Project description:Analysis of the transcriptional correlates of FOXP3 expression in suppressive and non-suppressive primary human Treg cell clones. Individual CD4+CD25High or Cd4+CD25- T cells were isolated from human PBMCs and expanded in vitro. After 3 weeks of expansion, individual clones were analysed for FOXP3 expression and in vitro suppressive activity against freshly sorted allogeneic effector T cells. This study analyses the total RNA isolated from FOXP3+ clones with suppressive potency to their non-suppressive counterparts. The resutls of this study should provide insights into the molecular pathways linking FOXP3 expression to distinct aspects of Treg phenotype and function. Total RNA obtained from individual clones of primary human regulatory and effector CD4+T cells.
Project description:Alterations in distal regulatory elements that control gene expression underlie many diseases, including cancer. Epigenomic analyses of normal and diseased cells have produced correlative predictions for connections between dysregulated enhancers and target genes involved in pathogenesis. However, with few exceptions, these predicted cis-regulatory circuits remain untested. Here, we dissect cis-regulatory circuits that lead to overexpression of NEK6, a mitosis-associated kinase, in human B cell lymphoma. We find that only a minor subset of predicted enhancers is required for NEK6 expression. Indeed, an annotated super-enhancer is dispensable for NEK6 overexpression and for maintaining the architecture of a B cell-specific regulatory hub. A CTCF cluster serves as a chromatin and architectural boundary to block communication of the NEK6 regulatory hub with neighboring genes. Our findings emphasize that validation of predicted cis-regulatory circuits and super-enhancers is needed to prioritize transcriptional control elements as therapeutic targets.
Project description:Alterations in distal regulatory elements that control gene expression underlie many diseases, including cancer. Epigenomic analyses of normal and diseased cells have produced correlative predictions for connections between dysregulated enhancers and target genes involved in pathogenesis. However, with few exceptions, these predicted cis-regulatory circuits remain untested. Here, we dissect cis-regulatory circuits that lead to overexpression of NEK6, a mitosis-associated kinase, in human B cell lymphoma. We find that only a minor subset of predicted enhancers is required for NEK6 expression. Indeed, an annotated super-enhancer is dispensable for NEK6 overexpression and for maintaining the architecture of a B cell-specific regulatory hub. A CTCF cluster serves as a chromatin and architectural boundary to block communication of the NEK6 regulatory hub with neighboring genes. Our findings emphasize that validation of predicted cis-regulatory circuits and super-enhancers is needed to prioritize transcriptional control elements as therapeutic targets.
Project description:How neuronal connections are established and organized in functional networks determines brain function. In the mouse cerebral cortex, different classes of GABAergic interneurons exhibit specific connectivity patterns that underlie their ability to shape temporal dynamics and information processing. Much progress has been made parsing interneuron diversity, yet the molecular mechanisms by which interneuron subtype-specific connectivity motifs emerge remain unclear. Here we investigate transcriptional dynamics in different classes of interneurons during the formation of cortical inhibitory circuits. We found that whether the interneurons synapse with pyramidal neurons on their dendrites, soma, or axon initial segment is determined by synaptic molecules that are expressed in a subtype-specific manner. Thus cell-specific molecular programs that unfold during early postnatal development underlie the connectivity patterns of cortical interneurons.
Project description:The present study aims to characterise proteins bound to circuits collected from children on extracorporeal membrane oxygenation (ECMO). ECMO circuits were collected from 6 patients. Quantification of concentrations for proteins bound to the ECMO circuit samples was performed using bicinchoninic acid (BCA) protein assay, whilst characterisation of the bound proteome was performed using Data-independent acquisition Mass Spectrometry (DIA-MS). Reactome Over-representation Pathway Analyses tool was used to identify functional pathways corresponding to the common proteins bound to circuits across all patients.
Project description:Neuronal diversification is a fundamental step in the construction of functional neural circuits, yet how neurons generated from single progenitor domains acquire diverse subtype identities remains poorly understood. Here, we developed a stem cell-based system to model subtype diversification of V1 interneurons, a class of spinal neurons comprising four clades, each containing dozens of molecularly distinct neuronal subtypes. We demonstrate that V1 subtype diversity is not hard-wired and can be modified by extrinsic signals. Inhibition of Notch and activation of retinoid signaling results in a switch to MafA clade identity and enriches differentiation of Renshaw cells, a specialized MafA subtype that mediates recurrent inhibition of spinal motor neurons. We show that in vitro-generated Renshaw cells migrate into appropriate spinal laminae upon transplantation and form subtype-specific synapses with motor neurons. Our results demonstrate that stem cell-derived neuronal subtypes can be used to investigate mechanisms underlying neuronal subtype specification and circuit assembly.
Project description:Multiple Myeloma (MM) is the second most prevalent blood cancer (10%) after non-Hodgkin's lymphoma and represents approximately 1% of all cancers and 2% of all cancer deaths. MM is a complex disease characterized by numerous genetic alterations and recent mRNA profiling studies have attempted to subclassify the disease to build pathogenetic and prognostic models Correct classification of cancer patients into subtypes is a prerequisite for acute diagnosis and effective treatment. Here we use high accuracy, quantitative proteomics to segregate cancer subtypes directly at the level of expressed proteins. Multiple myeloma is a heterogeneous disease in its initial clinical features as well as its outcome. We investigated two subtypes of Multiple Myeloma: multiple myeloma associated with t(4;14) chromosomal translocation as well as t(4;14)-negative MM subtype. The t(4;14) translocation, found in 15% of multiple myeloma cases, indicates a poor prognosis. Super-SILAC mix was combined of cell lysates from 8 diverse cell lines labelled with heavy amino acids (Lys8 and Arg10). The Super-SILAC library is mixed with samples (lysates), and quantitative mass spectrometric analysis is performed using a setup consisting of LC and on a linear ion trap Orbitrap mass spectrometer with high mass accuracy at the MS and MS/MS levels. This way we have analysed 20 patient samples from present MM. Shotgun proteomic analysis yielded a proteome of more than 5300 quantified proteins overall (3000 on an average per individual sample). High accuracy of quantification allowed robust separation of subtypes by hierarchical clustering on the protein level.