Interleukin-10 Prevents Pathological Microglia Hyperactivation following Peripheral Endotoxin Challenge.

Microglia, the resident macrophages of the brain parenchyma, are key players in central nervous system (CNS) development, homeostasis, and disorders. Distinct brain pathologies seem associated with discrete microglia activation modules. How microglia regain quiescence following challenges remains less understood. Here, we explored the role of the interleukin-10 (IL-10) axis in restoring murine microglia homeostasis following a peripheral endotoxin challenge. Specifically, we show that lipopolysaccharide (LPS)-challenged mice harboring IL-10 receptor-deficient microglia displayed neuronal impairment and succumbed to fatal sickness. Addition of a microglial tumor necrosis factor (TNF) deficiency rescued these animals, suggesting a microglia-based circuit driving pathology. Single cell transcriptome analysis revealed various IL-10 producing immune cells in the CNS, including most prominently Ly49D+ NK cells and neutrophils, but not microglia. Collectively, we define kinetics of the microglia response to peripheral endotoxin challenge, including their activation and robust silencing, and highlight the critical role of non-microglial IL-10 in preventing deleterious microglia hyperactivation.

Thymic mimetic cells function beyond self-tolerance.

Development of immunocompetent T cells in the thymus is required for effective defence against all types of pathogens, including viruses, bacteria and fungi. To this end, T cells undergo a very strict educational program in the thymus, during which both non-functional and self-reactive T cell clones are eliminated by means of positive and negative selection1.Thymic epithelial cells (TECs) have an indispensable role in these processes, and previous studies have shown the notable heterogeneity of these cells2-7. Here, using multiomic analysis, we provide further insights into the functional and developmental diversity of TECs in mice, and reveal a detailed atlas of the TEC compartment according to cell transcriptional states and chromatin landscapes. Our analysis highlights unconventional TEC subsets that are similar to functionally well-defined parenchymal populations, including endocrine cells, microfold cells and myocytes. By focusing on the endocrine and microfold TEC populations, we show that endocrine TECs require Insm1 for their development and are crucial to maintaining thymus cellularity in a ghrelin-dependent manner; by contrast, microfold TECs require Spib for their development and are essential for the generation of thymic IgA+ plasma cells. Collectively, our study reveals that medullary TECs have the potential to differentiate into various types of molecularly distinct and functionally defined cells, which not only contribute to the induction of central tolerance, but also regulate the homeostasis of other thymus-resident populations.

Enhanced proteostasis, lipid remodeling, and nitrogen remobilization define barley flag leaf senescence.

Leaf senescence is a developmental process allowing nutrient remobilization to sink organs. We characterized flag leaf senescence at 7, 14, and 21 d past anthesis in two near-isogenic barley lines varying in the allelic state of the HvNAM1 transcription factor gene, which influences senescence timing. Metabolomics and microscopy indicated that, as senescence progressed, thylakoid lipids were transiently converted to neutral lipids accumulating in lipid droplets. Senescing leaves also exhibited an accumulation of sugars including glucose, while nitrogen compounds (nucleobases, nucleotides, and amino acids) decreased. RNA-Seq analysis suggested lipid catabolism via ß-oxidation and the glyoxylate cycle, producing carbon skeletons and feeding respiration as a replacement of the diminished carbon supply from photosynthesis. Comparison of the two barley lines highlighted a more prominent up-regulation of heat stress transcription factor- and chaperone-encoding genes in the late-senescing line, suggesting a role for these genes in the control of leaf longevity. While numerous genes with putative roles in nitrogen remobilization were up-regulated in both lines, several peptidases, nucleases, and nitrogen transporters were more highly induced in the early-senescing line; this finding identifies processes and specific candidates which may affect nitrogen remobilization from senescing barley leaves, downstream of the HvNAM1 transcription factor.

Broad phylogeny and functionality of cellulosomal components in the bovine rumen microbiome.

The cellulosome is an extracellular multi-enzyme complex that is considered one of the most efficient plant cell wall-degrading strategies devised by nature. Its unique modular architecture, achieved by high affinity and specific interaction between protein modules (cohesins and dockerins) enables formation of various enzyme combinations. Extensive research has been dedicated to the mechanistic nature of the cellulosome complex. Nevertheless, little is known regarding its distribution and abundance among microbes in natural plant fibre-rich environments. Here, we explored these questions in bovine rumen microbial communities, specialized in efficient degradation of lignocellulosic plant material. We bioinformatically screened for cellulosomal modules in this complex environment using a previously published ultra-deep fibre-adherent rumen metagenome. Intriguingly, a large portion of the functions of the dockerin-containing proteins were related to alternative biological processes, and not necessarily to the classic fibre degradation function. Our analysis was experimentally validated by characterizing specific interactions between selected cohesins and dockerins and revealed that cellulosome is a more generalized strategy used by diverse bacteria, some of which were not previously associated with cellulosome production. Remarkably, our results provide additional proof of similarity among rumen microbial communities worldwide. This study suggests a broader and widespread role for the cellulosomal machinery in nature.

Ruminococcal cellulosome systems from rumen to human.

A cellulolytic fiber-degrading bacterium, Ruminococcus champanellensis, was isolated from human faecal samples, and its genome was recently sequenced. Bioinformatic analysis of the R. champanellensis genome revealed numerous cohesin and dockerin modules, the basic elements of the cellulosome, and manual sequencing of partially sequenced genomic segments revealed two large tandem scaffoldin-coding genes that form part of a gene cluster. Representative R. champanellensis dockerins were tested against putative cohesins, and the results revealed three different cohesin-dockerin binding profiles which implied two major types of cellulosome architectures: (i) an intricate cell-bound system and (ii) a simplistic cell-free system composed of a single cohesin-containing scaffoldin. The cell-bound system can adopt various enzymatic architectures, ranging from a single enzyme to a large enzymatic complex comprising up to 11 enzymes. The variety of cellulosomal components together with adaptor proteins may infer a very tight regulation of its components. The cellulosome system of the human gut bacterium R. champanellensis closely resembles that of the bovine rumen bacterium Ruminococcus flavefaciens. The two species contain orthologous gene clusters comprising fundamental components of cellulosome architecture. Since R. champanellensis is the only human colonic bacterium known to degrade crystalline cellulose, it may thus represent a keystone species in the human gut.

Spatio-temporal coherence of circadian clocks and temporal control of differentiation in Anabaena filaments.

Circadian clock arrays in multicellular filaments of the heterocyst-forming cyanobacterium Anabaena sp. strain PCC 7120 display remarkable spatio-temporal coherence under nitrogen-replete conditions. To shed light on the interplay between circadian clocks and the formation of developmental patterns, we followed the expression of a clock-controlled gene under nitrogen deprivation, at the level of individual cells. Our experiments showed that differentiation into heterocysts took place preferentially within a limited interval of the circadian clock cycle, that gene expression in different vegetative intervals along a developed filament was discoordinated, and that the circadian clock was active in individual heterocysts. Furthermore, Anabaena mutants lacking the kaiABC genes encoding the circadian clock core components produced heterocysts but failed in diazotrophy. Therefore, genes related to some aspect of nitrogen fixation, rather than early or mid-heterocyst differentiation genes, are likely affected by the absence of the clock. A bioinformatics analysis supports the notion that RpaA may play a role as master regulator of clock outputs in Anabaena, the temporal control of differentiation by the circadian clock and the involvement of the clock in proper diazotrophic growth. Together, these results suggest that under nitrogen-deficient conditions, the clock coherent unit in Anabaena is reduced from a full filament under nitrogen-rich conditions to the vegetative cell interval between heterocysts.IMPORTANCECircadian clocks, from unicellular organisms to animals, temporally align biological processes to day and night cycles. We study the dynamics of a circadian clock-controlled gene at the individual cell level in the multicellular filamentous cyanobacterium Anabaena, under nitrogen-stress conditions. Under these conditions, some cells along filaments differentiate to carry out atmospheric nitrogen fixation and lose their capability for oxygenic photosynthesis. We found that clock synchronization is limited to organismic units of contiguous photosynthetic cells, contrary to nitrogen-replete conditions in which clocks are synchronized over a whole filament. We provided evidence that the circadian clock regulates the process of differentiation, allowing it to occur preferentially within a limited time window during the circadian clock period. Lastly, we present evidence that the signal from the core clock to clock-regulated genes is conveyed in Anabaena as in unicellular cyanobacteria.

Molecular mechanisms underlying the modulation of T-cell proliferation and cytotoxicity by immobilized CCL21 and ICAM1.

BACKGROUND: Adoptive cancer immunotherapy, using engineered T-cells, expressing chimeric antigen receptor or autologous tumor infiltrating lymphocytes became, in recent years, a major therapeutic approach for diverse types of cancer. However, despite the transformative potential of adoptive cancer immunotherapy, this field still faces major challenges, manifested by the apparent decline of the cytotoxic capacity of effector CD8+ T cells upon their expansion. To address these challenges, we have developed an ex vivo "synthetic immune niche" (SIN), composed of immobilized CCL21 and ICAM1, which synergistically induce an efficient expansion of antigen-specific CD8+ T cells while retaining, and even enhancing their cytotoxic potency. METHODS: To explore the molecular mechanisms through which a CCL21+ICAM1-based SIN modulates the interplay between the proliferation and cytotoxic potency of antigen-activated and CD3/CD28-activated effector CD8+ T cells, we performed integrated analysis of specific differentiation markers via flow cytometry, together with gene expression profiling. RESULTS: On day 3, the transcriptomic effect induced by the SIN was largely similar for both dendritic cell (DC)/ovalbumin (OVA)-activated and anti-CD3/CD28-activated cells. Cell proliferation increased and the cells exhibited high killing capacity. On day 4 and on, the proliferation/cytotoxicity phenotypes became radically "activation-specific"; The DC/OVA-activated cells lost their cytotoxic activity, which, in turn, was rescued by the SIN treatment. On longer incubation, the cytotoxic activity further declined, and on day7, could not be rescued by the SIN. SIN stimulation following activation with anti-CD3/CD28 beads induced a major increase in the proliferative phenotype while transiently suppressing their cytotoxicity for 2-3 days and fully regaining their killing activity on day 7. Potential molecular regulatory pathways of the SIN effects were identified, based on transcriptomic and multispectral imaging profiling. CONCLUSIONS: These data indicate that cell proliferation and cytotoxicity are negatively correlated, and the interplay between them is differentially regulated by the mode of initial activation. The SIN stimulation greatly enhances the cell expansion, following both activation modes, while displaying high survival and cytotoxic potency at specific time points following stimulation, suggesting that it could effectively reinforce adoptive cancer immunotherapy.

Stability and gene strand bias of lambda prophages and chromosome organization in Escherichia coli.

Temperate phage-mediated horizontal gene transfer is a potent driver of genetic diversity in the evolution of bacteria. Most lambdoid prophages in Escherichia coli are integrated into the chromosome with the same orientation with respect to the direction of chromosomal replication, and their location on the chromosome is far from homogeneous. To better understand these features, we studied the interplay between lysogenic and lytic states of phage lambda in both native and inverted integration orientations at the wild-type integration site as well as at other sites on the bacterial chromosome. Measurements of free phage released by spontaneous induction showed that the stability of lysogenic states is affected by location and orientation along the chromosome, with stronger effects near the origin of replication. Competition experiments and range expansions between lysogenic strains with opposite orientations and insertion loci indicated that there are no major differences in growth. Moreover, measurements of the level of transcriptional bursts of the cI gene coding for the lambda phage repressor using single-molecule fluorescence in situ hybridization resulted in similar levels of transcription for both orientations and prophage location. We postulate that the preference for a given orientation and location is a result of a balance between the maintenance of lysogeny and the ability to lyse.IMPORTANCEThe integration of genetic material of temperate bacterial viruses (phages) into the chromosomes of bacteria is a potent evolutionary force, allowing bacteria to acquire in one stroke new traits and restructure the information in their chromosomes. Puzzlingly, this genetic material is preferentially integrated in a particular orientation and at non-random sites on the bacterial chromosome. The work described here reveals that the interplay between the maintenance of the stability of the integrated phage, its ability to excise, and its localization along the chromosome plays a key role in setting chromosomal organization in Escherichia coli.

Competitive fungal commensalism mitigates candidiasis pathology.

The mycobiota are a critical part of the gut microbiome, but host-fungal interactions and specific functional contributions of commensal fungi to host fitness remain incompletely understood. Here, we report the identification of a new fungal commensal, Kazachstania heterogenica var. weizmannii, isolated from murine intestines. K. weizmannii exposure prevented Candida albicans colonization and significantly reduced the commensal C. albicans burden in colonized animals. Following immunosuppression of C. albicans colonized mice, competitive fungal commensalism thereby mitigated fatal candidiasis. Metagenome analysis revealed K. heterogenica or K. weizmannii presence among human commensals. Our results reveal competitive fungal commensalism within the intestinal microbiota, independent of bacteria and immune responses, that could bear potential therapeutic value for the management of C. albicans-mediated diseases.

Oriented covalent immobilization of antibodies for measurement of intermolecular binding forces between zipper-like contact surfaces of split inteins.

In order to measure the intermolecular binding forces between two halves (or partners) of naturally split protein splicing elements called inteins, a novel thiol-hydrazide linker was designed and used to orient immobilized antibodies specific for each partner. Activation of the surfaces was achieved in one step, allowing direct intermolecular force measurement of the binding of the two partners of the split intein (called protein trans-splicing). Through this binding process, a whole functional intein is formed resulting in subsequent splicing. Atomic force microscopy (AFM) was used to directly measure the split intein partner binding at 1 µm/s between native (wild-type) and mixed pairs of C- and N-terminal partners of naturally occurring split inteins from three cyanobacteria. Native and mixed pairs exhibit similar binding forces within the error of the measurement technique (~52 pN). Bioinformatic sequence analysis and computational structural analysis discovered a zipper-like contact between the two partners with electrostatic and nonpolar attraction between multiple aligned ion pairs and hydrophobic residues. Also, we tested the Jarzynski's equality and demonstrated, as expected, that nonequilibrium dissipative measurements obtained here gave larger energies of interaction as compared with those for equilibrium. Hence, AFM coupled with our immobilization strategy and computational studies provides a useful analytical tool for the direct measurement of intermolecular association of split inteins and could be extended to any interacting protein pair.

Deciphering the involvement of the Hippo pathway co-regulators, YAP/TAZ in invadopodia formation and matrix degradation.

Invadopodia are adhesive, actin-rich protrusions formed by metastatic cancer cells that degrade the extracellular matrix and facilitate invasion. They support the metastatic cascade by a spatially and temporally coordinated process whereby invading cells bind to the matrix, degrade it by specific metalloproteinases, and mechanically penetrate diverse tissue barriers by forming actin-rich extensions. However, despite the apparent involvement of invadopodia in the metastatic process, the molecular mechanisms that regulate invadopodia formation and function are still largely unclear. In this study, we have explored the involvement of the key Hippo pathway co-regulators, namely YAP, and TAZ, in invadopodia formation and matrix degradation. Toward that goal, we tested the effect of depletion of YAP, TAZ, or both on invadopodia formation and activity in multiple human cancer cell lines. We report that the knockdown of YAP and TAZ or their inhibition by verteporfin induces a significant elevation in matrix degradation and invadopodia formation in several cancer cell lines. Conversely, overexpression of these proteins strongly suppresses invadopodia formation and matrix degradation. Proteomic and transcriptomic profiling of MDA-MB-231 cells, following co-knockdown of YAP and TAZ, revealed a significant change in the levels of key invadopodia-associated proteins, including the crucial proteins Tks5 and MT1-MMP (MMP14). Collectively, our findings show that YAP and TAZ act as negative regulators of invadopodia formation in diverse cancer lines, most likely by reducing the levels of essential invadopodia components. Dissecting the molecular mechanisms of invadopodia formation in cancer invasion may eventually reveal novel targets for therapeutic applications against invasive cancer.

Molecular characterization of the intact mouse muscle spindle using a multi-omics approach.

The proprioceptive system is essential for the control of coordinated movement, posture, and skeletal integrity. The sense of proprioception is produced in the brain using peripheral sensory input from receptors such as the muscle spindle, which detects changes in the length of skeletal muscles. Despite its importance, the molecular composition of the muscle spindle is largely unknown. In this study, we generated comprehensive transcriptomic and proteomic datasets of the entire muscle spindle isolated from the murine deep masseter muscle. We then associated differentially expressed genes with the various tissues composing the spindle using bioinformatic analysis. Immunostaining verified these predictions, thus establishing new markers for the different spindle tissues. Utilizing these markers, we identified the differentiation stages the spindle capsule cells undergo during development. Together, these findings provide comprehensive molecular characterization of the intact spindle as well as new tools to study its development and function in health and disease.

Kynurenic acid, a key L-tryptophan-derived metabolite, protects the heart from an ischemic damage.

BACKGROUND: Renal injury induces major changes in plasma and cardiac metabolites. Using a small- animal in vivo model, we sought to identify a key metabolite whose levels are significantly modified following an acute kidney injury (AKI) and to analyze whether this agent could offer cardiac protection once an ischemic event has occurred. METHODS AND RESULTS: Metabolomics profiling of cardiac lysates and plasma samples derived from rats that underwent AKI 1 or 7 days earlier by 5/6 nephrectomy versus sham-operated controls was performed. We detected 26 differential metabolites in both heart and plasma samples at the two selected time points, relative to sham. Out of which, kynurenic acid (kynurenate, KYNA) seemed most relevant. Interestingly, KYNA given at 10 mM concentration significantly rescued the viability of H9C2 cardiac myoblast cells grown under anoxic conditions and largely increased their mitochondrial content and activity as determined by flow cytometry and cell staining with MitoTracker dyes. Moreover, KYNA diluted in the drinking water of animals induced with an acute myocardial infarction, highly enhanced their cardiac recovery according to echocardiography and histopathology. CONCLUSION: KYNA may represent a key metabolite absorbed by the heart following AKI as part of a compensatory mechanism aiming at preserving the cardiac function. KYNA preserves the in vitro myocyte viability following exposure to anoxia in a mechanism that is mediated, at least in part, by protection of the cardiac mitochondria. A short-term administration of KYNA may be highly beneficial in the treatment of the acute phase of kidney disease in order to attenuate progression to reno-cardiac syndrom and to reduce the ischemic myocardial damage following an ischemic event.

Bacterial lifestyle switch in response to algal metabolites.

Unicellular algae, termed phytoplankton, greatly impact the marine environment by serving as the basis of marine food webs and by playing central roles in the biogeochemical cycling of elements. The interactions between phytoplankton and heterotrophic bacteria affect the fitness of both partners. It is becoming increasingly recognized that metabolic exchange determines the nature of such interactions, but the underlying molecular mechanisms remain underexplored. Here, we investigated the molecular and metabolic basis for the bacterial lifestyle switch, from coexistence to pathogenicity, in Sulfitobacter D7 during its interaction with Emiliania huxleyi, a cosmopolitan bloom-forming phytoplankter. To unravel the bacterial lifestyle switch, we analyzed bacterial transcriptomes in response to exudates derived from algae in exponential growth and stationary phase, which supported the Sulfitobacter D7 coexistence and pathogenicity lifestyles, respectively. In pathogenic mode, Sulfitobacter D7 upregulated flagellar motility and diverse transport systems, presumably to maximize assimilation of E. huxleyi-derived metabolites released by algal cells upon cell death. Algal dimethylsulfoniopropionate (DMSP) was a pivotal signaling molecule that mediated the transition between the lifestyles, supporting our previous findings. However, the coexisting and pathogenic lifestyles were evident only in the presence of additional algal metabolites. Specifically, we discovered that algae-produced benzoate promoted the growth of Sulfitobacter D7 and hindered the DMSP-induced lifestyle switch to pathogenicity, demonstrating that benzoate is important for maintaining the coexistence of algae and bacteria. We propose that bacteria can sense the physiological state of the algal host through changes in the metabolic composition, which will determine the bacterial lifestyle during interaction.

Genome-wide analysis of acetivibrio cellulolyticus provides a blueprint of an elaborate cellulosome system.

BACKGROUND: Microbial degradation of plant cell walls and its conversion to sugars and other byproducts is a key step in the carbon cycle on Earth. In order to process heterogeneous plant-derived biomass, specialized anaerobic bacteria use an elaborate multi-enzyme cellulosome complex to synergistically deconstruct cellulosic substrates. The cellulosome was first discovered in the cellulolytic thermophile, Clostridium thermocellum, and much of our knowledge of this intriguing type of protein composite is based on the cellulosome of this environmentally and biotechnologically important bacterium. The recently sequenced genome of the cellulolytic mesophile, Acetivibrio cellulolyticus, allows detailed comparison of the cellulosomes of these two select cellulosome-producing bacteria. RESULTS: Comprehensive analysis of the A. cellulolyticus draft genome sequence revealed a very sophisticated cellulosome system. Compared to C. thermocellum, the cellulosomal architecture of A. cellulolyticus is much more extensive, whereby the genome encodes for twice the number of cohesin- and dockerin-containing proteins. The A. cellulolyticus genome has thus evolved an inflated number of 143 dockerin-containing genes, coding for multimodular proteins with distinctive catalytic and carbohydrate-binding modules that play critical roles in biomass degradation. Additionally, 41 putative cohesin modules distributed in 16 different scaffoldin proteins were identified in the genome, representing a broader diversity and modularity than those of Clostridium thermocellum. Although many of the A. cellulolyticus scaffoldins appear in unconventional modular combinations, elements of the basic structural scaffoldins are maintained in both species. In addition, both species exhibit similarly elaborate cell-anchoring and cellulosome-related gene- regulatory elements. CONCLUSIONS: This work portrays a particularly intricate, cell-surface cellulosome system in A. cellulolyticus and provides a blueprint for examining the specific roles of the various cellulosomal components in the degradation of complex carbohydrate substrates of the plant cell wall by the bacterium.

Exploring differential exon usage via short- and long-read RNA sequencing strategies.

Alternative splicing produces various mRNAs, and thereby various protein products, from one gene, impacting a wide range of cellular activities. However, accurate reconstruction and quantification of full-length transcripts using short-reads is limited, due to their length. Long-reads sequencing technologies may provide a solution by sequencing full-length transcripts. We explored the use of both Illumina short-reads and two long Oxford Nanopore Technology (cDNA and Direct RNA) RNA-Seq reads for detecting global differential splicing during mouse embryonic stem cell differentiation, applying several bioinformatics strategies: gene-based, isoform-based and exon-based. We detected the strongest similarity among the sequencing platforms at the gene level compared to exon-based and isoform-based. Furthermore, the exon-based strategy discovered many differential exon usage (DEU) events, mostly in a platform-dependent manner and in non-differentially expressed genes. Thus, the platforms complemented each other in the ability to detect DEUs (i.e. long-reads exhibited an advantage in detecting DEUs at the UTRs, and short-reads detected more DEUs). Exons within 20 genes, detected in one or more platforms, were here validated by PCR, including key differentiation genes, such as Mdb3 and Aplp1. We provide an important analysis resource for discovering transcriptome changes during stem cell differentiation and insights for analysing such data.

H3-K27M-mutant nucleosomes interact with MLL1 to shape the glioma epigenetic landscape.

Cancer-associated mutations in genes encoding histones dramatically reshape chromatin and support tumorigenesis. Lysine to methionine substitution of residue 27 on histone H3 (K27M) is a driver mutation in high-grade pediatric gliomas, known to abrogate polycomb repressive complex 2 (PRC2) activity. We applied single-molecule systems to image individual nucleosomes and delineate the combinatorial epigenetic patterns associated with H3-K27M expression. We found that chromatin marks on H3-K27M-mutant nucleosomes are dictated both by their incorporation preferences and by intrinsic properties of the mutation. Mutant nucleosomes not only preferentially bind PRC2 but also directly interact with MLL1, leading to genome-wide redistribution of H3K4me3. H3-K27M-mediated deregulation of repressive and active chromatin marks leads to unbalanced "bivalent" chromatin, which may support a poorly differentiated cellular state. This study provides evidence for a direct effect of H3-K27M oncohistone on the MLL1-H3K4me3 pathway and highlights the capability of single-molecule tools to reveal mechanisms of chromatin deregulation in cancer.

Transcriptional regulation of the thymus master regulator Foxn1.

FOXN1 is a transcription factor critical for the development of both thymic epithelial cell (TEC) and hair follicle cell (HFC) compartments. However, mechanisms controlling its expression remain poorly understood. To address this question, we performed thorough analyses of the evolutionary conservation and chromatin status of the Foxn1 locus in different tissues and states and identified several putative cis-regulatory regions unique to TECs versus HFCs. Furthermore, experiments using genetically modified mice with specific deletions in the Foxn1 locus and additional bioinformatic analyses helped us identify key regions and transcription factors involved in either positive or negative regulation of Foxn1 in both TECs and HFCs. Specifically, we identified SIX1 and FOXN1 itself as key factors inducing Foxn1 expression in embryonic and neonatal TECs. Together, our data provide important mechanistic insights into the transcriptional regulation of the Foxn1 gene in TEC versus HFC and highlight the role of FOXN1 in its autoregulation.

Splicing of the mycobacteriophage Bethlehem DnaB intein: identification of a new mechanistic class of inteins that contain an obligate block F nucleophile.

Inteins are single turnover enzymes that splice out of protein precursors during maturation of the host protein (extein). The Cys or Ser at the N terminus of most inteins initiates a four-step protein splicing reaction by forming a (thio)ester bond at the N-terminal splice junction. Several recently identified inteins cannot perform this acyl rearrangement because they do not begin with Cys, Thr, or Ser. This study analyzes one of these, the mycobacteriophage Bethlehem DnaB intein, which we describe here as the prototype for a new class of inteins based on sequence comparisons, reactivity, and mechanism. These Class 3 inteins are characterized by a non-nucleophilic N-terminal residue that co-varies with a non-contiguous Trp, Cys, Thr triplet (WCT) and a Thr or Ser as the first C-extein residue. Several mechanistic differences were observed when compared with standard inteins or previously studied atypical KlbA Ala(1) inteins: (a) cleavage at the N-terminal splice junction in the absence of all standard N- and C-terminal splice junction nucleophiles, (b) activation of the N-terminal splice junction by a variant Block B motif that includes the WCT triplet Trp, (c) decay of the branched intermediate by thiols or Cys despite an ester linkage at the C-extein branch point, and (d) an absolute requirement for the WCT triplet Block F Cys. Based on biochemical data and confirmed by molecular modeling, we propose roles for these newly identified conserved residues, a novel protein splicing mechanism that includes a second branched intermediate, and an intein classification with three mechanistic categories.