Ageing is a major risk factor for development of metabolic diseases such as type 2 diabetes. Identification of the mechanisms underlying this association could help to elucidate the relationship between age-associated progressive loss of metabolic health and development of type 2 diabetes. We aimed to determine molecular signatures during ageing in the endocrine pancreas.

The microRNA-29 (miR-29) family is among the most abundantly expressed microRNA in the pancreas and liver. Here, we investigated the function of miR-29 in glucose regulation using miR-29a/b-1 (miR-29a)-deficient mice and newly generated miR-29b-2/c (miR-29c)-deficient mice. We observed multiple independent functions of the miR-29 family, which can be segregated into a hierarchical physiologic regulation of glucose handling. miR-29a, and not miR-29c, was observed to be a positive regulator of insulin secretion in vivo, with dysregulation of the exocytotic machinery sensitizing β-cells to overt diabetes after unfolded protein stress. By contrast, in the liver both miR-29a and miR-29c were important negative regulators of insulin signaling via phosphatidylinositol 3-kinase regulation. Global or hepatic insufficiency of miR-29 potently inhibited obesity and prevented the onset of diet-induced insulin resistance. These results demonstrate strong regulatory functions for the miR-29 family in obesity and diabetes, culminating in a hierarchical and dose-dependent effect on premature lethality.

Regenerative medicine, ranging from stem cell therapy to organ regeneration, is promising to revolutionize treatments of diseases and aging. These approaches require a perfect understanding of cell reprogramming and differentiation. Predictive modeling of cellular systems has the potential to provide insights about the dynamics of cellular processes, and guide their control. Moreover in many cases, it provides alternative to experimental tests, difficult to perform for practical or ethical reasons. The variety and accuracy of biological processes represented in mathematical models grew in-line with the discovery of underlying molecular mechanisms. High-throughput data generation led to the development of models based on data analysis, as an alternative to more established modeling based on prior mechanistic knowledge. In this chapter, we give an overview of existing mathematical models of pluripotency and cell fate, to illustrate the variety of methods and questions. We conclude that current approaches are yet to overcome a number of limitations: Most of the computational models have so far focused solely on understanding the regulation of pluripotency, and the differentiation of selected cell lineages. In addition, models generally interrogate only a few biological processes. However, a better understanding of the reprogramming process leading to ESCs and iPSCs is required to improve stem-cell therapies. One also needs to understand the links between signaling, metabolism, regulation of gene expression, and the epigenetics machinery.

Long-term survival and antitumor immunity of adoptively transferred CD8(+) T cells is dependent on their metabolic fitness, but approaches to isolate therapeutic T cells based on metabolic features are not well established. Here we utilized a lipophilic cationic dye tetramethylrhodamine methyl ester (TMRM) to identify and isolate metabolically robust T cells based on their mitochondrial membrane potential (ΔΨm). Comprehensive metabolomic and gene expression profiling demonstrated global features of improved metabolic fitness in low-ΔΨm-sorted CD8(+) T cells. Transfer of these low-ΔΨm T cells was associated with superior long-term in vivo persistence and an enhanced capacity to eradicate established tumors compared with high-ΔΨm cells. Use of ΔΨm-based sorting to enrich for cells with superior metabolic features was observed in CD8(+), CD4(+) T cell subsets, and long-term hematopoietic stem cells. This metabolism-based approach to cell selection may be broadly applicable to therapies involving the transfer of HSC or lymphocytes for the treatment of viral-associated illnesses and cancer.

The production of high-affinity antibodies by B cells is essential for pathogen clearance. Antibody affinity for antigen is increased through the affinity maturation in germinal centers (GCs). This is an iterative process in which B cells cycle between proliferation coupled with the acquisition of mutations and antigen-based positive selection, resulting in retention of the highest-affinity B cell clones. The posttranscriptional regulator microRNA-155 (miR-155) is critical for efficient affinity maturation and the maintenance of the GCs; however, the cellular and molecular mechanism by which miR-155 regulates GC responses is not well understood. Here, we utilized a miR-155 reporter mouse strain and showed that miR-155 is coexpressed with the proto-oncogene encoding c-MYC in positively selected B cells. Functionally, miR-155 protected positively selected c-MYC+ B cells from apoptosis, allowing clonal expansion of this population, providing an explanation as to why Mir155 deletion impairs affinity maturation and promotes the premature collapse of GCs. We determined that miR-155 directly inhibits the Jumonji family member JARID2, which enhances B cell apoptosis when overexpressed, and thereby promotes GC B cell survival. Our findings also suggest that there is cooperation between c-MYC and miR-155 during the normal GC response, a cooperation that may explain how c-MYC and miR-155 can collaboratively function as oncogenes.

Adoptive cell transfer (ACT) of purified naive, stem cell memory, and central memory T cell subsets results in superior persistence and antitumor immunity compared with ACT of populations containing more-differentiated effector memory and effector T cells. Despite a clear advantage of the less-differentiated populations, the majority of ACT trials utilize unfractionated T cell subsets. Here, we have challenged the notion that the mere presence of less-differentiated T cells in starting populations used to generate therapeutic T cells is sufficient to convey their desirable attributes. Using both mouse and human cells, we identified a T cell-T cell interaction whereby antigen-experienced subsets directly promote the phenotypic, functional, and metabolic differentiation of naive T cells. This process led to the loss of less-differentiated T cell subsets and resulted in impaired cellular persistence and tumor regression in mouse models following ACT. The T memory-induced conversion of naive T cells was mediated by a nonapoptotic Fas signal, resulting in Akt-driven cellular differentiation. Thus, induction of Fas signaling enhanced T cell differentiation and impaired antitumor immunity, while Fas signaling blockade preserved the antitumor efficacy of naive cells within mixed populations. These findings reveal that T cell subsets can synchronize their differentiation state in a process similar to quorum sensing in unicellular organisms and suggest that disruption of this quorum-like behavior among T cells has potential to enhance T cell-based immunotherapies.

In contrast to the class I phosphoinositide 3-kinases (PI3Ks), the organismal roles of the kinase activity of the class II PI3Ks are less clear. Here, we report that class II PI3K-C2β kinase-dead mice are viable and healthy but display an unanticipated enhanced insulin sensitivity and glucose tolerance, as well as protection against high-fat-diet-induced liver steatosis. Despite having a broad tissue distribution, systemic PI3K-C2β inhibition selectively enhances insulin signaling only in metabolic tissues. In a primary hepatocyte model, basal PI3P lipid levels are reduced by 60% upon PI3K-C2β inhibition. This results in an expansion of the very early APPL1-positive endosomal compartment and altered insulin receptor trafficking, correlating with an amplification of insulin-induced, class I PI3K-dependent Akt signaling, without impacting MAPK activity. These data reveal PI3K-C2β as a critical regulator of endosomal trafficking, specifically in insulin signaling, and identify PI3K-C2β as a potential drug target for insulin sensitization.

Spatial computer simulations are becoming more feasible and relevant for studies of signaling pathways due to technical advances in experimental techniques yielding better high resolution data. However, many common single particle simulation environments used in computational systems biology lack the functionality to easily implement spatially heterogeneous membrane environments.

Phosphoinositides in primary mammalian tissue are highly enriched in a stearoyl/arachidonyl (C38:4) diacylgycerol backbone. However, mammalian cells grown in culture typically contain more diverse molecular species of phosphoinositides, characterised by a reduction in arachidonyl content in the sn-2 position. We have analysed the phosphoinositide species in MCF10a cells grown in culture by mass spectrometry. Under either serum or serum starved conditions the most abundant species of PI, PIP, PIP2 and PIP3 had masses which corresponded to C36:2, C38:4, C38:3, C38:2 and C36:1 diacylglycerol backbones and the relative proportions of each molecular species were broadly similar between each phosphoinositide class (approx. 50%, 25%, 10%, 10% and 10% respectively, for the species listed above). Supplementing the culture medium with BSA-loaded arachidonic acid promoted a rapid increase in the proportion of the C38:4 species in all phosphoinositide classes (from approx. 25%-60% of total species within 24 h), but the total amount of all combined species for each class remained remarkably constant. Stimulation of cells, cultured in either normal or arachidonate-enriched conditions, with 2 ng/ml EGF for 90 s caused substantial activation of Class I PI3K and accumulation of PIP3. Despite the increased proportion of C38:4 PIP3 under the arachidonate-supplemented conditions, the total amount of all combined PIP3 species accumulating in response to EGF was the same, with or without arachidonate supplementation; there were however small but significant preferences for the conversion of some PIP2 species to PIP3, with the polyunsaturated C38:4 and C38:3 species being more favoured over other species. These results suggest the enzymes which interconvert phosphoinositides are able to act on several different molecular species and homoeostatic mechanisms are in place to deliver similar phosphoinositide pool sizes under quite different conditions of arachidonate availability. They also suggest enzymes regulating PIP3 levels downstream of growth factor stimulation (i.e. PI3Ks and PIP3-phosphatases) show some acyl selectivity and further work should be directed at assessing whether different acyl species of PIP3 exhibit differing signalling potential.

Serum-to-2i interconversion of mouse embryonic stem cells (mESCs) is a valuable in vitro model for early embryonic development. To assess whether 3D chromatin organization changes during this transition, we established Capture Hi-C with target-sequence enrichment of DNase I hypersensitive sites. We detected extremely long-range intra- and inter-chromosomal interactions between a small subset of H3K27me3 marked bivalent promoters involving the Hox clusters in serum-grown cells. Notably, these promoter-mediated interactions are not present in 2i ground-state pluripotent mESCs but appear upon their further development into primed-like serum mESCs. Reverting serum mESCs to ground-state 2i mESCs removes these promoter-promoter interactions in a spatiotemporal manner. H3K27me3, which is largely absent at bivalent promoters in ground-state 2i mESCs, is necessary, but not sufficient, to establish these interactions, as confirmed by Capture Hi-C on Eed(-/-) serum mESCs. Our results implicate H3K27me3 and PRC2 as critical players in chromatin alteration during priming of ESCs for differentiation.

Synthetic Biology Open Language (SBOL) Visual is a graphical standard for genetic engineering. It consists of symbols representing DNA subsequences, including regulatory elements and DNA assembly features. These symbols can be used to draw illustrations for communication and instruction, and as image assets for computer-aided design. SBOL Visual is a community standard, freely available for personal, academic, and commercial use (Creative Commons CC0 license). We provide prototypical symbol images that have been used in scientific publications and software tools. We encourage users to use and modify them freely, and to join the SBOL Visual community: http://www.sbolstandard.org/visual.

An effective immune system depends upon regulation of lymphocyte function and homeostasis. In recent years, members of the GTPases of the immunity associated protein (GIMAP) family were proposed to regulate T cell homeostasis. In contrast, little is known about their function and mode of action in B cells. We used a combination of transgenic mice and in vivo and in vitro techniques to conditionally and electively ablate GIMAP1 in resting and activated peripheral B cells. Our data suggest that GIMAP1 is absolutely essential for the survival of peripheral B cells, irrespective of their activation state. Together with recent data showing increased expression of GIMAP1 in B cell lymphomas, our work points to the possible potential of GIMAP1 as a target for manipulation in a variety of B cell-mediated diseases.

The review describes the roles of Rho- and Rap-guanosine triphosphatases (GTPases) and of their activators, guanine-nucleotide exchange factors (GEFs), and inhibitors, GTPase activating proteins (GAPs), in neutrophil recruitment from the blood stream into inflamed tissues, with a focus on recently identified roles in neutrophils, endothelial cells, and platelets.

Genome-wide association studies have been tremendously successful in identifying genetic variants associated with complex diseases. The majority of association signals are intergenic and evidence is accumulating that a high proportion of signals lie in enhancer regions. We use Capture Hi-C to investigate, for the first time, the interactions between associated variants for four autoimmune diseases and their functional targets in B- and T-cell lines. Here we report numerous looping interactions and provide evidence that only a minority of interactions are common to both B- and T-cell lines, suggesting interactions may be highly cell-type specific; some disease-associated SNPs do not interact with the nearest gene but with more compelling candidate genes (for example, FOXO1, AZI2) often situated several megabases away; and finally, regions associated with different autoimmune diseases interact with each other and the same promoter suggesting common autoimmune gene targets (for example, PTPRC, DEXI and ZFP36L1).

Regulatory T cells (Tregs) are characterized by the expression of the master transcription factor forkhead box P3 (Foxp3). Although Foxp3 expression is widely used as a marker of the Treg lineage, recent data show that the Treg fate is determined by a multifactorial signaling pathway, involving cytokines, nuclear factors, and epigenetic modifications. Foxp3 expression and the Treg phenotype can be acquired by T cells in the periphery, illustrating that the Treg fate is not necessarily conferred during thymic development. The two main Treg populations in vivo, thymic Tregs and peripheral Tregs, differ in the pathways followed for their maturation. This chapter discusses the molecular control of Treg induction, in the thymus as well as the periphery.

Maintenance of energy homeostasis depends on the highly regulated storage and release of triacylglycerol primarily in adipose tissue and excessive storage is a feature of common metabolic disorders. CIDEA is a lipid droplet (LD)-protein enriched in brown adipocytes promoting the enlargement of LDs which are dynamic, ubiquitous organelles specialized for storing neutral lipids. We demonstrate an essential role in this process for an amphipathic helix in CIDEA, which facilitates embedding in the LD phospholipid monolayer and binds phosphatidic acid (PA). LD pairs are docked by CIDEA trans-complexes through contributions of the N-terminal domain and a C-terminal dimerization region. These complexes, enriched at the LD-LD contact site, interact with the cone-shaped phospholipid PA and likely increase phospholipid barrier permeability, promoting LD fusion by transference of lipids. This physiological process is essential in adipocyte differentiation as well as serving to facilitate the tight coupling of lipolysis and lipogenesis in activated brown fat.

Class I phosphoinositide 3-kinases (PI3Ks) are important regulators of neutrophil migration in response to a range of chemoattractants. Their primary lipid products PtdIns(3,4,5)P3 and PtdIns(3,4)P2 preferentially accumulate near to the leading edge of migrating cells and are thought to act as an important cue organizing molecular and morphological polarization. We have investigated the distribution and accumulation of these lipids independently in mouse neutrophils using eGFP-PH reportersand electron microscopy (EM). We found that authentic mouse neutrophils rapidly polarized their Class I PI3K signalling, as read-out by eGFP-PH reporters, both at the up-gradient leading edge in response to local stimulation with fMLP as well as spontaneously and randomly in response to uniform stimulation. EM studies revealed these events occurred at the plasma membrane, were dominated by accumulation of PtdIns(3,4,5)P3, but not PtdIns(3,4)P2, and were dependent on PI3Kγ and its upstream activation by both Ras and Gβγs.

Elf5 is a transcription factor with pivotal roles in the trophoblast compartment, where it reinforces a trophoblast stem cell (TSC)-specific transcriptional circuit. However, Elf5 is also present in differentiating trophoblast cells that have ceased to express other TSC genes such as Cdx2 and Eomes. In the present study, we aimed to elucidate the context-dependent role of Elf5 at the interface between TSC self-renewal and the onset of differentiation. We demonstrate that precise levels of Elf5 are critical for normal expansion of the TSC compartment and embryonic survival, as Elf5 overexpression triggers precocious trophoblast differentiation. Through integration of protein interactome, transcriptome, and genome-wide chromatin immunoprecipitation data, we reveal that this abundance-dependent function is mediated through a shift in preferred Elf5-binding partners; in TSCs, Elf5 interaction with Eomes recruits Tfap2c to triply occupied sites at TSC-specific genes, driving their expression. In contrast, the Elf5 and Tfap2c interaction becomes predominant as their protein levels increase. This triggers binding to double- and single-occupancy sites that harbor the cognate Tfap2c motif, causing activation of the associated differentiation-promoting genes. These data place Elf5 at the center of a stoichiometry-sensitive transcriptional network, where it acts as a molecular switch governing the balance between TSC proliferation and differentiation.

Erasure and subsequent reinstatement of DNA methylation in the germline, especially at imprinted CpG islands (CGIs), is crucial to embryogenesis in mammals. The mechanisms underlying DNA methylation establishment remain poorly understood, but a number of post-translational modifications of histones are implicated in antagonizing or recruiting the de novo DNA methylation complex. In mouse oogenesis, DNA methylation establishment occurs on a largely unmethylated genome and in nondividing cells, making it a highly informative model for examining how histone modifications can shape the DNA methylome. Using a chromatin immunoprecipitation (ChIP) and genome-wide sequencing (ChIP-seq) protocol optimized for low cell numbers and novel techniques for isolating primary and growing oocytes, profiles were generated for histone modifications implicated in promoting or inhibiting DNA methylation. CGIs destined for DNA methylation show reduced protective H3K4 dimethylation (H3K4me2) and trimethylation (H3K4me3) in both primary and growing oocytes, while permissive H3K36me3 increases specifically at these CGIs in growing oocytes. Methylome profiling of oocytes deficient in H3K4 demethylase KDM1A or KDM1B indicated that removal of H3K4 methylation is necessary for proper methylation establishment at CGIs. This work represents the first systematic study performing ChIP-seq in oocytes and shows that histone remodeling in the mammalian oocyte helps direct de novo DNA methylation events.

Gene therapy holds promise to cure a wide range of genetic and acquired diseases. Recent successes in recombinant adeno-associated viral vector (rAAV)-based gene therapy in the clinic for hereditary disorders such as Leber's congenital amaurosis and hemophilia B encouraged us to reexplore an rAAV approach for pulmonary gene transfer. Only limited clinical successes have been achieved for airway gene transfer so far, underscoring the need for further preclinical development of rAAV-based gene therapy for pulmonary disorders. We sought to determine the preclinical potential of an airway-tropic serotype, rAAV2/5, encoding reporter genes when delivered to mouse airways. Although several groups have assessed the stability of gene transfer using a nonintegrating rAAV in mouse airways, long-term stability for more than a year has not been reported. Additionally, an extensive quantitative analysis of the specific cell types targeted by rAAV2/5 using cell-specific markers is lacking. We obtained sustained gene expression in upper and lower airways up to 15 months after vector administration, a substantial proportion of the lifespan of a laboratory mouse. In addition, we demonstrated that readministration of rAAV2/5 to the airways is feasible and increases gene expression 14 months after primary vector administration, despite the presence of circulating neutralizing antibodies. Finally, identification of transduced cell types revealed different subpopulations being targeted by rAAV2/5, with 64% of β-galactosidase-positive cells being ciliated cells, 34% club cells in the conducting airways, and 75% alveolar type II cells in the alveoli at 1 month postinjection. This underscores the therapeutic potential of a nonintegrating rAAV vector to develop a gene therapeutic drug for a variety of pulmonary disorders, such as cystic fibrosis, primary ciliary dyskinesia, and surfactant deficiencies.

We used high-resolution mass spectrometry to map the cytotoxic T lymphocyte (CTL) proteome and the effect of the metabolic checkpoint kinase mTORC1 on CTLs. The CTL proteome was dominated by metabolic regulators and granzymes, and mTORC1 selectively repressed and promoted expression of a subset of CTL proteins (~10%). These included key CTL effector molecules, signaling proteins and a subset of metabolic enzymes. Proteomic data highlighted the potential for negative control of the production of phosphatidylinositol (3,4,5)-trisphosphate (PtdIns(3,4,5)P3) by mTORC1 in CTLs. mTORC1 repressed PtdIns(3,4,5)P3 production and determined the requirement for mTORC2 in activation of the kinase Akt. Our unbiased proteomic analysis thus provides comprehensive understanding of CTL identity and the control of CTL function by mTORC1.

The integrity of chromatin, which provides a dynamic template for all DNA-related processes in eukaryotes, is maintained through replication-dependent and -independent assembly pathways. To address the role of histone deposition in the absence of DNA replication, we deleted the H3.3 chaperone Hira in developing mouse oocytes. We show that chromatin of non-replicative developing oocytes is dynamic and that lack of continuous H3.3/H4 deposition alters chromatin structure, resulting in increased DNase I sensitivity, the accumulation of DNA damage, and a severe fertility phenotype. On the molecular level, abnormal chromatin structure leads to a dramatic decrease in the dynamic range of gene expression, the appearance of spurious transcripts, and inefficient de novo DNA methylation. Our study thus unequivocally shows the importance of continuous histone replacement and chromatin homeostasis for transcriptional regulation and normal developmental progression in a non-replicative system in vivo.

Hi-C is a powerful method that provides pairwise information on genomic regions in spatial proximity in the nucleus. Hi-C requires millions of cells as input and, as genome organization varies from cell to cell, a limitation of Hi-C is that it only provides a population average of genome conformations. We developed single-cell Hi-C to create snapshots of thousands of chromatin interactions that occur simultaneously in a single cell. To adapt Hi-C to single-cell analysis, we modified the protocol to include in-nucleus ligation. This enables the isolation of single nuclei carrying Hi-C-ligated DNA into separate tubes, followed by reversal of cross-links, capture of biotinylated ligation junctions on streptavidin-coated magnetic beads and PCR amplification of single-cell Hi-C libraries. The entire laboratory protocol can be carried out in 1 week, and although we have demonstrated its use in mouse T helper (TH1) cells, it should be applicable to any cell type or species for which standard Hi-C has been successful. We also developed an analysis pipeline to filter noise and assess the quality of data sets in a few hours. Although the interactome maps produced by single-cell Hi-C are sparse, the data provide useful information to understand cellular variability in nuclear genome organization and chromosome structure. Standard wet and dry laboratory skills in molecular biology and computational analysis are required.