Identifying a novel role for the master regulator Tal1 in the Endothelial to Hematopoietic Transition.

Progress in the generation of Hematopoietic Stem and Progenitor Cells (HSPCs) in vitro and ex vivo has been built on the knowledge of developmental hematopoiesis, underscoring the importance of understanding this process. HSPCs emerge within the embryonic vasculature through an Endothelial-to-Hematopoietic Transition (EHT). The transcriptional regulator Tal1 exerts essential functions in the earliest stages of blood development, but is considered dispensable for the EHT. Nevertheless, Tal1 is expressed with its binding partner Lmo2 and it homologous Lyl1 in endothelial and transitioning cells at the time of EHT. Here, we investigated the function of these genes using a mouse embryonic-stem cell (mESC)-based differentiation system to model hematopoietic development. We showed for the first time that the expression of TAL1 in endothelial cells is crucial to ensure the efficiency of the EHT process and a sustained hematopoietic output. Our findings uncover an important function of Tal1 during the EHT, thus filling the current gap in the knowledge of the role of this master gene throughout the whole process of hematopoietic development.

Operation of a TCA cycle subnetwork in the mammalian nucleus.

Nucleic acid and histone modifications critically depend on the tricarboxylic acid (TCA) cycle for substrates and cofactors. Although a few TCA cycle enzymes have been reported in the nucleus, the corresponding pathways are considered to operate in mitochondria. Here, we show that a part of the TCA cycle is operational also in the nucleus. Using 13C-tracer analysis, we identified activity of glutamine-to-fumarate, citrate-to-succinate, and glutamine-to-aspartate routes in the nuclei of HeLa cells. Proximity labeling mass spectrometry revealed a spatial vicinity of the involved enzymes with core nuclear proteins. We further show nuclear localization of aconitase 2 and 2-oxoglutarate dehydrogenase in mouse embryonic stem cells. Nuclear localization of the latter enzyme, which produces succinyl-CoA, changed from pluripotency to a differentiated state with accompanying changes in the nuclear protein succinylation. Together, our results demonstrate operation of an extended metabolic pathway in the nucleus, warranting a revision of the canonical view on metabolic compartmentalization.

Single-cell transcriptomics identifies CD44 as a marker and regulator of endothelial to haematopoietic transition.

The endothelial to haematopoietic transition (EHT) is the process whereby haemogenic endothelium differentiates into haematopoietic stem and progenitor cells (HSPCs). The intermediary steps of this process are unclear, in particular the identity of endothelial cells that give rise to HSPCs is unknown. Using single-cell transcriptome analysis and antibody screening, we identify CD44 as a marker of EHT enabling us to isolate robustly the different stages of EHT in the aorta-gonad-mesonephros (AGM) region. This allows us to provide a detailed phenotypical and transcriptional profile of CD44-positive arterial endothelial cells from which HSPCs emerge. They are characterized with high expression of genes related to Notch signalling, TGFbeta/BMP antagonists, a downregulation of genes related to glycolysis and the TCA cycle, and a lower rate of cell cycle. Moreover, we demonstrate that by inhibiting the interaction between CD44 and its ligand hyaluronan, we can block EHT, identifying an additional regulator of HSPC development.

Human cyclin T1 expression ameliorates a T-cell-specific transcriptional limitation for HIV in transgenic rats, but is not sufficient for a spreading infection of prototypic R5 HIV-1 strains ex vivo.

BACKGROUND: Cells derived from native rodents have limits at distinct steps of HIV replication. Rat primary CD4 T-cells, but not macrophages, display a profound transcriptional deficit that is ameliorated by transient trans-complementation with the human Tat-interacting protein Cyclin T1 (hCycT1). RESULTS: Here, we generated transgenic rats that selectively express hCycT1 in CD4 T-cells and macrophages. hCycT1 expression in rat T-cells boosted early HIV gene expression to levels approaching those in infected primary human T-cells. hCycT1 expression was necessary, but not sufficient, to enhance HIV transcription in T-cells from individual transgenic animals, indicating that endogenous cellular factors are critical co-regulators of HIV gene expression in rats. T-cells from hCD4/hCCR5/hCycT1-transgenic rats did not support productive infection of prototypic wild-type R5 HIV-1 strains ex vivo, suggesting one or more significant limitation in the late phase of the replication cycle in this primary rodent cell type. Remarkably, we identify a replication-competent HIV-1 GFP reporter strain (R7/3 YU-2 Env) that displays characteristics of a spreading, primarily cell-to-cell-mediated infection in primary T-cells from hCD4/hCCR5-transgenic rats. Moreover, the replication of this recombinant HIV-1 strain was significantly enhanced by hCycT1 transgenesis. The viral determinants of this so far unique replicative ability are currently unknown. CONCLUSION: Thus, hCycT1 expression is beneficial to de novo HIV infection in a transgenic rat model, but additional genetic manipulations of the host or virus are required to achieve full permissivity.

The Nef protein of human immunodeficiency virus is a broad-spectrum modulator of chemokine receptor cell surface levels that acts independently of classical motifs for receptor endocytosis and Galphai signaling.

Chemokine receptors (CKRs) are important physiological mediators of immune defense, inflammatory responses, and angiogenesis, and they have also been implicated in a number of viral disease processes. Here, we report that the Nef protein of human immunodeficiency virus (HIV) reduces cell surface levels of eight different members of the CC- and CXC-family of CKRs by up to 92%. This broad-range activity required specific elements in HIV(SF2) Nef, including the proline-rich motif P73P76P79P82 as well as the acidic cluster motif E66E67E68E69, and Nef expression induced a marked perinuclear accumulation of CKRs. Surprisingly, receptor mutagenesis demonstrated that the cytoplasmic tail of CCR5 and CXCR4, which is critical for basal and ligand-mediated endocytosis, was completely dispensable for this Nef activity. In contrast, triple-mutation of the highly conserved DRY motif in the second intracellular CKR loop abolished the Nef-mediated down-regulation of CXCR4 independently of this motif's role in CKR binding to heterotrimeric G proteins and signaling via the Galphai subunit. Thus, we identify the lentiviral pathogenicity factor Nef as a unique and broad-range modulator of CKR cell surface levels. Nef uses a mechanism that is distinct from well-established pathways orchestrating CKR metabolism and offers an interesting tool to study the multifaceted biology of CKRs.

Single-cell transcriptomics reveals a new dynamical function of transcription factors during embryonic hematopoiesis.

Recent advances in single-cell transcriptomics techniques have opened the door to the study of gene regulatory networks (GRNs) at the single-cell level. Here, we studied the GRNs controlling the emergence of hematopoietic stem and progenitor cells from mouse embryonic endothelium using a combination of single-cell transcriptome assays. We found that a heptad of transcription factors (Runx1, Gata2, Tal1, Fli1, Lyl1, Erg and Lmo2) is specifically co-expressed in an intermediate population expressing both endothelial and hematopoietic markers. Within the heptad, we identified two sets of factors of opposing functions: one (Erg/Fli1) promoting the endothelial cell fate, the other (Runx1/Gata2) promoting the hematopoietic fate. Surprisingly, our data suggest that even though Fli1 initially supports the endothelial cell fate, it acquires a pro-hematopoietic role when co-expressed with Runx1. This work demonstrates the power of single-cell RNA-sequencing for characterizing complex transcription factor dynamics.

Activation of the TGFß pathway impairs endothelial to haematopoietic transition.

The endothelial to haematopoietic transition (EHT) is a key developmental process where a drastic change of endothelial cell morphology leads to the formation of blood stem and progenitor cells during embryogenesis. As TGFß signalling triggers a similar event during embryonic development called epithelial to mesenchymal transition (EMT), we hypothesised that TGFß activity could play a similar role in EHT as well. We used the mouse embryonic stem cell differentiation system for in vitro recapitulation of EHT and performed gain and loss of function analyses of the TGFß pathway. Quantitative proteomics analysis showed that TGFß treatment during EHT increased the secretion of several proteins linked to the vascular lineage. Live cell imaging showed that TGFß blocked the formation of round blood cells. Using gene expression profiling we demonstrated that the TGFß signalling activation decreased haematopoietic genes expression and increased the transcription of endothelial and extracellular matrix genes as well as EMT markers. Finally we found that the expression of the transcription factor Sox17 was up-regulated upon TGFß signalling activation and showed that its overexpression was enough to block blood cell formation. In conclusion we showed that triggering the TGFß pathway does not enhance EHT as we hypothesised but instead impairs it.

Microfluidic-based enzymatic on-chip labeling of miRNAs.

Small noncoding RNAs (sncRNAs) have moved from oddity to recognized important players in gene regulation. Next generation sequencing approaches discover more and more such molecules from a variety of different groups, but flexible tools translating this sequence information into affordable high-throughput assays are missing. Here we describe a microfluidic primer extension assay (MPEA) for the detection of sncRNAs on highly flexible microfluidic microarrays which combines several beneficial parameters: it can effortless incorporate any new sequence information; it is sensitive enough to work with as little as 20ng of total RNA and has a high level of specificity owing to a combination of a conventional hybridization assay and an enzymatic elongation step. Importantly, no labeling step is needed before hybridization and - because of its high sensitivity - no amplification is required. Both aspects ensure that no bias is introduced by such processes. Although the assay is exemplified with miRNAs, the flexibility of the technology platform allows the analysis of any type of sncRNA, such as piRNAs.

Polymorphisms of the TGF-beta1 gene are not associated with bronchial asthma in Caucasian children.

The chromosomal region 19q13 has been found in linkage to allergic diseases in several genome-wide linkage screens. One candidate gene within this region is the gene coding for TGF-beta1. Transforming growth factor (TGF)-beta acts as an anti-inflammatory cytokine suppressing allergic inflammation and hyper-reactivity. However, in ongoing inflammation of the lungs it can induce fibrosis and airway remodelling as seen in chronic asthma. Several polymorphisms within TGF-beta1 have been identified and one, -C509T, has been shown to be in association with elevated immunoglobulin E levels and severe bronchial asthma in different populations. However, other studies failed to confirm the association. The present study investigated two polymorphisms within the gene coding for TGF-beta1, -C509T and G915C, and for their potential association with bronchial asthma in Caucasian children. Genotyping of these polymorphisms was performed by means of restriction fragment length polymorphisms in a population of 231 asthmatic children and a control population of 269 individuals. Statistical analyses made use of the Armitage's trend test. In addition haplotypes were calculated by arlequin. None of the two polymorphisms showed association with bronchial asthma. They were found to be in linkage disequilibrium. We conclude from our data that TGF-beta1 is unlikely to represent a major gene in the development of bronchial asthma in the Caucasian population.

Association study of the IL13 variant Arg110Gln in atopic diseases and juvenile idiopathic arthritis.

BACKGROUND: It has previously been shown that various inflammatory diseases, such as diabetes mellitus, bronchial asthma, chronic inflammatory bowel diseases, and rheumatoid arthritis, are in some circumstances genetically linked to the same chromosomal regions. Consequently, common genes underlying the pathogenetics of these diseases have been proposed. Chronic inflammatory disorders can be subdivided by their predominant immune response, either TH1 or TH2. For example, juvenile idiopathic arthritis (JIA) is a TH1 disease, and bronchial asthma is a TH2 disease. OBJECTIVES: The present study investigated the polymorphism Arg110Gln within the IL13 gene, a strong TH2 cytokine. We attempted to determine whether it is associated with these 2 diseases and whether this would reflect the TH1/TH2 paradigm. METHODS: Arg110Gln was typed in 4 different populations: asthmatic children, atopic children, children with JIA, and a control population. Statistical analysis was performed by using logistic and linear regression analysis of serum IgE levels and the Armitage trend test. RESULTS: The variant Gln110 was shown to be associated with increased total serum IgE levels in our atopic population (P =.006) and was weakly associated with bronchial asthma (P =.04). There was no association of the variant with JIA when compared with the control population. However, the variant Gln110 was significantly less frequent in children with JIA compared with its presence in children with bronchial asthma (P =.007). CONCLUSION: This is the first study to compare the same gene variant in TH1 and TH2 chronic inflammatory diseases. The results suggest that the same gene variant might protect from one disease and make an individual susceptible to the other.