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Generation of chimeric antigen receptor (CAR) T cells from pluripotent stem cells (PSCs) will enable advances in cancer immunotherapy. Understanding how CARs affect T cell differentiation from PSCs is important for this effort. The recently described artificial thymic organoid (ATO) system supports in vitro differentiation of PSCs to T cells. Unexpectedly, PSCs transduced with a CD19-targeted CAR resulted in diversion of T cell differentiation to the innate lymphoid cell 2 (ILC2) lineage in ATOs. T cells and ILC2s are closely related lymphoid lineages with shared developmental and transcriptional programs. Mechanistically, we show that antigen-independent CAR signaling during lymphoid development enriched for ILC2-primed precursors at the expense of T cell precursors. We applied this understanding to modulate CAR signaling strength through expression level, structure, and presentation of cognate antigen to demonstrate that the T cell-versus-ILC lineage decision can be rationally controlled in either direction, providing a framework for achieving CAR-T cell development from PSCs.

In the version of this article initially published, the SCD1 lane in Figure 4e was inverted and has now been reoriented, while in the Source Data for Figure 2a, the PD1 lane labeled image now replaces the Source Data online. Original and corrected figures (Figs. 1 and 2) are shown below. Additionally, the “Gene expression and immunoblot analysis” section in Methods has been amended to clarify that “Proteins of interest and loading controls were run on separate membranes.” The changes have been made in the HTML and PDF versions of the article.

BackgroundAcute lymphoblastic leukemia (ALL) is the most common pediatric hematological malignancy, with ETV6::RUNX1 being the most prevalent translocation whose exact pathogenesis remains unclear. IGF2BP1 (Insulin-like Growth Factor 2 Binding Protein 1) is an oncofetal RNA binding protein seen to be specifically overexpressed in ETV6::RUNX1 positive B-ALL. In this study, we have studied the mechanistic role of IGF2BP1 in leukemogenesis and its synergism with the ETV6::RUNX1 fusion protein.MethodsGene expression was analyzed from patient bone marrow RNA using Real Time RT-qPCR. Knockout cell lines were created using CRISPR-Cas9 based lentiviral vectors. RNA-Seq and RNA Immunoprecipitation sequencing (RIP-Seq) after IGF2BP1 pulldown were performed using the Illumina platform. Mouse experiments were done by retroviral overexpression of donor HSCs followed by lethal irradiation of recipients using a bone marrow transplant model.ResultsWe observed specific overexpression of IGF2BP1 in ETV6::RUNX1 positive patients in an Indian cohort of pediatric ALL (n=167) with a positive correlation with prednisolone resistance. IGF2BP1 expression was essential for tumor cell survival in multiple ETV6::RUNX1 positive B-ALL cell lines. Integrated analysis of transcriptome sequencing after IGF2BP1 knockout and RIP-Seq after IGF2BP1 pulldown in Reh cell line revealed that IGF2BP1 targets encompass multiple pro-oncogenic signalling pathways including TNFα/NFκB and PI3K-Akt pathways. These pathways were also dysregulated in primary ETV6::RUNX1 positive B-ALL patient samples from our center as well as in public B-ALL patient datasets. IGF2BP1 showed binding and stabilization of the ETV6::RUNX1 fusion transcript itself. This positive feedback loop led to constitutive dysregulation of several oncogenic pathways. Enforced co-expression of ETV6::RUNX1 and IGF2BP1 in mouse bone marrow resulted in marrow hypercellularity which was characterized by multi-lineage progenitor expansion and strong Ki67 positivity. This pre-leukemic phenotype confirmed their synergism in-vivo. Clonal expansion of cells overexpressing both ETV6::RUNX1 and IGF2BP1 was clearly observed. These mice also developed splenomegaly indicating extramedullary hematopoiesis.ConclusionOur data suggest a combined impact of the ETV6::RUNX1 fusion protein and RNA binding protein, IGF2BP1 in activating multiple oncogenic pathways in B-ALL which makes IGF2BP1 and these pathways as attractive therapeutic targets and biomarkers.

In the version of this article initially published, there were graphical presentation errors in Fig. 3g, where the center panels of the “m6A writers” columns duplicated other images in the figure; in Fig. 4c, where the lane for “PDI” was an incorrect version; and in Extended Data Fig. 3j, where the lower-left “PDI” lane was inverted. The images have been replaced in the HTML and PDF versions of the article and the file for Fig. 4 Source Data: Unprocessed blots has also been replaced online.

The nuclear receptors liver X receptor (LXR) α and β play crucial roles in hepatic metabolism. Many genes induced in response to pharmacologic LXR agonism have been defined; however, the transcriptional consequences of loss of LXR binding to its genomic targets are less well characterized. Here, we addressed how deletion of both LXRα and LXRβ from mouse liver (LXR double knockout [DKO]) affects the transcriptional regulatory landscape by integrating changes in LXR binding, chromatin accessibility, and gene expression. Many genes involved in fatty acid metabolism showed reduced expression and chromatin accessibility at their intergenic and intronic regions in LXRDKO livers. Genes that were up-regulated with LXR deletion had increased chromatin accessibility at their promoter regions and were enriched for functions not linked to lipid metabolism. Loss of LXR binding in liver reduced the activity of a broad set of hepatic transcription factors, inferred through changes in motif accessibility. By contrast, accessibility at promoter nuclear factor Y (NF-Y) motifs was increased in the absence of LXR. Unexpectedly, we also defined a small set of LXR targets for direct ligand-dependent repression. These genes have LXR-binding sites but showed increased expression in LXRDKO liver and reduced expression in response to the LXR agonist. In summary, the binding of LXRs to the hepatic genome has broad effects on the transcriptional landscape that extend beyond its canonical function as an activator of lipid metabolic genes.

The microRNA, miR-146a, is a negative feedback regulator of the central immune transcription factor, nuclear factor kappa B (NFkB). MiR-146a plays important roles in the immune system, and miR-146a deficient mice show a complex phenotype with features of chronic inflammation and autoimmune disease. In this study, we examined the role of miR-146a in extrafollicular B-cell responses, finding that miR-146a suppresses cellular responses in vivo and in vitro. Gene expression profiling revealed that miR-146a-deficient B-cells showed upregulation of interferon pathway genes, including Traf6, a known miR-146a target. We next interrogated the role of TRAF6 in these B-cell responses, finding that TRAF6 is required for proliferation by genetic and pharmacologic inhibition. Together, our findings demonstrate a novel role for miR-146a and TRAF6 in the extrafollicular B-cell responses, which have recently been tied to autoimmune disease pathogenesis. Our work highlights the pathogenetic role of miR-146a and the potential of pharmacologic inhibition of TRAF6 in autoimmune diseases in which miR-146a is deregulated.

The particularly interdisciplinary nature of human microbiome research makes the organization and reporting of results spanning epidemiology, biology, bioinformatics, translational medicine and statistics a challenge. Commonly used reporting guidelines for observational or genetic epidemiology studies lack key features specific to microbiome studies. Therefore, a multidisciplinary group of microbiome epidemiology researchers adapted guidelines for observational and genetic studies to culture-independent human microbiome studies, and also developed new reporting elements for laboratory, bioinformatics and statistical analyses tailored to microbiome studies. The resulting tool, called 'Strengthening The Organization and Reporting of Microbiome Studies' (STORMS), is composed of a 17-item checklist organized into six sections that correspond to the typical sections of a scientific publication, presented as an editable table for inclusion in supplementary materials. The STORMS checklist provides guidance for concise and complete reporting of microbiome studies that will facilitate manuscript preparation, peer review, and reader comprehension of publications and comparative analysis of published results.

Males and females exhibit striking differences in the prevalence of metabolic traits including hepatic steatosis, a key driver of cardiometabolic morbidity and mortality. RNA methylation is a widespread regulatory mechanism of transcript turnover. Here, we show that presence of the RNA modification N6-methyladenosine (m6A) triages lipogenic transcripts for degradation and guards against hepatic triglyceride accumulation. In male but not female mice, this protective checkpoint stalls under lipid-rich conditions. Loss of m6A control in male livers increases hepatic triglyceride stores, leading to a more 'feminized' hepatic lipid composition. Crucially, liver-specific deletion of the m6A complex protein Mettl14 from male and female mice significantly diminishes sex-specific differences in steatosis. We further surmise that the m6A installing machinery is subject to transcriptional control by the sex-responsive BCL6-STAT5 axis in response to dietary conditions. These data show that m6A is essential for precise and synchronized control of lipogenic enzyme activity and provide insights into the molecular basis for the existence of sex-specific differences in hepatic lipid traits.

Post-transcriptional gene regulation, including that by RNA binding proteins (RBPs), has recently been described as an important mechanism in cancer. We had previously identified a set of RBPs that were highly dysregulated in B-cell acute lymphoblastic leukemia (B-ALL) with MLL translocations, which carry a poor prognosis. Here, we sought to functionally characterize these dysregulated RBP genes by performing a focused CRISPR dropout screen in B-ALL cell lines, finding dependencies on several genes including EIF3E, EPRS and USO1. Validating our findings, CRISPR/Cas9-mediated disruption of USO1 in MLL-translocated B-ALL cells reduced cell growth, promoted cell death, and altered the cell cycle. Transcriptomic analysis of USO1-deficient cells revealed alterations in pathways related to mTOR signaling, RNA metabolism, and targets of MYC. In addition, USO1-regulated genes from these experimental samples were significantly and concordantly correlated with USO1 expression in primary samples collected from B-ALL patients. Lastly, we found that loss of Uso1 inhibited colony formation of MLL-transformed in primary bone marrow cells from Cas9-EGFP mice. Together, our findings demonstrate an approach to performing focused sub-genomic CRISPR screens and highlight a putative RBP vulnerability in MLL-translocated B-ALL, thus identifying potential therapeutic targets in this disease.

BACKGROUND AND AIMS: The host receptor for severe acute respiratory syndrome coronavirus 2, angiotensin-converting enzyme 2 (ACE2), is highly expressed in small bowel (SB). Our aim was to identify factors influencing intestinal ACE2 expression in Crohns disease (CD), ulcerative colitis (UC), and non-inflammatory bowel disease (IBD) controls. METHODS: Using bulk RNA sequencing or microarray transcriptomics from tissue samples (4 SB and 2 colonic cohorts; n = 495; n = 387 UC; n = 94 non-IBD), we analyzed the relationship between ACE2 with demographics and disease activity and prognosis. We examined the outcome of anti-tumor necrosis factor and anti-interleukin-12/interleukin-23 treatment on SB and colonic ACE2 expression in 3 clinical trials. Univariate and multivariate regression models were fitted. RESULTS: ACE2 levels were consistently reduced in SB CD and elevated in colonic UC compared with non-IBD controls. Elevated SB ACE2 was also associated with demographic features (age and elevated body mass index) associated with poor coronavirus disease 2019 outcomes. Within CD, SB ACE2 was reduced in patients subsequently developing complicated disease. Within UC, colonic ACE2 was elevated in active disease and in patients subsequently requiring anti-tumor necrosis factor rescue therapy. SB and colonic ACE2 expression in active CD and UC were restored by anti-cytokine therapy, most notably in responders. CONCLUSIONS: Reduced SB but elevated colonic ACE2 levels in IBD are associated with inflammation and severe disease, but normalized after anti-cytokine therapy, suggesting compartmentalization of ACE2-related biology in SB and colonic inflammation. The restoration of ACE2 expression with anti-cytokine therapy might be important in the context of severe acute respiratory syndrome coronavirus 2 infection and potentially explain reports of reduced morbidity from coronavirus disease 2019 in IBD patients treated with anti-cytokines.

Although metabolic pathways have been shown to control differentiation and activation in peripheral T cells, metabolic studies on thymic T cell development are still lacking, especially in human tissue. In this study, we use transcriptomics and extracellular flux analyses to investigate the metabolic profiles of primary thymic and in vitro-derived mouse and human thymocytes. Core metabolic pathways, specifically glycolysis and oxidative phosphorylation, undergo dramatic changes between the double-negative (DN), double-positive (DP), and mature single-positive (SP) stages in murine and human thymus. Remarkably, despite the absence of the complex multicellular thymic microenvironment, in vitro murine and human T cell development recapitulated the coordinated decrease in glycolytic and oxidative phosphorylation activity between the DN and DP stages seen in primary thymus. Moreover, by inducing in vitro T cell differentiation from Rag1-/- mouse bone marrow, we show that reduced metabolic activity at the DP stage is independent of TCR rearrangement. Thus, our findings suggest that highly conserved metabolic transitions are critical for thymic T cell development.

Neonatal life marks the apogee of murine thymic growth. Over the first few days after birth, growth slows and the murine thymus switches from fetal to adult morphology and function; little is known about the cues driving this dramatic transition. In this study, we show for the first time (to our knowledge) the critical role of vascular endothelial growth factor (VEGF) on thymic morphogenesis beyond its well-known role in angiogenesis. During a brief window a few days after birth, VEGF inhibition induced rapid and profound remodeling of the endothelial, mesenchymal and epithelial thymic stromal compartments, mimicking changes seen during early adult maturation. Rapid transcriptional changes were seen in each compartment after VEGF inhibition, including genes involved in migration, chemotaxis, and cell adhesion as well as induction of a proinflammatory and proadipogenic signature in endothelium, pericytes, and mesenchyme. Thymocyte numbers fell subsequent to the stromal changes. Expression patterns and functional blockade of the receptors VEGFR2 and NRP1 demonstrated that VEGF mediates its pleiotropic effects through distinct receptors on each microenvironmental compartment of the developing mouse thymus.

Colon mucus segregates the intestinal microbiota from host tissues, but how it organizes to function throughout the colon is unclear. In mice, we found that colon mucus consists of two distinct O-glycosylated entities of Muc2: a major form produced by the proximal colon, which encapsulates the fecal material including the microbiota, and a minor form derived from the distal colon, which adheres to the major form. The microbiota directs its own encapsulation by inducing Muc2 production from proximal colon goblet cells. In turn, O-glycans on proximal colon-derived Muc2 modulate the structure and function of the microbiota as well as transcription in the colon mucosa. Our work shows how proximal colon control of mucin production is an important element in the regulation of host-microbiota symbiosis.

We report a serum-free, 3D murine artificial thymic organoid (M-ATO) system that mimics normal murine thymopoiesis with the production of all T cell stages, from early thymic progenitors to functional single-positive (CD8SP and CD4SP) TCRαβ and TCRγδ cells. RNA sequencing aligns M-ATO-derived populations with phenotypically identical primary thymocytes. M-ATOs initiated with Rag1-/- marrow produce the same differentiation block as seen in the endogenous thymus, and Notch signaling patterns in M-ATOs mirror primary thymopoiesis. M-ATOs initiated with defined hematopoietic stem cells (HSCs) and lymphoid progenitors from marrow and thymus generate each of the downstream differentiation stages, allowing the kinetics of T cell differentiation to be tracked. Remarkably, single HSCs deposited into each M-ATO generate the complete trajectory of T cell differentiation, producing diverse TCR repertoires across clones that largely match endogenous thymus. M-ATOs represent a highly reproducible and efficient experimental platform for the interrogation of clonal thymopoiesis from HSCs.

The finding that transformed mouse B-1 and B-2 progenitors give rise to B-cell acute lymphoblastic leukemias (B-ALLs) with varied aggressiveness suggests that B-cell lineage might also be a factor in the initiation and progression of pediatric B-ALLs in humans. If this is the case, we hypothesized that human pediatric B-ALLs would share gene expression patterns with mouse B-1 or B-2 progenitors. We tested this premise by deriving a distinct 30-gene B-1 and B-2 progenitor signature that was applied to a microarray data set of human pediatric ALLs. Cluster analysis revealed that CRLF2, E2A-PBX1, ERG, and ETV6-RUNX1 leukemias were B-1-like, whereas BCR-ABL1, hyperdiploid, and MLL leukemias were B-2-like. Examination of the 30-gene signature in two independent data sets of pediatric ALLs supported this result. Our data suggest that common genetic subtypes of human ALL have their origin in the B-1 or B-2 lineage.

PurposeRetinopathy of prematurity (ROP) is a leading cause of childhood blindness. ROP occurs as a consequence of postnatal hyperoxia exposure in premature infants, resulting in vasoproliferation in the retina. The tetraspan protein epithelial membrane protein-2 (EMP2) is highly expressed in the retinal pigment epithelium (RPE) in adults, and it controls vascular endothelial growth factor (VEGF) production in the ARPE-19 cell line. We, therefore, hypothesized that Emp2 knockout (Emp2 KO) protects against neovascularization in murine oxygen-induced retinopathy (OIR).MethodsEyes were obtained from wildtype (WT) and Emp2 KO mouse pups at P7, P12, P17, and P21 after normoxia or hyperoxia (P7-P12) exposure. Following hyperoxia exposure, RNA sequencing was performed using the retina/choroid layers obtained from WT and Emp2 KO at P17. Retinal sections from P7, P12, P17, and P21 were evaluated for Emp2, hypoxia-inducible factor 1α (Hif1α), and VEGF expression. Whole mount images were generated to assess vaso-obliteration at P12 and neovascularization at P17.ResultsEmp2 KO OIR mice demonstrated a decrease in pathologic neovascularization at P17 compared with WT OIR mice through evaluation of retinal vascular whole mount images. This protection was accompanied by a decrease in Hif1α at P12 and VEGFA expression at P17 in Emp2 KO animals compared with the WT animals in OIR conditions. Collectively, our results suggest that EMP2 enhances the effects of neovascularization through modulation of angiogenic signaling.ConclusionsThe protection of Emp2 KO mice against pathologic neovascularization through attenuation of HIF and VEGF upregulation in OIR suggests that hypoxia-induced upregulation of EMP2 expression in the neuroretina modulates HIF-mediated neuroretinal VEGF expression.

Aging results in increased myelopoiesis, which is linked to the increased incidence of myeloid leukemias and production of myeloid-derived suppressor cells. Here, we examined the contribution of plasma cells (PCs) to age-related increases in myelopoiesis, as PCs exhibit immune regulatory function and sequester in bone marrow (BM). PC number was increased in old BM, and they exhibited high expression of genes encoding inflammatory cytokines and pathogen sensors. Antibody-mediated depletion of PCs from old mice reduced the number of myeloid-biased hematopoietic stem cells and mature myeloid cells to levels in young animals, but lymphopoiesis was not rejuvenated, indicating that redundant mechanisms inhibit that process. PCs also regulated the production of inflammatory factors from BM stromal cells, and disruption of the PC-stromal cell circuitry with inhibitors of the cytokines IL-1 and TNF-α attenuated myelopoiesis in old mice. Thus, the age-related increase in myelopoiesis is driven by an inflammatory network orchestrated by PCs.

Inflammatory bowel diseases, which include Crohn's disease and ulcerative colitis, affect several million individuals worldwide. Crohn's disease and ulcerative colitis are complex diseases that are heterogeneous at the clinical, immunological, molecular, genetic, and microbial levels. Individual contributing factors have been the focus of extensive research. As part of the Integrative Human Microbiome Project (HMP2 or iHMP), we followed 132 subjects for one year each to generate integrated longitudinal molecular profiles of host and microbial activity during disease (up to 24 time points each; in total 2,965 stool, biopsy, and blood specimens). Here we present the results, which provide a comprehensive view of functional dysbiosis in the gut microbiome during inflammatory bowel disease activity. We demonstrate a characteristic increase in facultative anaerobes at the expense of obligate anaerobes, as well as molecular disruptions in microbial transcription (for example, among clostridia), metabolite pools (acylcarnitines, bile acids, and short-chain fatty acids), and levels of antibodies in host serum. Periods of disease activity were also marked by increases in temporal variability, with characteristic taxonomic, functional, and biochemical shifts. Finally, integrative analysis identified microbial, biochemical, and host factors central to this dysregulation. The study's infrastructure resources, results, and data, which are available through the Inflammatory Bowel Disease Multi'omics Database ( http://ibdmdb.org ), provide the most comprehensive description to date of host and microbial activities in inflammatory bowel diseases.

Current models propose that reductions in the number of lymphoid-biased hematopoietic stem cells (Ly-HSCs) underlie age-related declines in lymphopoiesis. We show that Ly-HSCs do not decline in number with age. Old Ly-HSCs exhibit changes in gene expression and a myeloid-biased genetic profile, but we demonstrate that they retain normal lymphoid potential when removed from the old in vivo environment. Additional studies showing that interleukin-1 inhibits Ly-HSC lymphoid potential provide support for the hypothesis that increased production of inflammatory cytokines during aging underlies declines in lymphocyte production. These results indicate that current models proposing that lymphopoiesis declines with age due to loss of Ly-HSCs require revision and provide an additional perspective on why lymphocyte development in the elderly is attenuated.

The ability to generate T cells from pluripotent stem cells (PSCs) has the potential to transform autologous T cell immunotherapy by facilitating universal, off-the-shelf cell products. However, differentiation of human PSCs into mature, conventional T cells has been challenging with existing methods. We report that a continuous 3D organoid system induced an orderly sequence of commitment and differentiation from PSC-derived embryonic mesoderm through hematopoietic specification and efficient terminal differentiation to naive CD3+CD8αβ+ and CD3+CD4+ conventional T cells with a diverse T cell receptor (TCR) repertoire. Introduction of an MHC class I-restricted TCR in PSCs produced naive, antigen-specific CD8αβ+ T cells that lacked endogenous TCR expression and showed anti-tumor efficacy in vitro and in vivo. Functional assays and RNA sequencing aligned PSC-derived T cells with primary naive CD8+ T cells. The PSC-artificial thymic organoid (ATO) system presented here is an efficient platform for generating functional, mature T cells from human PSCs.

Microbial metabolites are an emerging class of mediators influencing CD4+ T-cell function. To advance the understanding of direct causal microbial factors contributing to Crohn's disease, we screened 139 predicted Crohn's disease-associated microbial metabolites for their bioactivity on human CD4+ T-cell functions induced by disease-associated T helper 17 (Th17) polarizing conditions. We observed 15 metabolites with CD4+ T-cell bioactivity, 3 previously reported, and 12 unprecedented. A deeper investigation of the microbe-derived metabolite, ascorbate, revealed its selective inhibition on activated human CD4+ effector T cells, including IL-17A-, IL-4-, and IFNγ-producing cells. Mechanistic assessment suggested the apoptosis of activated human CD4+ T cells associated with selective inhibition of energy metabolism. These findings suggest a substantial rate of relevant T-cell bioactivity among Crohn's disease-associated microbial metabolites, and evidence for novel modes of bioactivity, including targeting of T-cell energy metabolism.

Intrauterine growth restriction (IUGR) enhances risk for adult onset cardiovascular disease (CVD). The mechanisms underlying IUGR are poorly understood, though inadequate blood flow and oxygen/nutrient provision are considered common endpoints. Based on evidence in humans linking IUGR to adult CVD, we hypothesized that in murine pregnancy, maternal late gestational hypoxia (LG-H) exposure resulting in IUGR would result in (1) placental transcriptome changes linked to risk for later CVD, and 2) adult phenotypes of CVD in the IUGR offspring. After subjecting pregnant mice to hypoxia (10.5% oxygen) from gestational day (GD) 14.5 to 18.5, we undertook RNA sequencing from GD19 placentas. Functional analysis suggested multiple changes in structural and functional genes important for placental health and function, with maximal dysregulation involving vascular and nutrient transport pathways. Concordantly, a ~10% decrease in birthweights and ~30% decrease in litter size was observed, supportive of placental insufficiency. We also found that the LG-H IUGR offspring exhibit increased risk for CVD at 4 months of age, manifesting as hypertension, increased abdominal fat, elevated leptin and total cholesterol concentrations. In summary, this animal model of IUGR links the placental transcriptional response to the stressor of gestational hypoxia to increased risk of developing cardiometabolic disease.

The PU.1 transcription factor plays a critical role in the regulation of T cell development, so a report that it is dispensable for fetal thymopoiesis is puzzling. To understand this paradox, we examined the requirement for PU.1, encoded by Spi1, during fetal, neonatal, and adult thymopoiesis in a PU.1 hypomorphic mouse generated by deletion of the Spi1 14-kb upstream regulatory element and by analysis of patterns of gene expression in fetal and adult T cell progenitors. Our data demonstrate that the initiation of thymopoiesis during early gestation is less dependent on PU.1 compared with T cell differentiation in adults and that fetal T cell progenitors express lower levels of Spi1 compared with their adult counterparts. We also show that expression of the core network of T lineage transcription factors regulated by PU.1 differs in fetal and adult T cell progenitors. In particular, PU.1-regulated genes that promote T cell differentiation are differentially expressed in fetal versus adult early T lineage progenitors. These results indicate that the transcriptional differences between the fetal and adult T cell developmental programs are driven in part by differential levels of PU.1 expression and that this likely underlies the differences in the properties of fetal and adult T cell progenitors.

Nuclear receptors regulate gene expression in response to environmental cues, but the molecular events governing the cell type specificity of nuclear receptors remain poorly understood. Here we outline a role for a long noncoding RNA (lncRNA) in modulating the cell type-specific actions of liver X receptors (LXRs), sterol-activated nuclear receptors that regulate the expression of genes involved in cholesterol homeostasis and that have been causally linked to the pathogenesis of atherosclerosis. We identify the lncRNA MeXis as an amplifier of LXR-dependent transcription of the gene Abca1, which is critical for regulation of cholesterol efflux. Mice lacking the MeXis gene show reduced Abca1 expression in a tissue-selective manner. Furthermore, loss of MeXis in mouse bone marrow cells alters chromosome architecture at the Abca1 locus, impairs cellular responses to cholesterol overload, and accelerates the development of atherosclerosis. Mechanistic studies reveal that MeXis interacts with and guides promoter binding of the transcriptional coactivator DDX17. The identification of MeXis as a lncRNA modulator of LXR-dependent gene expression expands understanding of the mechanisms underlying cell type-selective actions of nuclear receptors in physiology and disease.

Various mesenchymal cell types have been identified as critical components of the hematopoietic stem/progenitor cell (HSPC) niche. Although several groups have described the generation of mesenchyme from human pluripotent stem cells (hPSCs), the capacity of such cells to support hematopoiesis has not been reported. Here, we demonstrate that distinct mesenchymal subpopulations co-emerge from mesoderm during hPSC differentiation. Despite co-expression of common mesenchymal markers (CD73, CD105, CD90, and PDGFRβ), a subset of cells defined as CD146hiCD73hi expressed genes associated with the HSPC niche and supported the maintenance of functional HSPCs ex vivo, while CD146loCD73lo cells supported differentiation. Stromal support of HSPCs was contact dependent and mediated in part through high JAG1 expression and low WNT signaling. Molecular profiling revealed significant transcriptional similarity between hPSC-derived CD146++ and primary human CD146++ perivascular cells. The derivation of functionally diverse types of mesenchyme from hPSCs opens potential avenues to model the HSPC niche and develop PSC-based therapies.

BackgroundLong non-coding RNAs (lncRNAs) play a variety of cellular roles, including regulation of transcription and translation, leading to alterations in gene expression. Some lncRNAs modulate the expression of chromosomally adjacent genes. Here, we assess the roles of the lncRNA CASC15 in regulation of a chromosomally nearby gene, SOX4, and its function in RUNX1/AML translocated leukemia.ResultsCASC15 is a conserved lncRNA that was upregulated in pediatric B-acute lymphoblastic leukemia (B-ALL) with t (12; 21) as well as pediatric acute myeloid leukemia (AML) with t (8; 21), both of which are associated with relatively better prognosis. Enforced expression of CASC15 led to a myeloid bias in development, and overall, decreased engraftment and colony formation. At the cellular level, CASC15 regulated cellular survival, proliferation, and the expression of its chromosomally adjacent gene, SOX4. Differentially regulated genes following CASC15 knockdown were enriched for predicted transcriptional targets of the Yin and Yang-1 (YY1) transcription factor. Interestingly, we found that CASC15 enhances YY1-mediated regulation of the SOX4 promoter.ConclusionsOur findings represent the first characterization of this CASC15 in RUNX1-translocated leukemia, and point towards a mechanistic basis for its action.

Epithelial membrane protein-2 (EMP2) is a tetraspan protein predicted to regulate placental development. Highly expressed in secretory endometrium and trophectoderm cells, previous studies suggest that it may regulate implantation by orchestrating the surface expression of integrins and other membrane proteins. In order to test the role of EMP2 in pregnancy, mice lacking EMP2 (Emp2-/- ) were generated. Emp2-/- females are fertile but have reduced litter sizes when carrying Emp2-/- but not Emp2+/- fetuses. Placentas of Emp2-/- fetuses exhibit dysregulation in pathways related to neoangiogenesis, coagulation, and oxidative stress, and have increased fibrin deposition and altered vasculature. Given that these findings often occur due to placental insufficiency resulting in an oxygen-poor environment, the expression of hypoxia-inducible factor-1 alpha (HIF-1α) was examined. Placentas from Emp2-/- fetuses had increased total HIF-1α expression in large part through an increase in uterine NK (uNK) cells, demonstrating a unique interplay between uNK cells and trophoblasts modulated through EMP2. To determine if these results translated to human pregnancy, placentas from normal, term deliveries or those complicated by placental insufficiency resulting in intrauterine growth restriction (IUGR) were stained for EMP2. EMP2 was significantly reduced in both villous and extravillous trophoblast populations in IUGR placentas. Experiments in vitro using human trophoblast cells lines indicate that EMP2 modulates angiogenesis by altering HIF-1α expression. Our results reveal a novel role for EMP2 in regulating trophoblast function and vascular development in mice and humans, and suggest that it may be a new biomarker for placental insufficiency. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

B cell development is often depicted as a linear process initiating in the fetus and continuing postnatally. Using a PU.1 hypomorphic mouse model, we found that B-1 and B-2 lymphopoiesis occurred in distinct fetal and adult waves differentially dependent on the Sfpi1 14 kB upstream regulatory element. The initial wave of fetal B-1 development was absent in PU.1 hypomorphic mice, while subsequent fetal and adult waves emerged. In contrast, B-2 lymphopoiesis occurred in distinct fetal and adult waves. Whole-transcriptome profiling of fetal and adult B cell progenitors supported the existence of three waves of B-1 and two waves of B-2 development and revealed that the network of transcription factors governing B lineage specification and commitment was highly divergent between B-1 and B-2 progenitors. These findings support the view that the B-1 and B-2 lineages are distinct and provide a genetic basis for layering of immune system development.

Liver X receptors (LXRs) are transcriptional regulators of cellular and systemic cholesterol homeostasis. Under conditions of excess cholesterol, LXR activation induces the expression of several genes involved in cholesterol efflux, facilitates cholesterol esterification by promoting fatty acid synthesis, and inhibits cholesterol uptake by the low-density lipoprotein receptor. The fact that sterol content is maintained in a narrow range in most cell types and in the organism as a whole suggests that extensive crosstalk between regulatory pathways must exist. However, the molecular mechanisms that integrate LXRs with other lipid metabolic pathways are incompletely understood. Here we show that ligand activation of LXRs in mouse liver not only promotes cholesterol efflux, but also simultaneously inhibits cholesterol biosynthesis. We further identify the long non-coding RNA LeXis as a mediator of this effect. Hepatic LeXis expression is robustly induced in response to a Western diet (high in fat and cholesterol) or to pharmacological LXR activation. Raising or lowering LeXis levels in the liver affects the expression of genes involved in cholesterol biosynthesis and alters the cholesterol levels in the liver and plasma. LeXis interacts with and affects the DNA interactions of RALY, a heterogeneous ribonucleoprotein that acts as a transcriptional cofactor for cholesterol biosynthetic genes in the mouse liver. These findings outline a regulatory role for a non-coding RNA in lipid metabolism and advance our understanding of the mechanisms that coordinate sterol homeostasis.

Although clonal studies of lineage potential have been extensively applied to organ specific stem and progenitor cells, much less is known about the clonal origins of lineages formed from the germ layers in early embryogenesis. We applied lentiviral tagging followed by vector integration site analysis (VISA) with high-throughput sequencing to investigate the ontogeny of the hematopoietic, endothelial and mesenchymal lineages as they emerge from human embryonic mesoderm. In contrast to studies that have used VISA to track differentiation of self-renewing stem cell clones that amplify significantly over time, we focused on a population of progenitor clones with limited self-renewal capability. Our analyses uncovered the critical influence of sampling on the interpretation of lentiviral tag sharing, particularly among complex populations with minimal clonal duplication. By applying a quantitative framework to estimate the degree of undersampling we revealed the existence of tripotent mesodermal progenitors derived from pluripotent stem cells, and the subsequent bifurcation of their differentiation into bipotent endothelial/hematopoietic or endothelial/mesenchymal progenitors. Stem Cells 2016;34:1239-1250.

Cellular lipid requirements are achieved through a combination of biosynthesis and import programs. Using isotope tracer analysis, we show that type I interferon (IFN) signaling shifts the balance of these programs by decreasing synthesis and increasing import of cholesterol and long chain fatty acids. Genetically enforcing this metabolic shift in macrophages is sufficient to render mice resistant to viral challenge, demonstrating the importance of reprogramming the balance of these two metabolic pathways in vivo. Unexpectedly, mechanistic studies reveal that limiting flux through the cholesterol biosynthetic pathway spontaneously engages a type I IFN response in a STING-dependent manner. The upregulation of type I IFNs was traced to a decrease in the pool size of synthesized cholesterol and could be inhibited by replenishing cells with free cholesterol. Taken together, these studies delineate a metabolic-inflammatory circuit that links perturbations in cholesterol biosynthesis with activation of innate immunity.

To elucidate the transcriptional 'landscape' that regulates human lymphoid commitment during postnatal life, we used RNA sequencing to assemble the long non-coding transcriptome across human bone marrow and thymic progenitor cells spanning the earliest stages of B lymphoid and T lymphoid specification. Over 3,000 genes encoding previously unknown long non-coding RNAs (lncRNAs) were revealed through the analysis of these rare populations. Lymphoid commitment was characterized by lncRNA expression patterns that were highly stage specific and were more lineage specific than those of protein-coding genes. Protein-coding genes co-expressed with neighboring lncRNA genes showed enrichment for ontologies related to lymphoid differentiation. The exquisite cell-type specificity of global lncRNA expression patterns independently revealed new developmental relationships among the earliest progenitor cells in the human bone marrow and thymus.

UnlabelledLong noncoding RNAs (lncRNA) have been found to play a role in gene regulation with dysregulated expression in various cancers. The precise role that lncRNA expression plays in the pathogenesis of B-acute lymphoblastic leukemia (B-ALL) is unknown. Therefore, unbiased microarray profiling was performed on human B-ALL specimens, and it was determined that lncRNA expression correlates with cytogenetic abnormalities, which was confirmed by qRT-PCR in a large set of B-ALL cases. Importantly, high expression of BALR-2 correlated with poor overall survival and diminished response to prednisone treatment. In line with a function for this lncRNA in regulating cell survival, BALR-2 knockdown led to reduced proliferation, increased apoptosis, and increased sensitivity to prednisolone treatment. Conversely, overexpression of BALR-2 led to increased cell growth and resistance to prednisone treatment. Interestingly, BALR-2 expression was repressed by prednisolone treatment and its knockdown led to upregulation of the glucocorticoid response pathway in both human and mouse B cells. Together, these findings indicate that BALR-2 plays a functional role in the pathogenesis and/or clinical responsiveness of B-ALL, and that altering the levels of particular lncRNAs may provide a future direction for therapeutic development.ImplicationslncRNA expression has the potential to segregate the common subtypes of B-ALL, predict the cytogenetic subtype, and indicate prognosis.

miR-146a is a NF-κB induced microRNA that serves as a feedback regulator of this critical pathway. In mice, deficiency of miR-146a results in hematolymphoid cancer at advanced ages as a consequence of constitutive NF-κB activity. In this study, we queried whether the deficiency of miR-146a contributes to B-cell oncogenesis. Combining miR-146a deficiency with transgenic expression of c-Myc led to the development of highly aggressive B-cell malignancies. Mice transgenic for c-Myc and deficient for miR-146a were characterized by significantly shortened survival, increased lymph node involvement, differential involvement of the spleen and a mature B-cell phenotype. High-throughput sequencing of the tumors revealed significant dysregulation of approximately 250 genes. Amongst these, the transcription factor Egr1 was consistently upregulated in mice deficient for miR-146a. Interestingly, transcriptional targets of Egr1 were enriched in both the high-throughput dataset and in a larger set of miR-146a-deficient tumors. miR-146a overexpression led to downregulation of Egr1 and downstream targets with concomitant decrease in cell growth. Direct targeting of the human EGR1 by miR-146a was seen by luciferase assay. Together our findings illuminate a bona fide role for miR-146a in the modulation of B-cell oncogenesis and reveal the importance of understanding microRNA function in a cell- and disease-specific context.

Cell number in the mouse thymus increases steadily during the first two weeks after birth. It then plateaus and begins to decline by seven weeks after birth. The factors governing these dramatic changes in cell production are not well understood. The data herein correlate levels of High mobility group A 2 protein (Hmga2) expression with these temporal changes in thymopoiesis. Hmga2 is expressed at high levels in murine fetal and neonatal early T cell progenitors (ETP), which are the most immature intrathymic precursors, and becomes almost undetectable in these progenitors after 5 weeks of age. Hmga2 expression is critical for the initial, exponential expansion of thymopoiesis, as Hmga2 deficient mice have a deficit of ETPs within days after birth, and total thymocyte number is repressed compared to wild type littermates. Finally, our data raise the possibility that similar events occur in humans, because Hmga2 expression is high in human fetal thymic progenitors and falls in these cells during early infancy.

Singlet oxygen is a highly toxic and inevitable byproduct of oxygenic photosynthesis. The unicellular green alga Chlamydomonas reinhardtii is capable of acclimating specifically to singlet oxygen stress, but the retrograde signaling pathway from the chloroplast to the nucleus mediating this response is unknown. Here we describe a mutant, singlet oxygen acclimation knocked-out 1 (sak1), that lacks the acclimation response to singlet oxygen. Analysis of genome-wide changes in RNA abundance during acclimation to singlet oxygen revealed that SAK1 is a key regulator of the gene expression response during acclimation. The SAK1 gene encodes an uncharacterized protein with a domain conserved among chlorophytes and present in some bZIP transcription factors. The SAK1 protein is located in the cytosol, and it is induced and phosphorylated upon exposure to singlet oxygen, suggesting that it is a critical intermediate component of the retrograde signal transduction pathway leading to singlet oxygen acclimation.DOI: http://dx.doi.org/10.7554/eLife.02286.001.

When the sta6 (starch-null) strain of the green microalga Chlamydomonas reinhardtii is nitrogen starved in acetate and then "boosted" after 2 days with additional acetate, the cells become "obese" after 8 days, with triacylglyceride (TAG)-filled lipid bodies filling their cytoplasm and chloroplasts. To assess the transcriptional correlates of this response, the sta6 strain and the starch-forming cw15 strain were subjected to RNA-Seq analysis during the 2 days prior and 2 days after the boost, and the data were compared with published reports using other strains and growth conditions. During the 2 h after the boost, ∼425 genes are upregulated ≥2-fold and ∼875 genes are downregulated ≥2-fold in each strain. Expression of a small subset of "sensitive" genes, encoding enzymes involved in the glyoxylate and Calvin-Benson cycles, gluconeogenesis, and the pentose phosphate pathway, is responsive to culture conditions and genetic background as well as to boosting. Four genes-encoding a diacylglycerol acyltransferase (DGTT2), a glycerol-3-P dehydrogenase (GPD3), and two candidate lipases (Cre03.g155250 and Cre17.g735600)-are selectively upregulated in the sta6 strain. Although the bulk rate of acetate depletion from the medium is not boost enhanced, three candidate acetate permease-encoding genes in the GPR1/FUN34/YaaH superfamily are boost upregulated, and 13 of the "sensitive" genes are strongly responsive to the cell's acetate status. A cohort of 64 autophagy-related genes is downregulated by the boost. Our results indicate that the boost serves both to avert an autophagy program and to prolong the operation of key pathways that shuttle carbon from acetate into storage lipid, the combined outcome being enhanced TAG accumulation, notably in the sta6 strain.

Chlamydomonas reinhardtii cells exposed to abiotic stresses (e.g. nitrogen, zinc, or phosphorus deficiency) accumulate triacylglycerols (TAG), which are stored in lipid droplets. Here, we report that iron starvation leads to formation of lipid droplets and accumulation of TAGs. This occurs between 12 and 24 h after the switch to iron-starvation medium. C. reinhardtii cells deprived of iron have more saturated fatty acid (FA), possibly due to the loss of function of FA desaturases, which are iron-requiring enzymes with diiron centers. The abundance of a plastid acyl-ACP desaturase (FAB2) is decreased to the same degree as ferredoxin. Ferredoxin is a substrate of the desaturases and has been previously shown to be a major target of the iron deficiency response. The increase in saturated FA (C16:0 and C18:0) is concomitant with the decrease in unsaturated FA (C16:4, C18:3, or C18:4). This change was gradual for diacylglyceryl-N,N,N-trimethylhomoserine (DGTS) and digalactosyldiacylglycerol (DGDG), whereas the monogalactosyldiacylglycerol (MGDG) FA profile remained stable during the first 12 h, whereas MGDG levels were decreasing over the same period of time. These changes were detectable after only 2 h of iron starvation. On the other hand, DGTS and DGDG contents gradually decreased until a minimum was reached after 24-48 h. RNA-Seq analysis of iron-starved C. reinhardtii cells revealed notable changes in many transcripts coding for enzymes involved in FA metabolism. The mRNA abundances of genes coding for components involved in TAG accumulation (diacylglycerol acyltransferases or major lipid droplet protein) were increased. A more dramatic increase at the transcript level has been observed for many lipases, suggesting that major remodeling of lipid membranes occurs during iron starvation in C. reinhardtii.

Abstract Background Progress in genome sequencing is proceeding at an exponential pace, and several new algal genomes are becoming available every year. One of the challenges facing the community is the association of protein sequences encoded in the genomes with biological function. While most genome assembly projects generate annotations for predicted protein sequences, they are usually limited and integrate functional terms from a limited number of databases. Another challenge is the use of annotations to interpret large lists of 'interesting' genes generated by genome-scale datasets. Previously, these gene lists had to be analyzed across several independent biological databases, often on a gene-by-gene basis. In contrast, several annotation databases, such as DAVID, integrate data from multiple functional databases and reveal underlying biological themes of large gene lists. While several such databases have been constructed for animals, none is currently available for the study of algae. Due to renewed interest in algae as potential sources of biofuels and the emergence of multiple algal genome sequences, a significant need has arisen for such a database to process the growing compendiums of algal genomic data. Description The Algal Functional Annotation Tool is a web-based comprehensive analysis suite integrating annotation data from several pathway, ontology, and protein family databases. The current version provides annotation for the model alga Chlamydomonas reinhardtii, and in the future will include additional genomes. The site allows users to interpret large gene lists by identifying associated functional terms, and their enrichment. Additionally, expression data for several experimental conditions were compiled and analyzed to provide an expression-based enrichment search. A tool to search for functionally-related genes based on gene expression across these conditions is also provided. Other features include dynamic visualization of genes on KEGG pathway maps and batch gene identifier conversion. Conclusions The Algal Functional Annotation Tool aims to provide an integrated data-mining environment for algal genomics by combining data from multiple annotation databases into a centralized tool. This site is designed to expedite the process of functional annotation and the interpretation of gene lists, such as those derived from high-throughput RNA-seq experiments. The tool is publicly available at http://pathways.mcdb.ucla.edu.