisms for compartmentalization within rigid nuclei have been explored, we study a chromatin model consisting of an active, crosslinked polymer tethered to a deformable, polymeric lamina shell. Contractile motors, the deformability of the shell, and the spatial distribution of crosslinks all play pivotal roles in this compartmentalization. We find that a radial crosslink density distribution, even with a small linear differential of higher crosslinking density at the edge of the nucleus, combined with contractile motor activity, drives genomic segregation, in agreement with experimental observations. This arises from contractile motors preferentially drawing crosslinks into their vicinity at the nuclear periphery, forming high-density domains that promote heterochromatin formation. We also find an increased stiffness of nuclear wrinkles given the preferential heterochromatin compaction below the lamina shell, which is consistent with instantaneous nuclear stiffening under applied nanoindentation. We conclude with the potential for experimental validation of our model predictions.

Journal Article↗

Simulation of the kinetics of a sphere attached to a fluctuating polymer: Implications for target search by DNA-binding proteins.

We simulate the dynamics of a sphere, bound with a certain probability onto the beads of a harmonic bead-spring polymer in a confined geometry. The sphere hops from ith to the spatially closest i+/-kth bead. There is a crossover between kinetics dominated by different mechanisms with increasing binding probability. We relate these observations to the context of a biologically important problem of target location by DNA-binding proteins within a genome.

Binding Sites↗

Single-cell gene expression profiling.

A key goal of biology is to relate the expression of specific genes to a particular cellular phenotype. However, current assays for gene expression destroy the structural context. By combining advances in computational fluorescence microscopy with multiplex probe design, we devised technology in which the expression of many genes can be visualized simultaneously inside single cells with high spatial and temporal resolution. Analysis of 11 genes in serum-stimulated cultured cells revealed unique patterns of gene expression within individual cells. Using the nucleus as the substrate for parallel gene analysis, we provide a platform for the fusion of genomics and cell biology: "cellular genomics."

Adenocarcinoma↗

[The fibroblast, definition of its phenotype by its "programme" of biosynthesis of the extracellular matrix].

This presentation is an introduction to a symposium focusing on the fibroblast, a virtually ubiquitous cell which is cultured in many laboratories. The traditional definition of the fibroblast based solely on morphological criteria, which has satisfied most biologists for years, now needs reappraisal. The papers delivered at this symposium addressed this issue. Investigations ranged from the phylogenetic origin of fibroblasts in sponges, studied by R. Garrone, to the phenotypic modulations leading to the "myofibroblast" reported by G. Gabbiani, whose manuscript was unfortunately received too late for the conference. The present article describes how the fibroblast can be defined on the basis of its programme for producing extracellular matrix macromolecules. This programme includes a qualitative aspect, i.e., the selection of genes to be expressed: collagen types, variations in alternative splicing patterns of matrix proteins--and a quantitative aspect, i.e., the precise spatial and temporal regulation of production of "selected" macromolecules in order to meet the body's needs. This programme can be studied experimentally, and data are accumulating on the mechanisms involved and on its regulation through intrinsic factors (acting on the genome) and extrinsic and epigenetic factors (vitamins, hormones, nutrition...). Several French laboratories have supplied useful contributions to this research. The results of their investigations, which cover part of this field, are reproduced in this issue of this revue.

Collagen↗

Differential Crosslinking and Contractile Motors Drive Nuclear Chromatin Compaction.

During interphase, a typical cell nucleus features spatial compartmentalization of transcriptionally active euchromatin and repressed heterochromatin domains. In conventional nuclear organization, euchromatin predominantly occupies the nuclear interior, while heterochromatin, which is approximately 50% more dense than euchromatin, is positioned near the nuclear periphery. Peripheral chromatin organization can be further modulated by the nuclear lamina, which is itself a deformable structure. While a number of biophysical mechanisms for compartmentalization within rigid nuclei have been explored, we study a chromatin model consisting of an active, crosslinked polymer tethered to a deformable, polymeric lamina shell. Contractile motors, the deformability of the shell, and the spatial distribution of crosslinks all play pivotal roles in this compartmentalization. We find that a radial crosslink density distribution, even with a small linear differential of higher crosslinking density at the edge of the nucleus, combined with contractile motor activity, drives genomic segregation, in agreement with experimental observations. This arises from contractile motors preferentially drawing crosslinks into their vicinity at the nuclear periphery, forming high-density domains that promote heterochromatin formation. We also find an increased stiffness of nuclear wrinkles given the preferential heterochromatin compaction below the lamina shell, which is consistent with instantaneous nuclear stiffening under applied nanoindentation. We conclude with the potential for experimental validation of our model predictions.

Journal Article↗

Multi-omics and spatial transcriptomics reveal that S100A10 drives CD8+ T-cell exhaustion and immune evasion in hepatocellular carcinoma through cPLA2-5-LOX-mediated arachidonic acid metabolism and ferroptosis.

Immune evasion in hepatocellular carcinoma (HCC) represents a major biological barrier limiting the efficacy of immunotherapy, yet its molecular basis remains incompletely understood. Increasing evidence indicates that tumor metabolic reprogramming and ferroptosis-related signaling play critical roles in shaping an immunosuppressive tumor microenvironment (TME); however, the specific regulatory factors involved remain unclear. This study aims to systematically elucidate the functional role of S100 calcium-binding protein A10 (S100A10) in immune evasion in HCC, with a particular focus on the molecular mechanisms by which S100A10 regulates CD8+ T-cell exhaustion through arachidonic acid (AA) metabolism and ferroptosis, as well as its potential therapeutic implications. To this end, data from The Cancer Genome Atlas Liver Hepatocellular Carcinoma (TCGA-LIHC) cohort are integrated to analyze the expression patterns of S100A10, its prognostic value, and its association with the immune microenvironment. S100A10 overexpression and knockout models are established in HCCLM3 and MHCC97L cell lines, and S100A10-mediated metabolic pathway reprogramming is characterized using transcriptomic profiling, untargeted metabolomics, and ferroptosis-related functional assays. In parallel, single-cell RNA sequencing (scRNA-seq) and spatial transcriptomics are employed to delineate the cell-type specificity and spatial distribution of S100A10. Furthermore, human CD8+ T-cell co-culture systems and orthotopic mouse HCC models are used to evaluate the impact of S100A10 on immune function and responsiveness to anti-programmed cell death protein 1 (anti-PD-1) therapy. The results demonstrate that S100A10 is significantly upregulated in HCC and is closely associated with poor prognosis and an immunosuppressive state. Mechanistically, S100A10 activates cytosolic phospholipase A2-arachidonate 5-lipoxygenase (cPLA2-5-LOX)-mediated AA oxidative metabolism, leading to the accumulation of lipid peroxidation products and ferroptosis-associated signals, thereby driving CD8+ T-cell exhaustion and promoting immune evasion. Significantly, inhibition of S100A10 reshapes the tumor immune microenvironment (TIME) and enhances the therapeutic efficacy of anti-PD-1 treatment. Collectively, these findings identify S100A10 as a critical regulator of metabolic-immune coupling in HCC and provide a theoretical basis for combinatorial strategies targeting metabolism and immunotherapy.

Arachidonic acid metabolism↗

An Optimized Adaptation of DamID for NGS Applications.

Recent studies have implicated higher-order genome organization in the regulation of genes and cellular state. Lamina-Associated Domains (LADs) are regions of heterochromatin associated with the nuclear envelope and the nuclear lamina, a protein network involved in both nuclear organization and genome structure. LADs are developmentally regulated, and their dysregulation is associated with several diseases and pathological states, including cancer and premature aging. In addition to LADs, other nuclear protein compartments appear to scaffold or support unique chromatin environments to affect gene expression. These revelations carry profound implications for our comprehension of developmental processes and the pathogenesis of various diseases, especially given the numerous disorders already directly associated with, for example, mutations in lamin and INM proteins. This spatial compartmentalization of chromatin subtypes to unique protein compartments has led to the adoption of proximity-labeling methods, such as DamID (DNA Adenine Methyltransferase Identification), to identify these unique chromatin compartments.

Humans↗

Promoter accessibility within the environment of the MHC is affected in class II-deficient combined immunodeficiency.

Class II-deficient combined immunodeficiency (CID) is a hereditary disease resulting in abrogation of transcription of the class II genes of the major histocompatibility complex, due to a defect in a trans-acting regulatory factor. Cell lines from certain CID patients lack factor binding at multiple sites in class II promoters in vivo. A mutation in one of the promoter binding proteins could explain this 'bare' phenotype only if these factors bind cooperatively or in a temporal hierarchy. Alternatively, the mutation could affect the configuration of the promoter within the MHC locus. Here, we provide evidence that the factor(s) defective in class II-deficient CID controls the accessibility of class II promoters within the environment of the MHC. The in vivo occupancy of wild type and mutated class II promoter constructs was examined in stable transfectants of normal and CID-derived cell lines. The CID promoter phenotype could not be reproduced in a normal cell line by eliminating binding at any one promoter element, suggesting that these factors bind independently, both spatially and temporally. In contrast, promoter occupancy was partially restored in two CID lines at a randomly integrated wild type promoter, implying that the promoter is inaccessible to factors in its native environment, but accessible when moved to another location in the genome.

Base Sequence↗

Divergent evolution of an "orphon" histone gene cluster in Chironomus.

The histone genes of the midge Chironomus thummi thummi are organized in tandemly repeated gene groups, each containing the four core histone genes plus an H1 gene. These repetitive gene groups are found at five different loci, linked on one chromosomal arm. In addition to the clustered gene groups an isolated histone gene group exists which is found spatially separated on a different chromosome ("orphon" gene group). These orphon genes have been cloned and analysed in detail. Nucleotide sequence and in situ hybridization data suggest that the orphon gene group was established early during chironomid speciation, possibly by a transposition-like mechanism. This allowed the genes to be moved as an integer group. The comparison of orphon and "clustered" histone genes in C. thummi thummi indicates that the early spatial separation of the orphon genes from their tandemly organized relatives may have stimulated divergent sequence evolution. This is particularly true for the orphon H1 gene, which has diverged considerably by unusual mutation mechanisms. The translocation of normally clustered genes to new genomic sites may favour the generation of sequence variants, which could fulfill specialized functional tasks.

Animals↗

Sea urchin Forkhead gene family: phylogeny and embryonic expression.

Transcription factors of the Forkhead (Fox) family have been identified in many metazoans, and play important roles in diverse biological processes. Here we define the set of fox genes present in the sea urchin genome, and survey their usage during development. This genome includes 22 fox genes, only three of which were previously known. Of the 23 fox gene subclasses identified in vertebrate genomes, the Strongylocentrotus purpuratus genome has orthologues of all but four (E, H, R and S). Phylogenetic analysis suggests that one S. purpuratus fox gene is equally related to foxA and foxB of vertebrates; this gene defines a new class. Two other genes appear to be specific to the sea urchin, with respect to the genomes so far sequenced. Fox genes orthologous with those of vertebrates but lacking in arthropod or nematode genomes may be deuterostome-specific (subclasses I, J1, J2, L1, M and Q1), while the majority are pan-bilaterian. All but one of the S. purpuratus fox genes (SpfoxQ1) are expressed during embryogenesis, most in a very specific temporal and spatial manner. The sea urchin fox genes clearly execute many different regulatory functions, and almost all of them participate in the process of embryonic development.

Animals↗

Maternal versus paternal expression of a LacZ transgene in preimplantation mouse embryos: effects of genetic background and 2-cell block.

The expression of a transgene NI-ROSA LacZ (LacZtg) trapped into the genes for two presumably untranslated, ubiquitously expressed RNAs, was studied in preimplantation mouse embryos with respect to penetrance (fraction of expressing embryos) and to localisation of beta-galactosidase activity. With maternal origin in NMRI mice beta-galactosidase was first detected within one dot in the cytoplasm of zygotes at 30 h post-hCG. The staining pattern progressed to small clusters and to dense, homogeneous staining of the entire cytoplasm during further development. Within the NMRI background, penetrance in utero was delayed by at least 6 h when the transgene was of paternal as compared with maternal origin. Paternal transgene expression increased marginally during culture to 50 h after explantation of embryos at 30-48 h post-hCG and remained low or decreased in the '2-cell block'. Expression of a paternal transgene in preimplantation embryos developing in utero was further delayed in the maternal MF1 as compared with the NMRI background. In contrast to NMRI x NMRI embryos with paternally derived transgene, expression increased with time during the 2-cell block in MF1 x NMRI embryos. Thus, in the earliest phase of mammalian development expression of this LacZtg is influenced by parental origin, maternal genetic background and environment. The spatial distribution of the gene product is developmentally controlled.

Animals↗

The interaction between p53 and DNA topoisomerase I is regulated differently in cells with wild-type and mutant p53.

DNA topoisomerase I is a nuclear enzyme involved in transcription, recombination, and DNA damage recognition. Previous studies have shown that topoisomerase I interacts directly with the tumor-suppressor protein p53. p53 is a transcription factor that activates certain genes through binding to specific DNA sequences. We now report that topoisomerase I can be stimulated by both latent and activated wild-type p53 as well as by several mutant and truncated p53 proteins in vitro, indicating that sequence-specific DNA-binding and stimulation of topoisomerase I are distinct properties of p53. These assays also suggest that the binding site for topoisomerase I on p53 is between amino acids 302 and 321. In living cells, the interaction between p53 and topoisomerase I is strongly dependent on p53 status. In MCF-7 cells, which have wild-type p53, the association between the two proteins is tightly regulated in a spatial and temporal manner and takes place only during brief periods of genotoxic stress. In marked contrast, the two proteins are constitutively associated in HT-29 cells, which have mutant p53. These findings have important implications for both cellular stress response and genomic stability, given the ability of topoisomerase I to recognize DNA lesions as well as to cause illegitimate recombination.

Animals↗

Functional significance of the alternative transcript processing of the Arabidopsis floral promoter FCA.

The Arabidopsis gene FCA encodes an RNA binding protein that functions to promote the floral transition. The FCA transcript is alternatively processed to yield four transcripts, the most abundant of which is polyadenylated within intron 3. We have analyzed the role of the alternative processing on the floral transition. The introduction of FCA intronless transgenes resulted in increased FCA protein levels and accelerated flowering, but no role in flowering was found for products of the shorter transcripts. The consequences of the alternative processing on the FCA expression pattern were determined using a series of translational FCA-beta-glucuronidase fusions. The inclusion of FCA genomic sequence containing the alternatively processed intron 3 restricted the expression of the transgene predominantly to shoot and root apices and young flower buds. Expression of this fusion also was delayed developmentally. Therefore, the alternative processing of the FCA transcript limits, both spatially and temporally, the amount of functional FCA protein. Expression in roots prompted an analysis of root development, which indicated that FCA functions more generally than in the control of the floral transition.

Alternative Splicing↗

Multiple widely spaced elements determine the efficiency with which a distal cistron is expressed from the polycistronic pregenomic RNA of figwort mosaic caulimovirus.

The polycistronic expression mechanism of the plant pararetrovirus figwort mosaic caulimovirus (FMV) depends upon cis-acting elements present in its pregenomic RNA and a trans-acting protein (P6) which is expressed from a monocistronic subgenomic RNA. Using transient expression of FMV-derived polycistronic reporter constructs in Nicotiana edwardsonii cell suspension protoplasts, we further analyzed the cis-acting elements involved in polycistronic expression. A cis-acting element located within the first 74 nucleotides of the 7,954-nucleotide pregenomic RNA appears to be essential for P6 to transactivate expression of an internal cistron. Expression of this internal cistron, in the presence of P6, is greatly enhanced by the combined presence of two cis-acting elements located at the 3' end of the polycistronic RNA. Surprisingly, deletion of the most upstream of these two 3' cis-acting elements exposed a negative-acting element located internally on the polycistronic RNA, at the 3' end of open reading frame I. The action of both this negative-acting internal element and the positive-acting 3' elements is more pronounced when the large 5' untranslated leader region is present. This indicates that the 5' untranslated leader region is central to regulation of the FMV gene expression mechanism. Although a limited set of elements suffices to direct polycistronic expression in this eukaryotic system, a complex interplay between elements is involved in the spatial regulation of the genes present on the pregenomic RNA of FMV.

Caulimovirus↗

Multilocus sequence typing method for identification and genotypic classification of pathogenic Leptospira species.

BACKGROUND: Leptospira are the parasitic bacterial organisms associated with a broad range of mammalian hosts and are responsible for severe cases of human Leptospirosis. The epidemiology of leptospirosis is complex and dynamic. Multiple serovars have been identified, each adapted to one or more animal hosts. Adaptation is a dynamic process that changes the spatial and temporal distribution of serovars and clinical manifestations in different hosts. Serotyping based on repertoire of surface antigens is an ambiguous and artificial system of classification of leptospiral agents. Molecular typing methods for the identification of pathogenic leptospires up to individual genome species level have been highly sought after since the decipherment of whole genome sequences. Only a few resources exist for microbial genotypic data based on individual techniques such as Multiple Locus Sequence Typing (MLST), but unfortunately no such databases are existent for leptospires. RESULTS: We for the first time report development of a robust MLST method for genotyping of Leptospira. Genotyping based on DNA sequence identity of 4 housekeeping genes and 2 candidate genes was analyzed in a set of 120 strains including 41 reference strains representing different geographical areas and from different sources. Of the six selected genes, adk, icdA and secY were significantly more variable whereas the LipL32 and LipL41 coding genes and the rrs2 gene were moderately variable. The phylogenetic tree clustered the isolates according to the genome-based species. CONCLUSION: The main advantages of MLST over other typing methods for leptospires include reproducibility, robustness, consistency and portability. The genetic relatedness of the leptospires can be better studied by the MLST approach and can be used for molecular epidemiological and evolutionary studies and population genetics.

Animals↗

The role of stem cells in pituitary tumour formation.

Pituitary tumours are intracranial neoplasms that pose significant clinical challenges due to their potential for recurrence, therapeutic resistance and resultant endocrine dysfunction and mass effects. In the normal anterior pituitary, resident pituitary stem cells (PSCs) contribute to tissue homeostasis and cellular turnover. The extent to which PSCs contribute to tumourigenesis is not known, but an increasing number of studies have been aiming to address this. In this review, we summarise current evidence implicating PSCs and tumour stem-like populations in pituitary tumour biology, including potential roles in tumour initiation, maintenance and progression. We outline practical criteria for defining tumour stem cells and evaluate findings from functional studies of human tumours, emerging single-cell and spatial transcriptomic datasets and murine lineage-tracing models. We also provide a curated overview of published single-cell RNA sequencing studies of pituitary tumours, highlighting reported stem/progenitor populations and transcriptional signatures across tumour subtypes and propose a framework for future genomic analyses. Finally, we discuss the translational implications of these findings, including the potential for targeting stem-like populations and their associated signalling pathways.

Humans↗

Role of creatine kinase isoenzymes on muscular and cardiorespiratory endurance: genetic and molecular evidence.

The ability to perform well in activities that require muscular and cardiorespiratory endurance is a trait influenced, in a considerable part, by the genetic make-up of individuals. Early studies of performance and recent scans of the human genome have pointed at various candidate genes responsible for the heterogeneity of these phenotypes within the population. Among these are the genes for the various creatine kinase (CK) isoenzyme subunits. CK and phosphocreatine (PCr) form an important metabolic system for temporal and spatial energy buffering in cells with large variations in energy demand. The different CK isoenzyme subunits (CK-M and CK-B) are differentially expressed in the tissues of the body. Although CK-M is the predominant form in both skeletal and cardiac muscle, CK-B is expressed to a greater extent in heart than in skeletal muscle. Studies in humans and mice have shown that the expression of CK-B messenger RNA (mRNA) and the abundance and activity of the CK-MB dimer increase in response to cardiorespiratory endurance training. Increases in muscle tissue CK-B content can be energetically favourable because of its lower Michaelis constant (Km) for ADP. The activity of the mitochondrial isoform of CK (Scmit-CK) has also been significantly and positively correlated to oxidative capacity and to CK-MB activity in muscle. In mice where the CK-M gene has been knocked out, significant increases in fatigue resistance together with cellular adaptations increasing aerobic capacity have been observed. These observations have led to the notion that this enzyme may be responsible for fatigue under normal circumstances, most likely because of the local cell compartment increase in inorganic phosphate concentration. Studies where the Scmit-CK gene was knocked out have helped demonstrate that this isoenzyme is very important for the stimulation of aerobic respiration. Human studies of CK-M gene sequence variation have shown a significant association between a polymorphism, distinguished by the NcoI restriction enzyme, and an increase in cardiorespiratory endurance as indexed by maximal oxygen uptake following 20 weeks of training. In conclusion, there is now evidence at the tissue, cell and molecular level indicating that the CK-PCr system plays an important role in determining the phenotypes of muscular and cardiorespiratory endurance. It is envisioned that newer technologies will help determine how the genetic variability of these genes (and many others) impact on performance and health-related phenotypes.

Adaptation, Physiological↗

Structure-based active site profiles for genome analysis and functional family subclassification.

In previous work, structure-based functional site descriptors, fuzzy functional forms (FFFs), were developed to recognize structurally conserved active sites in proteins. These descriptors identify members of protein families according to active-site structural similarity, rather than overall sequence or structure similarity. FFFs are defined by a minimal number of highly conserved residues and their three-dimensional arrangement. This approach is advantageous for function assignment across broad families, but is limited when applied to detailed subclassification within these families. In the work described here, we developed a method of three-dimensional, or structure-based, active-site profiling that utilizes FFFs to identify residues located in the spatial environment around the active site. Three-dimensional active-site profiling reveals similarities and differences among active sites across protein families. Using this approach, active-site profiles were constructed from known structures for 193 functional families, and these profiles were verified as distinct and characteristic. To achieve this result, a scoring function was developed that discriminates between true functional sites and those that are geometrically most similar, but do not perform the same function. In a large-scale retrospective analysis of human genome sequences, this profile score was shown to identify specific functional families correctly. The method is effective at recognizing the likely subtype of structurally uncharacterized members of the diverse family of protein kinases, categorizing sequences correctly that were misclassified by global sequence alignment methods. Subfamily information provided by this three-dimensional active-site profiling method yields key information for specific and selective inhibitor design for use in the pharmaceutical industry.

Algorithms↗