P-TEFb Activation by RBM7 Shapes a Pro-survival Transcriptional Response to Genotoxic Stress

DNA damage response (DDR) involves dramatic transcriptional alterations, the mechanisms of which remain ill defined. Here, we show that following genotoxic stress, the RNA-binding motif protein 7 (RBM7) stimulates RNA polymerase II (Pol II) transcription and promotes cell viability by activating the positive transcription elongation factor b (P-TEFb) via its release from the inhibitory 7SK small nuclear ribonucleoprotein (7SK snRNP). This is mediated by activation of p38MAPK, which triggers enhanced binding of RBM7 with core subunits of 7SK snRNP. In turn, P-TEFb relocates to chromatin to induce transcription of short units, including key DDR genes and multiple classes of non-coding RNAs. Critically, interfering with the axis of RBM7 and P-TEFb provokes cellular hypersensitivity to DNA-damage-inducing agents due to activation of apoptosis. Our work uncovers the importance of stress-dependent stimulation of Pol II pause release, which enables a pro-survival transcriptional response that is crucial for cell fate upon genotoxic insult.Peer reviewe

Deciphering oligodendrocyte lineage heterogeneity in development and disease

Oligodendrocytes are the myelinating glia of the central nervous system (CNS). By forming lipid-rich myelin membranes that spirally enwrap axonal segments, oligodendrocytes ensure rapid saltatory impulse propagation, structural integrity, and metabolic and trophic support to the nerves. Oligodendrocyte lineage (OLG)(1) arises from oligodendrocyte precursor cells (OPCs), which emerge from radial glia during early embryonic development in an intricate, spatiotemporally defined fashion. As highly migratory, OPCs rapidly populate the brain and the spinal cord and begin transitioning to differentiation-committed oligodendrocyte precursors (COPs). Shortly after birth, myelin synthesis occurs with the newly-formed and myelin-forming oligodendrocytes (NFOLs and MFOLs), which finally differentiate into mature oligodendrocytes (MOLs), completing the lineage trajectory. These populations possess unique transcriptional signatures reflecting their lineage differentiation state. Moreover, MOLs, in particular, comprise several transcriptionally distinct cell clusters, the functional significance of which remains unclear. In multiple sclerosis (MS), myelinating oligodendrocytes become the primary targets of chronic CNS inflammation. Consequently, the accumulating myelin damage materializes in the form of multifocal lesions, the pathological hallmarks of MS. In parallel, OLG respond to pathological environments by promoting myelin repair but also by expressing genes involved in complement formation, interferon response, and major histocompatibility complex (MHC) I and II, among others. Thus, their transcriptional heterogeneity(2) expands, and the emergence of the disease-associated (DA) states suggests that OLG might not be just passive targets of the immune system but potentially active players in immunomodulation. Nevertheless, when and where the DA states emerge, how they affect the remyelination, and whether they carry protective or detrimental functions in the context of MS remains to be determined. This thesis aimed to bridge the gap between the transcriptional diversity of OLG and its significance in health and disease. The work can be divided into three central topics: 1) Deciphering OLG heterogeneity in development and health (Papers I-II, i, iii); 2) Adaptations of spatial transcriptomics tools to investigate physiological and pathological phenomena (Papers II-IV, ii-iv); and 3) Elucidating the dynamics and potential roles of OLG heterogeneity in pathologies (Papers I, IV, and v). (1) Oligodendrocyte lineage cells, i.e., oligodendroglia (OLG). (2) The terms heterogeneity and diversity are used interchangeably in this thesis

HEXIM1-Tat chimera inhibits HIV-1 replication.

Transcription of HIV provirus is a key step of the viral cycle, and depends on the recruitment of the cellular positive transcription elongation factor b (P-TEFb) to the HIV promoter. The viral transactivator Tat can displace P-TEFb from the 7SK small nuclear ribonucleoprotein, where it is bound and inactivated by HEXIM1, and bring it to TAR, which allows the stalled RNA polymerase II to transition to successful transcription elongation. In this study, we designed a chimeric inhibitor of HIV transcription by combining functional domains from HEXIM1 and Tat. The chimera (HT1) potently inhibited gene expression from the HIV promoter, by competing with Tat for TAR and P-TEFb binding, while keeping the latter inactive. HT1 inhibited spreading infection as well as viral reactivation in lymphocyte T cell line models of HIV latency, with little effect on cellular transcription and metabolism. This proof-of-concept study validates an innovative approach to interfering with HIV transcription via peptide mimicry and competition for RNA-protein interactions. HT1 represents a new candidate for HIV therapy, or HIV cure via the proposed block and lock strategy

Sequencing of freshly produced RNA following exposure of cells to DNA damage-inducing UV mimetic 4-hydroxyaminoquinolone (4-NQO)

We used Illumina-HiSeq4000 to sequence 4sU-labelled RNA samples isolated from unchallenged and DNA damaged HeLa Flp-In cells, which revealed the nature of transcriptional response folowing genotoxic stress and the contribution of P-TEFb kinase in DNA damage-induced gene transcription.We mock treated or treated HeLa Flp-In cells for 1 or 2 hr with DMSO, 4-NQO, or 4-NQO + flavopiridol (FP) as indicated below. During the last 30 minutes of the treatments, we labeled the RNA or not with the nucleoside analogue 4-thiouridine (500µM 4sU) for 30 minutes.Bugai A, Quaresma AJC, Friedel CC, Lenasi T et al. P-TEFb Activation by RBM7 Shapes a Pro-survival Transcriptional Response to Genotoxic Stress. Mol Cell 2019 Apr 18;74(2):254-267.e10. PMID: 3082437

Cellular architecture of evolving neuroinflammatory lesions and multiple sclerosis pathology

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

Distinct oligodendrocyte populations have spatial preference and different responses to spinal cord injury

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

Spatial epigenome–transcriptome co-profiling of mammalian tissues

Emerging spatial technologies, including spatial transcriptomics and spatial epigenomics, are becoming powerful tools for profiling of cellular states in the tissue context1-5. However, current methods capture only one layer of omics information at a time, precluding the possibility of examining the mechanistic relationship across the central dogma of molecular biology. Here, we present two technologies for spatially resolved, genome-wide, joint profiling of the epigenome and transcriptome by cosequencing chromatin accessibility and gene expression, or histone modifications (H3K27me3, H3K27ac or H3K4me3) and gene expression on the same tissue section at near-single-cell resolution. These were applied to embryonic and juvenile mouse brain, as well as adult human brain, to map how epigenetic mechanisms control transcriptional phenotype and cell dynamics in tissue. Although highly concordant tissue features were identified by either spatial epigenome or spatial transcriptome we also observed distinct patterns, suggesting their differential roles in defining cell states. Linking epigenome to transcriptome pixel by pixel allows the uncovering of new insights in spatial epigenetic priming, differentiation and gene regulation within the tissue architecture. These technologies are of great interest in life science and biomedical research

Developmental origin of oligodendrocytes determines their function in the adult brain.

In the mouse embryonic forebrain, developmentally distinct oligodendrocyte progenitor cell populations and their progeny, oligodendrocytes, emerge from three distinct regions in a spatiotemporal gradient from ventral to dorsal. However, the functional importance of this oligodendrocyte developmental heterogeneity is unknown. Using a genetic strategy to ablate dorsally derived oligodendrocyte lineage cells (OLCs), we show here that the areas in which dorsally derived OLCs normally reside in the adult central nervous system become populated and myelinated by OLCs of ventral origin. These ectopic oligodendrocytes (eOLs) have a distinctive gene expression profile as well as subtle myelination abnormalities. The failure of eOLs to fully assume the role of the original dorsally derived cells results in locomotor and cognitive deficits in the adult animal. This study reveals the importance of developmental heterogeneity within the oligodendrocyte lineage and its importance for homeostatic brain function.We thank Dr Daniel Morrison, Matthew Gratian and Mark Bowen for technical support. Funding: This work was supported by the UK Multiple Sclerosis Society (RJMF/CZ), and The Adelson Medical Research Foundation (RJMF/DHR/K-AN/MR/DEB); the Swedish Research Council (grant 2015-03558 and 2019-01360), the European Union (Horizon 2020 Research and Innovation Programme/European Research Council Consolidator Grant EPIScOPE, grant agreement number 681893), the Swedish Brain Foundation (FO2017-0075), Knut and Alice Wallenberg Foundation (grant 2019-0107 and 2019-0089), The Swedish Society for Medical Research (SSMF, grant JUB2019), the G.ran Gustafsson Foundation for Research in Natural Sciences and Medicine, Strategic Research Programme in Neuroscience (StratNeuro), Ming Wai Lau Centre for Reparative Medicine and Karolinska Institutet (GC-B); the Medical Research Council (G0800575) (NK and WDR); a Project Grant from the National Centre for the Replacement, Refinement, & Reduction of Animals in Research (NC/N001451/1) (CJH,LLC, TJB, LMS); the Korean Health Technology R&D Project, Ministry of Health & Welfare, Republic of Korea (HI14C2173) (EK, TJB, LMS); the NIH (AG072305) (DB); and a core support grant from the Wellcome Trust and MRC to the Wellcome Trust-Medical Research Council Cambridge Stem Cell Institute (203151/Z/16/Z). SF was supported by a PhD studentship from the Wellcome Trust. TB is supported by a Junior Fellowship from the Loulou Foundation

Developmental origin of oligodendrocytes determines their function in the adult brain.

In the mouse embryonic forebrain, developmentally distinct oligodendrocyte progenitor cell populations and their progeny, oligodendrocytes, emerge from three distinct regions in a spatiotemporal gradient from ventral to dorsal. However, the functional importance of this oligodendrocyte developmental heterogeneity is unknown. Using a genetic strategy to ablate dorsally derived oligodendrocyte lineage cells (OLCs), we show here that the areas in which dorsally derived OLCs normally reside in the adult central nervous system become populated and myelinated by OLCs of ventral origin. These ectopic oligodendrocytes (eOLs) have a distinctive gene expression profile as well as subtle myelination abnormalities. The failure of eOLs to fully assume the role of the original dorsally derived cells results in locomotor and cognitive deficits in the adult animal. This study reveals the importance of developmental heterogeneity within the oligodendrocyte lineage and its importance for homeostatic brain function.We thank Dr Daniel Morrison, Matthew Gratian and Mark Bowen for technical support. Funding: This work was supported by the UK Multiple Sclerosis Society (RJMF/CZ), and The Adelson Medical Research Foundation (RJMF/DHR/K-AN/MR/DEB); the Swedish Research Council (grant 2015-03558 and 2019-01360), the European Union (Horizon 2020 Research and Innovation Programme/European Research Council Consolidator Grant EPIScOPE, grant agreement number 681893), the Swedish Brain Foundation (FO2017-0075), Knut and Alice Wallenberg Foundation (grant 2019-0107 and 2019-0089), The Swedish Society for Medical Research (SSMF, grant JUB2019), the G.ran Gustafsson Foundation for Research in Natural Sciences and Medicine, Strategic Research Programme in Neuroscience (StratNeuro), Ming Wai Lau Centre for Reparative Medicine and Karolinska Institutet (GC-B); the Medical Research Council (G0800575) (NK and WDR); a Project Grant from the National Centre for the Replacement, Refinement, & Reduction of Animals in Research (NC/N001451/1) (CJH,LLC, TJB, LMS); the Korean Health Technology R&D Project, Ministry of Health & Welfare, Republic of Korea (HI14C2173) (EK, TJB, LMS); the NIH (AG072305) (DB); and a core support grant from the Wellcome Trust and MRC to the Wellcome Trust-Medical Research Council Cambridge Stem Cell Institute (203151/Z/16/Z). SF was supported by a PhD studentship from the Wellcome Trust. TB is supported by a Junior Fellowship from the Loulou Foundation

Developmental origin of oligodendrocytes determines their function in the adult brain

Acknowledgements: We thank D. Morrison, M. Gratian and M. Bowen for technical support. This work was supported by the UK Multiple Sclerosis Society (RJMF/CZ); the Adelson Medical Research Foundation (RJMF/DHR/K-AN/MR/DEB); the Swedish Research Council (grants 2015-03558 and 2019-01360); the European Union (Horizon 2020 Research and Innovation Program/European Research Council Consolidator Grant EPIScOPE, grant agreement number 681893); the Swedish Brain Foundation (FO2017-0075); the Knut and Alice Wallenberg Foundation (grants 2019-0107 and 2019-0089); the Swedish Society for Medical Research (grant JUB2019); the Göran Gustafsson Foundation for Research in Natural Sciences and Medicine, Strategic Research Programme in Neuroscience (StratNeuro); the Ming Wai Lau Centre for Reparative Medicine and Karolinska Institutet (GC-B); the Medical Research Council (G0800575) (N.K. and W.D.R.); a project grant from the National Centre for the Replacement, Refinement and Reduction of Animals in Research (NC/N001451/1) (C.J.H., L.L.-C., T.J.B. and L.S.); the Korean Health Technology R&D Project; the Ministry of Health & Welfare, Republic of Korea (HI14C2173) (E.K., T.J.B. and L.S.); the National Institutes of Health (AG072305) (D.E.B.); and a core support grant from the Wellcome Trust and Medical Research Council to the Wellcome Trust-Medical Research Council Cambridge Stem Cell Institute (203151/Z/16/Z). S.F. was supported by a PhD studentship from the Wellcome Trust. T.B. is supported by a Junior Fellowship from the Loulou Foundation.In the mouse embryonic forebrain, developmentally distinct oligodendrocyte progenitor cell populations and their progeny, oligodendrocytes, emerge from three distinct regions in a spatiotemporal gradient from ventral to dorsal. However, the functional importance of this oligodendrocyte developmental heterogeneity is unknown. Using a genetic strategy to ablate dorsally derived oligodendrocyte lineage cells (OLCs), we show here that the areas in which dorsally derived OLCs normally reside in the adult central nervous system become populated and myelinated by OLCs of ventral origin. These ectopic oligodendrocytes (eOLs) have a distinctive gene expression profile as well as subtle myelination abnormalities. The failure of eOLs to fully assume the role of the original dorsally derived cells results in locomotor and cognitive deficits in the adult animal. This study reveals the importance of developmental heterogeneity within the oligodendrocyte lineage and its importance for homeostatic brain function