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1.
Cell ; 184(10): 2618-2632.e17, 2021 05 13.
Article in English | MEDLINE | ID: mdl-33836156

ABSTRACT

The ongoing pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is currently affecting millions of lives worldwide. Large retrospective studies indicate that an elevated level of inflammatory cytokines and pro-inflammatory factors are associated with both increased disease severity and mortality. Here, using multidimensional epigenetic, transcriptional, in vitro, and in vivo analyses, we report that topoisomerase 1 (TOP1) inhibition suppresses lethal inflammation induced by SARS-CoV-2. Therapeutic treatment with two doses of topotecan (TPT), an FDA-approved TOP1 inhibitor, suppresses infection-induced inflammation in hamsters. TPT treatment as late as 4 days post-infection reduces morbidity and rescues mortality in a transgenic mouse model. These results support the potential of TOP1 inhibition as an effective host-directed therapy against severe SARS-CoV-2 infection. TPT and its derivatives are inexpensive clinical-grade inhibitors available in most countries. Clinical trials are needed to evaluate the efficacy of repurposing TOP1 inhibitors for severe coronavirus disease 2019 (COVID-19) in humans.


Subject(s)
COVID-19 Drug Treatment , DNA Topoisomerases, Type I/metabolism , SARS-CoV-2/metabolism , Topoisomerase I Inhibitors/pharmacology , Topotecan/pharmacology , Animals , COVID-19/enzymology , COVID-19/pathology , Chlorocebus aethiops , Humans , Inflammation/drug therapy , Inflammation/enzymology , Inflammation/pathology , Inflammation/virology , Mesocricetus , Mice , Mice, Transgenic , THP-1 Cells , Vero Cells
2.
Cell ; 181(7): 1502-1517.e23, 2020 06 25.
Article in English | MEDLINE | ID: mdl-32559462

ABSTRACT

RNA viruses are a major human health threat. The life cycles of many highly pathogenic RNA viruses like influenza A virus (IAV) and Lassa virus depends on host mRNA, because viral polymerases cleave 5'-m7G-capped host transcripts to prime viral mRNA synthesis ("cap-snatching"). We hypothesized that start codons within cap-snatched host transcripts could generate chimeric human-viral mRNAs with coding potential. We report the existence of this mechanism of gene origination, which we named "start-snatching." Depending on the reading frame, start-snatching allows the translation of host and viral "untranslated regions" (UTRs) to create N-terminally extended viral proteins or entirely novel polypeptides by genetic overprinting. We show that both types of chimeric proteins are made in IAV-infected cells, generate T cell responses, and contribute to virulence. Our results indicate that during infection with IAV, and likely a multitude of other human, animal and plant viruses, a host-dependent mechanism allows the genesis of hybrid genes.


Subject(s)
RNA Caps/genetics , RNA Virus Infections/genetics , Recombinant Fusion Proteins/genetics , 5' Untranslated Regions/genetics , Animals , Cattle , Cell Line , Cricetinae , Dogs , Humans , Influenza A virus/metabolism , Mice , Mutant Chimeric Proteins/genetics , Mutant Chimeric Proteins/metabolism , Open Reading Frames/genetics , RNA Caps/metabolism , RNA Virus Infections/metabolism , RNA Viruses/genetics , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA, Viral/metabolism , RNA-Dependent RNA Polymerase/genetics , RNA-Dependent RNA Polymerase/metabolism , Recombinant Fusion Proteins/metabolism , Transcription, Genetic/genetics , Viral Proteins/metabolism , Virus Replication/genetics
3.
Mol Cell ; 83(23): 4255-4271.e9, 2023 Dec 07.
Article in English | MEDLINE | ID: mdl-37995687

ABSTRACT

Endogenous retroviruses (ERVs) are remnants of ancient parasitic infections and comprise sizable portions of most genomes. Although epigenetic mechanisms silence most ERVs by generating a repressive environment that prevents their expression (heterochromatin), little is known about mechanisms silencing ERVs residing in open regions of the genome (euchromatin). This is particularly important during embryonic development, where induction and repression of distinct classes of ERVs occur in short temporal windows. Here, we demonstrate that transcription-associated RNA degradation by the nuclear RNA exosome and Integrator is a regulatory mechanism that controls the productive transcription of most genes and many ERVs involved in preimplantation development. Disrupting nuclear RNA catabolism promotes dedifferentiation to a totipotent-like state characterized by defects in RNAPII elongation and decreased expression of long genes (gene-length asymmetry). Our results indicate that RNA catabolism is a core regulatory module of gene networks that safeguards RNAPII activity, ERV expression, cell identity, and developmental potency.


Subject(s)
Endogenous Retroviruses , Endogenous Retroviruses/genetics , RNA, Nuclear , Epigenesis, Genetic , Heterochromatin , Gene Expression
4.
Nature ; 606(7916): 945-952, 2022 06.
Article in English | MEDLINE | ID: mdl-35732742

ABSTRACT

Amyotrophic lateral sclerosis (ALS) is a heterogenous neurodegenerative disorder that affects motor neurons and voluntary muscle control1. ALS heterogeneity includes the age of manifestation, the rate of progression and the anatomical sites of symptom onset. Disease-causing mutations in specific genes have been identified and define different subtypes of ALS1. Although several ALS-associated genes have been shown to affect immune functions2, whether specific immune features account for ALS heterogeneity is poorly understood. Amyotrophic lateral sclerosis-4 (ALS4) is characterized by juvenile onset and slow progression3. Patients with ALS4 show motor difficulties by the time that they are in their thirties, and most of them require devices to assist with walking by their fifties. ALS4 is caused by mutations in the senataxin gene (SETX). Here, using Setx knock-in mice that carry the ALS4-causative L389S mutation, we describe an immunological signature that consists of clonally expanded, terminally differentiated effector memory (TEMRA) CD8 T cells in the central nervous system and the blood of knock-in mice. Increased frequencies of antigen-specific CD8 T cells in knock-in mice mirror the progression of motor neuron disease and correlate with anti-glioma immunity. Furthermore, bone marrow transplantation experiments indicate that the immune system has a key role in ALS4 neurodegeneration. In patients with ALS4, clonally expanded TEMRA CD8 T cells circulate in the peripheral blood. Our results provide evidence of an antigen-specific CD8 T cell response in ALS4, which could be used to unravel disease mechanisms and as a potential biomarker of disease state.


Subject(s)
Amyotrophic Lateral Sclerosis , CD8-Positive T-Lymphocytes , Clone Cells , Amyotrophic Lateral Sclerosis/immunology , Amyotrophic Lateral Sclerosis/pathology , Animals , CD8-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/pathology , Clone Cells/pathology , DNA Helicases/genetics , DNA Helicases/metabolism , Gene Knock-In Techniques , Mice , Motor Neurons/pathology , Multifunctional Enzymes/genetics , Multifunctional Enzymes/metabolism , Mutation , RNA Helicases/genetics , RNA Helicases/metabolism
6.
Blood ; 129(14): 1901-1912, 2017 04 06.
Article in English | MEDLINE | ID: mdl-28179275

ABSTRACT

Granulocyte colony-stimulating factor (G-CSF) is used clinically to treat leukopenia and to enforce hematopoietic stem cell (HSC) mobilization to the peripheral blood (PB). However, G-CSF is also produced in response to infection, and excessive exposure reduces HSC repopulation capacity. Previous work has shown that dormant HSCs contain all the long-term repopulation potential in the bone marrow (BM), and that as HSCs accumulate a divisional history, they progressively lose regenerative potential. As G-CSF treatment also induces HSC proliferation, we sought to examine whether G-CSF-mediated repopulation defects are a result of increased proliferative history. To do so, we used an established H2BGFP label retaining system to track HSC divisions in response to G-CSF. Our results show that dormant HSCs are preferentially mobilized to the PB on G-CSF treatment. We find that this mobilization does not result in H2BGFP label dilution of dormant HSCs, suggesting that G-CSF does not stimulate dormant HSC proliferation. Instead, we find that proliferation within the HSC compartment is restricted to CD41-expressing cells that function with short-term, and primarily myeloid, regenerative potential. Finally, we show CD41 expression is up-regulated within the BM HSC compartment in response to G-CSF treatment. This emergent CD41Hi HSC fraction demonstrates no observable engraftment potential, but directly matures into megakaryocytes when placed in culture. Together, our results demonstrate that dormant HSCs mobilize in response to G-CSF treatment without dividing, and that G-CSF-mediated proliferation is restricted to cells with limited regenerative potential found within the HSC compartment.


Subject(s)
Cell Proliferation/drug effects , Gene Expression Regulation/drug effects , Granulocyte Colony-Stimulating Factor/pharmacology , Hematopoietic Stem Cell Mobilization , Hematopoietic Stem Cells/metabolism , Platelet Membrane Glycoprotein IIb/biosynthesis , Animals , Cell Proliferation/genetics , Gene Expression Regulation/genetics , Hematopoietic Stem Cells/cytology , Mice , Mice, Transgenic , Platelet Membrane Glycoprotein IIb/genetics
7.
J Exp Med ; 218(7)2021 07 05.
Article in English | MEDLINE | ID: mdl-33970190

ABSTRACT

DNMT3A encodes an enzyme that carries out de novo DNA methylation, which is essential for the acquisition of cellular identity and specialized functions during cellular differentiation. DNMT3A is the most frequently mutated gene in age-related clonal hematopoiesis. As such, mature immune cells harboring DNMT3A mutations can be readily detected in elderly persons. Most DNMT3A mutations associated with clonal hematopoiesis are heterozygous and predicted to cause loss of function, indicating that haploinsufficiency is the predominant pathogenic mechanism. Yet, the impact of DNMT3A haploinsufficiency on the function of mature immune cells is poorly understood. Here, we demonstrate that DNMT3A haploinsufficiency impairs the gain of DNA methylation at decommissioned enhancers, while simultaneously and unexpectedly impairing DNA demethylation of newly activated enhancers in mature human myeloid cells. The DNA methylation defects alter the activity of affected enhancers, leading to abnormal gene expression and impaired immune response. These findings provide insights into the mechanism of immune dysfunction associated with clonal hematopoiesis and acquired DNMT3A mutations.


Subject(s)
DNA (Cytosine-5-)-Methyltransferases/genetics , DNA Methylation/genetics , Haploinsufficiency/genetics , Immune System/immunology , Regulatory Sequences, Nucleic Acid/genetics , Cells, Cultured , DNA (Cytosine-5-)-Methyltransferases/immunology , DNA Methylation/immunology , DNA Methyltransferase 3A , Gene Expression/genetics , Gene Expression/immunology , Haploinsufficiency/immunology , Humans , Mutation/genetics , Mutation/immunology , Regulatory Sequences, Nucleic Acid/immunology
8.
bioRxiv ; 2020 Dec 01.
Article in English | MEDLINE | ID: mdl-33299999

ABSTRACT

The ongoing pandemic caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is currently affecting millions of lives worldwide. Large retrospective studies indicate that an elevated level of inflammatory cytokines and pro-inflammatory factors are associated with both increased disease severity and mortality. Here, using multidimensional epigenetic, transcriptional, in vitro and in vivo analyses, we report that Topoisomerase 1 (Top1) inhibition suppresses lethal inflammation induced by SARS-CoV-2. Therapeutic treatment with two doses of Topotecan (TPT), a FDA-approved Top1 inhibitor, suppresses infection-induced inflammation in hamsters. TPT treatment as late as four days post-infection reduces morbidity and rescues mortality in a transgenic mouse model. These results support the potential of Top1 inhibition as an effective host-directed therapy against severe SARS-CoV-2 infection. TPT and its derivatives are inexpensive clinical-grade inhibitors available in most countries. Clinical trials are needed to evaluate the efficacy of repurposing Top1 inhibitors for COVID-19 in humans.

9.
FEBS Lett ; 593(23): 3266-3287, 2019 12.
Article in English | MEDLINE | ID: mdl-31557312

ABSTRACT

Transcription factor (TF)-based reprogramming of somatic tissues holds great promise for regenerative medicine. Previously, we demonstrated that the TFs GATA2, GFI1B, and FOS convert mouse and human fibroblasts to hemogenic endothelial-like precursors that generate hematopoietic stem progenitor (HSPC)-like cells over time. This conversion is lacking in robustness both in yield and biological function. Herein, we show that inclusion of GFI1 to the reprogramming cocktail significantly expands the HSPC-like population. AFT024 coculture imparts functional potential to these cells and allows quantification of stem cell frequency. Altogether, we demonstrate an improved human hemogenic induction protocol that could provide a valuable human in vitro model of hematopoiesis for disease modeling and a platform for cell-based therapeutics. DATABASE: Gene expression data are available in the Gene Expression Omnibus (GEO) database under the accession number GSE130361.


Subject(s)
Cell Differentiation/genetics , Cellular Reprogramming/genetics , Hemangioblasts/cytology , Hematopoietic Stem Cells/cytology , Animals , Coculture Techniques/methods , Fibroblasts/cytology , Fibroblasts/metabolism , GATA2 Transcription Factor/genetics , Gene Expression Regulation, Developmental/genetics , Hemangioblasts/metabolism , Hematopoiesis/genetics , Hematopoietic Stem Cells/metabolism , Humans , Mice , Proto-Oncogene Proteins/genetics , Proto-Oncogene Proteins c-fos/genetics , Repressor Proteins/genetics , Transcription Factors/genetics
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