| 1. |
- Bárcenas-Walls, José Ramón, et al.
(författare)
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Nano-CUT&Tag for multimodal chromatin profiling at single-cell resolution
- 2024
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Ingår i: Nature Protocols. - 1754-2189 .- 1750-2799. ; 19:3, s. 791-830
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Tidskriftsartikel (refereegranskat)abstract
- The ability to comprehensively analyze the chromatin state with single-cell resolution is crucial for understanding gene regulatory principles in heterogenous tissues or during development. Recently, we developed a nanobody-based single-cell CUT&Tag (nano-CT) protocol to simultaneously profile three epigenetic modalities-two histone marks and open chromatin state-from the same single cell. Nano-CT implements a new set of secondary nanobody-Tn5 fusion proteins to direct barcoded tagmentation by Tn5 transposase to genomic targets labeled by primary antibodies raised in different species. Such nanobody-Tn5 fusion proteins are currently not commercially available, and their in-house production and purification can be completed in 3-4 d by following our detailed protocol. The single-cell indexing in nano-CT is performed on a commercially available platform, making it widely accessible to the community. In comparison to other multimodal methods, nano-CT stands out in data complexity, low sample requirements and the flexibility to choose two of the three modalities. In addition, nano-CT works efficiently with fresh brain samples, generating multimodal epigenomic profiles for thousands of brain cells at single-cell resolution. The nano-CT protocol can be completed in just 3 d by users with basic skills in standard molecular biology and bioinformatics, although previous experience with single-cell assay for transposase-accessible chromatin using sequencing (scATAC-seq) is beneficial for more in-depth data analysis. As a multimodal assay, nano-CT holds immense potential to reveal interactions of various chromatin modalities, to explore epigenetic heterogeneity and to increase our understanding of the role and interplay that chromatin dynamics has in cellular development. This protocol involves profiling gene regulatory dynamics in complex tissues at the single-cell level. This is achieved by generating two nanobody-Tn5 fusion proteins to recognize primary antibodies against widespread histone modifications.The nanobody-Tn5 fusions are combined with ATAC-seq to simultaneously profile three epigenetic modalities from the same single cell, thousands of cells at the same time. A bioinformatic pipeline, Nanoscope, for seamless analysis of nano-CT datasets is also described. Mapping of chromatin states at single-cell resolution is still challenging. This protocol describes nano-CT, a novel method to simultaneously characterize up to three epigenetic modalities at single-cell resolution.
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| 2. |
- Castelo-Branco, Gonçalo de Sá e Sousa de
(författare)
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Wnt signalling in the development of ventral midbrain dopaminergic neurons
- 2005
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Dopaminergic (DA) neurons in the ventral midbrain (VM) are one of the major cell types lost in Parkinson's disease (PD). Proof-of-principle exists for cell replacement therapies for PD, but wider application is halted by the unavailability of abundant sources of DA neurons. Stem cells might constitute one of these sources. However, efficient protocols promoting their specific differentiation into a DA neuronal phenotype are required. In this thesis, evidence is presented for a major contribution of the Wnt family of glycolipoproteins in the development of VM DA neurons. In the first study, we found that Wnt-1, Wnt-3a, and Wnt-5a expression is differentially regulated during VM development and that partially purified Wnts distinctively regulate DA neuron development. Wnt-1 and Wnt-5a increased the number of VM DA neurons in rat embryonic day (E) 14.5 precursor cultures by two distinct mechanisms. Wnt-1 predominantly increased the proliferation of nuclear receptor-related factor 1 (Nurr1) positive precursors. In contrast, Wnt-5a primarily increased the proportion of Nurr1 precursors that acquired a neuronal DA (tyrosine hydroxylase (TH) positive) phenotype. These findings indicate that Writs are key regulators of proliferation and differentiation of DA precursors during VM neurogenesis and that different Wnts have specific and unique activity profiles. In a second study, we found that VM glia, but not cortical glia, secrete high levels of Wnt-5a and increase the differentiation of Nurr1 precursors into DA neurons. Post-natal VM glial cells, including astrocytes and radial glia, were found to retain a specific regional transcriptional code and to be the source of instructive signals required by neural precursors or stem/ progenitor cells (neurospheres) to acquire a DA phenotype. Moreover, Wnt-5a and VM glia were able to induce a TH positive phenotype in cortical precursors expressing Nurr1. The Wnt signal transduction can be modulated by secreted ligands of the dickkopf (Dkk) family, which can bind with high specificity to the low density lipoprotein receptor related protein (LRP) -5 and -6. In the third study, we investigated the role of Dkks and LRPs in the induction of a DA phenotype. We found a dynamic expression pattern in the developing rat VM, with a sequential expression of Dkk-1, Dkk-2 and LRP-6 (both peaking at the time of DA neurogenesis) and Dkk-3. Dkks distinctively regulated DA differentiation from E14.5 VM precursors. Dkk-1, a Wnt/beta-catenin signalling inhibitor, decreased the number of TH positive neurons, while Dkk-2 increased the conversion of Nurr1 positive precursors into DA neurons. LRP-6 null mutant mice showed a delay in DA differentiation, with 50% less DA neurons at E11.5 and a 25% reduction at E13.5. Our results identified Dkk-2 and LRP-6 as key regulators of DA differentiation in the developing VM. Wnts and Dkks could thus serve as potential tools in the treatment of PD. However, Wnts are poorly soluble and act mainly as short range signalling molecules. In our fourth study, we investigated whether intracellular canonical Wnt signalling components could modulate the development of DA neurons. Two chemical inhibitors of glycogen synthase kinase-3 beta (GSK3beta), indirubin-3-monoxime (I3M) and kenpaullone (KP), were found to stabilize beta-catenin and to increase DA differentiation in VM precursor cultures, thereby mimicking an effect of Wnts. The three to five fold increase in DA differentiation of precursor cells by GSK-3beta inhibitors suggests that such compounds may contribute to improve stem/precursor cell therapy approaches to PD.
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| 3. |
- Floriddia, Elisa M., et al.
(författare)
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Distinct oligodendrocyte populations have spatial preference and different responses to spinal cord injury
- 2020
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Ingår i: Nature Communications. - : Springer Science and Business Media LLC. - 2041-1723. ; 11:1
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Tidskriftsartikel (refereegranskat)abstract
- Mature oligodendrocytes (MOLs) show transcriptional heterogeneity, the functional consequences of which are unclear. MOL heterogeneity might correlate with the local environment or their interactions with different neuron types. Here, we show that distinct MOL populations have spatial preference in the mammalian central nervous system (CNS). We found that MOL type 2 (MOL2) is enriched in the spinal cord when compared to the brain, while MOL types 5 and 6 (MOL5/6) increase their contribution to the OL lineage with age in all analyzed regions. MOL2 and MOL5/6 also have distinct spatial preference in the spinal cord regions where motor and sensory tracts run. OL progenitor cells (OPCs) are not specified into distinct MOL populations during development, excluding a major contribution of OPC intrinsic mechanisms determining MOL heterogeneity. In disease, MOL2 and MOL5/6 present different susceptibility during the chronic phase following traumatic spinal cord injury. Our results demonstrate that the distinct MOL populations have different spatial preference and different responses to disease.
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| 4. |
- Kukanja, Petra, et al.
(författare)
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Cellular architecture of evolving neuroinflammatory lesions and multiple sclerosis pathology
- 2024
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Ingår i: Cell. - : Cell Press. - 0092-8674 .- 1097-4172. ; 187:8, s. 1990-2009
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Tidskriftsartikel (refereegranskat)abstract
- Multiple sclerosis (MS) is a neurological disease characterized by multifocal lesions and smoldering pathology. Although single -cell analyses provided insights into cytopathology, evolving cellular processes underlying MS remain poorly understood. We investigated the cellular dynamics of MS by modeling temporal and regional rates of disease progression in mouse experimental autoimmune encephalomyelitis (EAE). By performing single -cell spatial expression profiling using in situ sequencing (ISS), we annotated disease neighborhoods and found centrifugal evolution of active lesions. We demonstrated that disease -associated (DA)-glia arise independently of lesions and are dynamically induced and resolved over the disease course. Single -cell spatial mapping of human archival MS spinal cords confirmed the differential distribution of homeostatic and DA-glia, enabled deconvolution of active and inactive lesions into sub -compartments, and identified new lesion areas. By establishing a spatial resource of mouse and human MS neuropathology at a single -cell resolution, our study unveils the intricate cellular dynamics underlying MS.
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| 5. |
- Marques, Sueli, et al.
(författare)
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Transcriptional Convergence of Oligodendrocyte Lineage Progenitors during Development
- 2018
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Ingår i: Developmental Cell. - : Elsevier BV. - 1878-1551 .- 1534-5807. ; 46:4, s. 504-517
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Tidskriftsartikel (refereegranskat)abstract
- Pdgfra+ oligodendrocyte precursor cells (OPCs) arise in distinct specification waves during embryogenesis in the central nervous system (CNS). It is unclear whether there is a correlation between these waves and different oligodendrocyte (OL) states at adult stages. Here, we present bulk and single-cell transcriptomics resources providing insights on how transitions between these states occur. We found that post-natal OPCs from brain and spinal cord present similar transcriptional signatures. Moreover, post-natal OPC progeny of E13.5 Pdgfra+ cells present electrophysiological and transcriptional profiles similar to OPCs derived from subsequent specification waves, indicating that Pdgfra+ pre-OPCs rewire their transcriptional network during development. Single-cell RNA-seq and lineage tracing indicates that a subset of E13.5 Pdgfra+ cells originates cells of the pericyte lineage. Thus, our results indicate that embryonic Pdgfra+ cells in the CNS give rise to distinct post-natal cell lineages, including OPCs with convergent transcriptional profiles in different CNS regions.
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| 6. |
- Meijer, Mandy, et al.
(författare)
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Epigenomic priming of immune genes implicates oligodendroglia in multiple sclerosis susceptibility
- 2022
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Ingår i: Neuron. - : Elsevier. - 0896-6273 .- 1097-4199. ; 110:7, s. 1193-1210
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Tidskriftsartikel (refereegranskat)abstract
- Multiple sclerosis (MS) is characterized by a targeted attack on oligodendroglia (OLG) and myelin by immune cells, which are thought to be the main drivers of MS susceptibility. We found that immune genes exhibit a primed chromatin state in single mouse and human OLG in a non-disease context, compatible with transitions to immune-competent states in MS. We identified BACH1 and STAT1 as transcription factors involved in immune gene regulation in oligodendrocyte precursor cells (OPCs). A subset of immune genes presents bivalency of H3K4me3/H3K27me3 in OPCs, with Polycomb inhibition leading to their increased activation upon interferon gamma (IFN-g) treatment. Some MS susceptibility single-nucleotide polymorphisms (SNPs) overlap with these regulatory regions in mouse and human OLG. Treatment of mouse OPCs with IFN-g leads to chromatin architecture remodeling at these loci and altered expression of interacting genes. Thus, the susceptibility for MS may involve OLG, which therefore constitutes novel targets for immunological based therapies for MS.
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| 7. |
- Samudyata, Samudyata, et al.
(författare)
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Interaction of Sox2 with RNA binding proteins in mouse embryonic stem cells
- 2019
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Ingår i: Experimental Cell Research. - : Elsevier BV. - 0014-4827 .- 1090-2422. ; 381:1, s. 129-138
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Tidskriftsartikel (refereegranskat)abstract
- Sox2 is a master transcriptional regulator of embryonic development. In this study, we determined the protein interactome of Sox2 in the chromatin and nucleoplasm of mouse embryonic stem (mES) cells. Apart from canonical interactions with pluripotency-regulating transcription factors, we identified interactions with several chromatin modulators, including members of the heterochromatin protein 1 (HP1) family, suggesting a role for Sox2 in chromatin-mediated transcriptional repression. Sox2 was also found to interact with RNA binding proteins (RBPs), including proteins involved in RNA processing. RNA immunoprecipitation followed by sequencing revealed that Sox2 associates with different messenger RNAs, as well as small nucleolar RNA Snord34 and the non-coding RNA 7SK. 7SK has been shown to regulate transcription at gene regulatory regions, which could suggest a functional interaction with Sox2 for chromatin recruitment. Nevertheless, we found no evidence of Sox2 modulating recruitment of 7SK to chromatin when examining 7SK chromatin occupancy by Chromatin Isolation by RNA Purification (ChIRP) in Sox2 depleted mES cells. In addition, knockdown of 7SK in mES cells did not lead to any change in Sox2 occupancy at 7SK-regulated genes. Thus, our results show that Sox2 extensively interacts with RBPs, and suggest that Sox2 and 7SK co-exist in a ribonucleoprotein complex whose function is not to regulate chromatin recruitment, but could rather regulate other processes in the nucleoplasm.
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| 8. |
- Vaid, Roshan, 1987-
(författare)
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Transcription regulation across levels of chromatin organization
- 2020
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Konstnärligt arbete (övrigt vetenskapligt/konstnärligt)abstract
- With advancements in high-throughput sequencing and high-resolution microscopy techniques, the significance of genome organization in transcription regulation is rapidly unveiling. Nonetheless, we are yet far from completely understanding this key relationship. In this thesis, I utilize some of the cutting-edge high-throughput sequencing techniques along with the power of Drosophila as a model to understand some of the molecular mechanisms active at various levels of chromatin organization that influence gene expression. At the level of DNA sequence, our analysis revealed occupancy of CBP/p300, a transcription co-activator and histone acetyltransferase at insulator regions genome wide, we also identified a novel role of CBP as a barrier to heterochromatin spreading at these insulator regions.To gain insight into transcription regulation at level of nucleosomes, we perturbed histone modifications either chemically, by inhibiting histone deacetylases (HDACs), or genetically, by creating Drosophila mutants for the H3K14 residue. We found that the immediate transcriptional response to HDAC inhibition is only up-regulation (96 genes). Moreover, our results suggest that histone acetylation stimulates transcription by releasing promoter-proximal paused Pol II into elongation. In another study we discovered that acetylation of histone 3 lysine 14, H3K14ac, decorates some genes that lack canonical histone marks, thereby constituting a unique chromatin state. Further, Drosophila mutants expressing only H3K14R histones revealed that this residue is vital for expression of these genes, for animal survival and for developmental patterning.To further understand gene regulation during tissue specification in early embryo development, we utilized dorsoventral (DV) patterning as a model along with PRO-seq, ATAC-seq and ChIP-seq. We identified zygotically transcribed DV patterning genes that are spatially and temporally resolved. Most interestingly, the DV patterning genes are all highly paused and promoter proximal paused Pol II is released into active elongation in a tissue-specific manner. Finally our single cell RNA-seq (scRNA-seq) and HiC data in DV mutants revealed that differential regulation of gene expression occurs independently of differences in higher-order chromatin organization.Collectively, we provide evidence that transcription is being modulated at various levels of chromatin organization and our results suggest that histone modifications but not higher order chromatin organization influence transcriptional output.
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| 9. |
- van Bruggen, David, et al.
(författare)
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Developmental landscape of human forebrain at a single-cell level identifies early waves of oligodendrogenesis
- 2022
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Ingår i: Developmental Cell. - : Elsevier BV. - 1534-5807 .- 1878-1551. ; 57:11, s. 1421-1436, 1421-1436.e1-e5
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Tidskriftsartikel (refereegranskat)abstract
- Oligodendrogenesis in the human central nervous system has been observed mainly at the second trimester of gestation, a much later developmental stage compared to oligodendrogenesis in mice. Here, we characterize the transcriptomic neural diversity in the human forebrain at post-conception weeks (PCW) 8–10. Using single-cell RNA sequencing, we find evidence of the emergence of a first wave of oligodendrocyte lineage cells as early as PCW 8, which we also confirm at the epigenomic level through the use of single-cell ATAC-seq. Using regulatory network inference, we predict key transcriptional events leading to the specification of oligodendrocyte precursor cells (OPCs). Moreover, by profiling the spatial expression of 50 key genes through the use of in situ sequencing (ISS), we identify regions in the human ventral fetal forebrain where oligodendrogenesis first occurs. Our results indicate evolutionary conservation of the first wave of oligodendrogenesis between mice and humans and describe regulatory mechanisms involved in human OPC specification.
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| 10. |
- Zeisel, Amit, et al.
(författare)
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Cell types in the mouse cortex and hippocampus revealed by single-cell RNA-seq
- 2015
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Ingår i: Science. - : American Association for the Advancement of Science (AAAS). - 0036-8075 .- 1095-9203. ; 347:6226, s. 1138-1142
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Tidskriftsartikel (refereegranskat)abstract
- The mammalian cerebral cortex supports cognitive functions such as sensorimotor integration, memory, and social behaviors. Normal brain function relies on a diverse set of differentiated cell types, including neurons, glia, and vasculature. Here, we have used large-scale single-cell RNA sequencing (RNA-seq) to classify cells in the mouse somatosensory cortex and hippocampal CA1 region. We found 47 molecularly distinct subclasses, comprising all known major cell types in the cortex. We identified numerous marker genes, which allowed alignment with known cell types, morphology, and location. We found a layer I interneuron expressing Pax6 and a distinct postmitotic oligodendrocyte subclass marked by Itpr2. Across the diversity of cortical cell types, transcription factors formed a complex, layered regulatory code, suggesting a mechanism for the maintenance of adult cell type identity.
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