Eukaryotic genomes are folded into loops. It is thought that these are formed by cohesin complexes extrusion, either until loop expansion is arrested by CTCF or until cohesin is removed from DNA by WAPL. Although WAPL limits cohesin's chromatin residence time to minutes, it has been reported that some loops exist for hours. How these loops can persist is unknown. We show that during G1-phase, mammalian cells contain acetylated cohesin which binds chromatin for hours, whereas cohesin binds chromatin for minutes. Our results indicate that CTCF and the acetyltransferase ESCO1 protect a subset of cohesin complexes from WAPL, thereby enable formation of long and presumably long-lived loops, and that ESCO1, like CTCF, contributes to boundary formation in chromatin looping. Our data are consistent with a model of nested loop extrusion, in which acetylated cohesin forms stable loops between CTCF sites, demarcating the boundaries of more transient cohesin extrusion activity.

Calmodulin sits at the center of molecular mechanisms underlying learning and memory. Its complex and sometimes opposite influences, mediated via the binding to various proteins, are yet to be fully understood. Calcium/calmodulin-dependent protein kinase II (CaMKII) and calcineurin (CaN) both bind open calmodulin, favoring Long-Term Potentiation (LTP) or Depression (LTD) respectively. Neurogranin binds to the closed conformation of calmodulin and its impact on synaptic plasticity is less clear. We set up a mechanistic computational model based on allosteric principles to simulate calmodulin state transitions and its interactions with calcium ions and the three binding partners mentioned above. We simulated calcium spikes at various frequencies and show that neurogranin regulates synaptic plasticity along three modalities. At low spike frequencies, neurogranin inhibits the onset of LTD by limiting CaN activation. At intermediate frequencies, neurogranin facilitates LTD, but limits LTP by precluding binding of CaMKII with calmodulin. Finally, at high spike frequencies, neurogranin promotes LTP by enhancing CaMKII autophosphorylation. While neurogranin might act as a calmodulin buffer, it does not significantly preclude the calmodulin opening by calcium. On the contrary, neurogranin synchronizes the opening of calmodulin's two lobes and promotes their activation at specific frequencies. Neurogranin suppresses basal CaN activity, thus increasing the chance of CaMKII trans-autophosphorylation at high-frequency calcium spikes. Taken together, our study reveals dynamic regulatory roles played by neurogranin on synaptic plasticity, which provide mechanistic explanations for opposing experimental findings.

Antibodies secreted within the intestinal tract provide protection from the invasion of microbes into the host tissues. Germinal center (GC) formation in lymph nodes and spleen strictly requires SLAM-associated protein (SAP)-mediated T cell functions; however, it is not known whether this mechanism plays a similar role in mucosal-associated lymphoid tissues. Here, we find that in Peyer's patches (PPs), SAP-mediated T cell help is required for promoting B cell selection in GCs, but not for clonal diversification. PPs of SAP-deficient mice host chronic GCs that are absent in T cell-deficient mice. GC B cells in SAP-deficient mice express AID and Bcl6 and generate plasma cells in proportion to the GC size. Single-cell IgA sequencing analysis reveals that these mice host few diversified clones that were subjected to mild selection forces. These findings demonstrate that T cell-derived help to B cells in PPs includes SAP-dependent and SAP-independent functions.

Amyloid beta peptides (Aβ) proteins play a key role in vascular pathology in Alzheimer's Disease (AD) including impairment of the blood-brain barrier and aberrant angiogenesis. Although previous work has demonstrated a pro-angiogenic role of Aβ, the exact mechanisms by which amyloid precursor protein (APP) processing and endothelial angiogenic signalling cascades interact in AD remain a largely unsolved problem. Here, we report that increased endothelial sprouting in human-APP transgenic mouse (TgCRND8) tissue is dependent on β-secretase (BACE1) processing of APP. Higher levels of Aβ processing in TgCRND8 tissue coincides with decreased NOTCH3/JAG1 signalling, overproduction of endothelial filopodia and increased numbers of vascular pericytes. Using a novel in vitro approach to study sprouting angiogenesis in TgCRND8 organotypic brain slice cultures (OBSCs), we find that BACE1 inhibition normalises excessive endothelial filopodia formation and restores NOTCH3 signalling. These data present the first evidence for the potential of BACE1 inhibition as an effective therapeutic target for aberrant angiogenesis in AD.

Children from low- and middle-income countries, where there is a high incidence of infectious disease, have the greatest need for the protection afforded by vaccination, but vaccines often show reduced efficacy in these populations. An improved understanding of how age, infection, nutrition, and genetics influence immune ontogeny and function is key to informing vaccine design for this at-risk population. We sought to identify factors that shape immune development in children under 5 years of age from Tanzania and Mozambique by detailed immunophenotyping of longitudinal blood samples collected during the RTS,S malaria vaccine phase 3 trial. In these cohorts, the composition of the immune system is dynamically transformed during the first years of life, and this was further influenced by geographical location, with some immune cell types showing an altered rate of development in Tanzanian children compared to Dutch children enrolled in the Generation R population-based cohort study. High-titer antibody responses to the RTS,S/AS01E vaccine were associated with an activated immune profile at the time of vaccination, including an increased frequency of antibody-secreting plasmablasts and follicular helper T cells. Anemic children had lower frequencies of recent thymic emigrant T cells, isotype-switched memory B cells, and plasmablasts; modulating iron bioavailability in vitro could recapitulate the B cell defects observed in anemic children. Our findings demonstrate that the composition of the immune system in children varies according to age, geographical location, and anemia status.

Effective T cell responses against infections and tumors require a swift and ample production of cytokines, chemokines, and cytotoxic molecules. The production of these effector molecules relies on rapid changes of gene expression, determined by cell-intrinsic signals and environmental cues. Here, we review our current understanding of gene-specific regulatory networks that define the magnitude and timing of cytokine production in CD8 T cells. We discuss the dynamic features of post-transcriptional control during CD8 T cell homeostasis and activation, and focus on the crosstalk between cell signaling and RNA-binding proteins. Elucidating gene-specific regulatory circuits may help in the future to rectify dysfunctional T cell responses.

In , the cold-induced epigenetic regulation of () involves distinct phases of Polycomb repressive complex 2 (PRC2) silencing. During cold, a PHD-PRC2 complex metastably and digitally nucleates H3K27me3 within On return to warm, PHD-PRC2 spreads across the locus delivering H3K27me3 to maintain long-term silencing. Here, we studied natural variation in this process in accessions, exploring Lov-1, which shows reactivation on return to warm, a feature characteristic of in perennial This analysis identifies an additional phase in this Polycomb silencing mechanism downstream from H3K27me3 spreading. In this long-term silencing (perpetuated) phase, the PHD proteins are lost from the nucleation region and silencing is likely maintained by the read-write feedbacks associated with H3K27me3. A combination of noncoding SNPs in the nucleation region mediates instability in this long-term silencing phase with the result that Lov-1 frequently digitally reactivates in individual cells, with a probability that diminishes with increasing cold duration. We propose that this decrease in reactivation probability is due to reduced DNA replication after flowering. Overall, this work defines an additional phase in the Polycomb mechanism instrumental in natural variation of silencing, and provides avenues to dissect broader evolutionary changes at .

We have previously developed and described a method for measuring RNA co-locations within cells, called Proximity RNA-seq, which promises insights into RNA expression, processing, storage and translation. Here, we describe transcriptome-wide proximity RNA-seq datasets obtained from human neuroblastoma SH-SY5Y cell nuclei. To aid future users of this method, we also describe and release our analysis pipeline, CloseCall, which maps cDNA to a custom transcript annotation and allocates cDNA-linked barcodes to barcode groups. CloseCall then performs Monte Carlo simulations on the data to identify pairs of transcripts, which are co-barcoded more frequently than expected by chance. Furthermore, derived co-barcoding frequencies for individual transcripts, dubbed valency, serve as proxies for RNA density or connectivity for that given transcript. We outline how this pipeline was applied to these sequencing datasets and openly share the processed data outputs and access to a virtual machine that runs CloseCall. The resulting data specify the spatial organization of RNAs and builds hypotheses for potential regulatory relationships between RNAs.

The microbiota in the human gut are an important component of normal physiology that has co-evolved from the earliest multicellular organisms. Therefore, it is unsurprising that there is intimate crosstalk between the microbial world in the gut and the host. Genome regulation through microbiota-host interactions not only affects the host's immunity, but also metabolic health and resilience against cancer. Chromatin dynamics of the host epithelium involving histone modifications and other facets of the epigenetic machinery play an important role in this process.

None available

After publication of the original article [1], we were notified that the software used to create the figures has exported them wrong so they display incomplete

The germinal center (GC) response is critical for generating high-affinity humoral immunity and immunological memory, which forms the basis of successful immunization. Control of the GC response is thought to require follicular regulatory T (Tfr) cells, a subset of suppressive Foxp3 regulatory T cells located within GCs. Relatively little is known about the exact role of Tfr cells within the GC and how they exert their suppressive function. A unique feature of Tfr cells is their reported CXCR5-dependent localization to the GC. Here, we show that the lack of CXCR5 on Foxp3 regulatory T cells results in a reduced frequency, but not an absence, of GC-localized Tfr cells. This reduction in Tfr cells is not sufficient to alter the magnitude or output of the GC response. This demonstrates that additional, CXCR5-independent mechanisms facilitate Treg cell homing to the GC.

Interpretation of metabolomics data in the context of biological pathways is important to gain knowledge about underlying metabolic processes. In this chapter we present methods to analyze genome-scale models (GSMs) and metabolomics data together. This includes reading and mining of GSMs using the SBTab format to retrieve information on genes, reactions, and metabolites. Furthermore, the chapter showcases the generation of metabolic pathway maps using the Escher tool, which can be used for data visualization. Lastly, approaches to constrain flux balance analysis (FBA) by metabolomics data are presented.

Autophagy is an important cellular degradation pathway with a central role in metabolism as well as basic quality control, two processes inextricably linked to ageing. A decrease in autophagy is associated with increasing age, yet it is unknown if this is causal in the ageing process, and whether autophagy restoration can counteract these ageing effects. Here we demonstrate that systemic autophagy inhibition induces the premature acquisition of age-associated phenotypes and pathologies in mammals. Remarkably, autophagy restoration provides a near complete recovery of morbidity and a significant extension of lifespan; however, at the molecular level this rescue appears incomplete. Importantly autophagy-restored mice still succumb earlier due to an increase in spontaneous tumour formation. Thus, our data suggest that chronic autophagy inhibition confers an irreversible increase in cancer risk and uncovers a biphasic role of autophagy in cancer development being both tumour suppressive and oncogenic, sequentially.

Recent studies have suggested that fatty acid oxidation (FAO) is a key metabolic pathway for the growth of triple negative breast cancers (TNBCs), particularly those that have high expression of MYC. However, the underlying mechanism by which MYC promotes FAO remains poorly understood.

Mitochondrial DNA (mtDNA) is widely used in several fields including medical genetics, forensic science, genetic genealogy, and evolutionary anthropology. In this study, mtDNA haplotype diversity was determined for 293 unrelated subjects from Jordanian population (Circassians, Chechens, and the original inhabitants of Jordan). A total of 102 haplotypes were identified and analyzed among the populations to describe the maternal lineage landscape. Our results revealed that the distribution of mtDNA haplotype frequencies among the three populations showed disparity and significant differences when compared to each other. We also constructed mitochondrial haplotype classification trees for the three populations to determine the phylogenetic relationship of mtDNA haplotype variants, and we observed clear differences in the distribution of maternal genetic ancestries, especially between Arab and the minority ethnic populations. To our knowledge, this study is the first, to date, to characterize mitochondrial haplotypes and haplotype distributions in a population-based sample from the Jordanian population. It provides a powerful reference for future studies investigating the contribution of mtDNA variation to human health and disease and studying population history and evolution by comparing the mtDNA haplotypes to other populations.

In vitro follicle culture (IFC), as applied in the mouse system, allows the growth and maturation of a large number of immature preantral follicles to become mature and competent oocytes. In the human oncofertility clinic, there is increasing interest in developing this technique as an alternative to ovarian cortical tissue transplantation and to preserve the fertility of prepubertal cancer patients. However, the effect of IFC and hormonal stimulation on DNA methylation in the oocyte is not fully known, and there is legitimate concern over epigenetic abnormalities that could be induced by procedures applied during assisted reproductive technology (ART).

Maternal effect mutations in the components of the subcortical maternal complex (SCMC) of the human oocyte can cause early embryonic failure, gestational abnormalities and recurrent pregnancy loss. Enigmatically, they are also associated with DNA methylation abnormalities at imprinted genes in conceptuses: in the devastating gestational abnormality biparental complete hydatidiform mole (BiCHM) or in multi-locus imprinting disease (MLID). However, the developmental timing, genomic extent and mechanistic basis of these imprinting defects are unknown. The rarity of these disorders and the possibility that methylation defects originate in oocytes have made these questions very challenging to address.

Disruption of axonal transport causes a number of rare, inherited axonopathies and is heavily implicated in a wide range of more common neurodegenerative disorders, many of them age-related. Acetylation of α-tubulin is one important regulatory mechanism, influencing microtubule stability and motor protein attachment. Of several strategies so far used to enhance axonal transport, increasing microtubule acetylation through inhibition of the deacetylase enzyme histone deacetylase 6 (HDAC6) has been one of the most effective. Several inhibitors have been developed and tested in animal and cellular models, but better drug candidates are still needed. Here we report the development and characterization of two highly potent HDAC6 inhibitors, which show low toxicity, promising pharmacokinetic properties, and enhance microtubule acetylation in the nanomolar range. We demonstrate their capacity to rescue axonal transport of mitochondria in a primary neuronal culture model of the inherited axonopathy Charcot-Marie-Tooth Type 2F, caused by a dominantly acting mutation in heat shock protein beta 1.

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Despite being discovered over 60 years ago, the precise role of Phospholipase D (PLD) is still being elucidated. PLD enzymes catalyze the hydrolysis of the phosphodiester bond of glycerophospholipids producing phosphatidic acid and the free headgroup. PLD family members are found in organisms ranging from viruses, and bacteria to plants, and mammals, and they display a range of substrate specificities, are regulated by a diverse range of molecules, and have been implicated in a broad range of cellular processes including receptor signaling, cytoskeletal regulation and membrane trafficking. Recent technological advances including: the development of PLD knockout mice, isoform-specific antibodies, and specific inhibitors are finally permitting a thorough analysis of the in vivo role of mammalian PLDs. These studies are facilitating increased recognition of PLD's role in disease states including cancers and Alzheimer's disease, offering potential as a target for therapeutic intervention.

Formation of the three primary germ layers during gastrulation is an essential step in the establishment of the vertebrate body plan and is associated with major transcriptional changes. Global epigenetic reprogramming accompanies these changes, but the role of the epigenome in regulating early cell-fate choice remains unresolved, and the coordination between different molecular layers is unclear. Here we describe a single-cell multi-omics map of chromatin accessibility, DNA methylation and RNA expression during the onset of gastrulation in mouse embryos. The initial exit from pluripotency coincides with the establishment of a global repressive epigenetic landscape, followed by the emergence of lineage-specific epigenetic patterns during gastrulation. Notably, cells committed to mesoderm and endoderm undergo widespread coordinated epigenetic rearrangements at enhancer marks, driven by ten-eleven translocation (TET)-mediated demethylation and a concomitant increase of accessibility. By contrast, the methylation and accessibility landscape of ectodermal cells is already established in the early epiblast. Hence, regulatory elements associated with each germ layer are either epigenetically primed or remodelled before cell-fate decisions, providing the molecular framework for a hierarchical emergence of the primary germ layers.

Patients with inflammatory bowel disease (IBD) often present poor bone health and are 40% more at risk of bone fracture. Studies have implicated autophagy in IBD pathology and drugs used to treat IBD stimulate autophagy in varying degrees, however, their effect on the skeleton is currently unknown. Here, we have utilised the dextran sulphate sodium (DSS) model of colitis in mice to examine the effects of the thiopurine drug azathioprine on the skeleton. Ten-week-old male mice ( = 6/group) received 3.0% DSS in their drinking water for four days, followed by a 14-day recovery period. Mice were treated with 10 mg/kg/day azathioprine or vehicle control. Histopathological analysis of the colon from DSS mice revealed significant increases in scores for inflammation severity, extent, and crypt damage ( < 0.05). Azathioprine provided partial protection to the colon, as reflected by a lack of significant difference in crypt damage and tissue regeneration with DSS treatment. MicroCT of vehicle-treated DSS mice revealed azathioprine treatment had a significant detrimental effect on the trabecular bone microarchitecture, independent of DSS treatment. Specifically, significant decreases were observed in bone volume/tissue volume ( < 0.01), and trabecular number ( < 0.05), with a concurrent significant increase in trabecular pattern factor ( < 0.01). Immunohistochemical labelling for LC3 revealed azathioprine to induce autophagy in the bone marrow. Together these data suggest that azathioprine treatment may have a deleterious effect on IBD patients who may already be at increased risk of osteoporotic bone fractures and thus will inform on future treatment strategies for patient stratification.

Extrachromosomal circular DNA (eccDNA) facilitates adaptive evolution by allowing rapid and extensive gene copy number variation and is implicated in the pathology of cancer and ageing. Here, we demonstrate that yeast aged under environmental copper accumulate high levels of eccDNA containing the copper-resistance gene CUP1. Transcription of the tandemly repeated CUP1 gene causes CUP1 eccDNA accumulation, which occurs in the absence of phenotypic selection. We have developed a sensitive and quantitative eccDNA sequencing pipeline that reveals CUP1 eccDNA accumulation on copper exposure to be exquisitely site specific, with no other detectable changes across the eccDNA complement. eccDNA forms de novo from the CUP1 locus through processing of DNA double-strand breaks (DSBs) by Sae2, Mre11 and Mus81, and genome-wide analyses show that other protein coding eccDNA species in aged yeast share a similar biogenesis pathway. Although abundant, we find that CUP1 eccDNA does not replicate efficiently, and high-copy numbers in aged cells arise through frequent formation events combined with asymmetric DNA segregation. The transcriptional stimulation of CUP1 eccDNA formation shows that age-linked genetic change varies with transcription pattern, resulting in gene copy number profiles tailored by environment.