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1.
Methods Mol Biol ; 2351: 165-179, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34382189

RESUMO

Targeted chromatin capture (T2C) is a 3C-based method and is used to study the 3D chromatin organization, interactomes and structural changes associated with gene regulation, progression through the cell cycle, and cell survival and development. Low input targeted chromatin capture (low-T2C) is an optimized version of the T2C protocol for low numbers of cells. Here, we describe the protocol for low-T2C, including all experimental steps and bioinformatics tools in detail.


Assuntos
Montagem e Desmontagem da Cromatina , Cromatina/genética , Biologia Computacional/métodos , Cromatina/química , Cromatina/metabolismo , Mapeamento Cromossômico , Regulação da Expressão Gênica , Biblioteca Gênica , Genômica/métodos , Reprodutibilidade dos Testes
2.
Nat Cell Biol ; 23(8): 881-893, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34326481

RESUMO

The 11 zinc finger (ZF) protein CTCF regulates topologically associating domain formation and transcription through selective binding to thousands of genomic sites. Here, we replaced endogenous CTCF in mouse embryonic stem cells with green-fluorescent-protein-tagged wild-type or mutant proteins lacking individual ZFs to identify additional determinants of CTCF positioning and function. While ZF1 and ZF8-ZF11 are not essential for cell survival, ZF8 deletion strikingly increases the DNA binding off-rate of mutant CTCF, resulting in reduced CTCF chromatin residence time. Loss of ZF8 results in widespread weakening of topologically associating domains, aberrant gene expression and increased genome-wide DNA methylation. Thus, important chromatin-templated processes rely on accurate CTCF chromatin residence time, which we propose depends on local sequence and chromatin context as well as global CTCF protein concentration.


Assuntos
Fator de Ligação a CCCTC/fisiologia , Cromatina/metabolismo , Metilação de DNA , Regulação da Expressão Gênica , Genoma , Células-Tronco Pluripotentes/fisiologia , Animais , Fator de Ligação a CCCTC/genética , Feminino , Proteínas de Fluorescência Verde/genética , Masculino , Camundongos , Mitose , Células-Tronco Embrionárias Murinas , Mutação , Células-Tronco Pluripotentes/metabolismo , Fatores de Tempo , Elongação da Transcrição Genética
3.
Annu Rev Genomics Hum Genet ; 22: 127-146, 2021 08 31.
Artigo em Inglês | MEDLINE | ID: mdl-33951408

RESUMO

Accurate control of gene expression in the right cell at the right moment is of fundamental importance to animal development and homeostasis. At the heart of gene regulation lie the enhancers, a class of gene regulatory elements that ensures precise spatiotemporal activation of gene transcription. Mammalian genomes are littered with enhancers, which are frequently organized in cooperative clusters such as locus control regions and superenhancers. Here, we discuss our current knowledge of enhancer biology, including an overview of the discovery of the various enhancer subsets and the mechanistic models used to explain their gene regulatory function.

4.
J Infect Dis ; 223(12): 2020-2028, 2021 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-34043806

RESUMO

Effective clinical intervention strategies for coronavirus disease 2019 (COVID-19) are urgently needed. Although several clinical trials have evaluated use of convalescent plasma containing virus-neutralizing antibodies, levels of neutralizing antibodies are usually not assessed and the effectiveness has not been proven. We show that hamsters treated prophylactically with a 1:2560 titer of human convalescent plasma or a 1:5260 titer of monoclonal antibody were protected against weight loss, had a significant reduction of virus replication in the lungs, and showed reduced pneumonia. Interestingly, this protective effect was lost with a titer of 1:320 of convalescent plasma. These data highlight the importance of screening plasma donors for high levels of neutralizing antibodies. Our data show that prophylactic administration of high levels of neutralizing antibody, either monoclonal or from convalescent plasma, prevent severe SARS-CoV-2 pneumonia in a hamster model, and could be used as an alternative or complementary to other antiviral treatments for COVID-19.


Assuntos
Anticorpos Monoclonais/uso terapêutico , Anticorpos Neutralizantes/uso terapêutico , COVID-19/terapia , Pulmão/patologia , SARS-CoV-2/imunologia , Replicação Viral/efeitos dos fármacos , Animais , Anticorpos Monoclonais/administração & dosagem , Anticorpos Neutralizantes/administração & dosagem , COVID-19/imunologia , Cricetinae , Modelos Animais de Doenças , Humanos , Imunização Passiva , Pulmão/efeitos dos fármacos , Eliminação de Partículas Virais/efeitos dos fármacos , Perda de Peso/efeitos dos fármacos
5.
Nat Commun ; 12(1): 1715, 2021 03 17.
Artigo em Inglês | MEDLINE | ID: mdl-33731724

RESUMO

The coronavirus spike glycoprotein, located on the virion surface, is the key mediator of cell entry and the focus for development of protective antibodies and vaccines. Structural studies show exposed sites on the spike trimer that might be targeted by antibodies with cross-species specificity. Here we isolated two human monoclonal antibodies from immunized humanized mice that display a remarkable cross-reactivity against distinct spike proteins of betacoronaviruses including SARS-CoV, SARS-CoV-2, MERS-CoV and the endemic human coronavirus HCoV-OC43. Both cross-reactive antibodies target the stem helix in the spike S2 fusion subunit which, in the prefusion conformation of trimeric spike, forms a surface exposed membrane-proximal helical bundle. Both antibodies block MERS-CoV infection in cells and provide protection to mice from lethal MERS-CoV challenge in prophylactic and/or therapeutic models. Our work highlights an immunogenic and vulnerable site on the betacoronavirus spike protein enabling elicitation of antibodies with unusual binding breadth.


Assuntos
Anticorpos Monoclonais Humanizados/imunologia , Betacoronavirus/imunologia , Epitopos/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Animais , Anticorpos Monoclonais Humanizados/uso terapêutico , Anticorpos Neutralizantes/imunologia , Anticorpos Neutralizantes/uso terapêutico , Anticorpos Antivirais/imunologia , Betacoronavirus/classificação , Camelus , Infecções por Coronavirus/tratamento farmacológico , Infecções por Coronavirus/virologia , Reações Cruzadas , Epitopos/química , Epitopos/genética , Humanos , Camundongos , Conformação Proteica , Subunidades Proteicas , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/genética
6.
Biomaterials ; 268: 120580, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33321292

RESUMO

Ex vivo gene editing of CD34+ hematopoietic stem and progenitor cells (HSPCs) offers great opportunities to develop new treatments for a number of malignant and non-malignant diseases. Efficient gene-editing in HSPCs has been achieved using electroporation and/or viral transduction to deliver the CRISPR-complex, but cellular toxicity is a drawback of currently used methods. Nanoparticle (NP)-based gene-editing strategies can further enhance the gene-editing potential of HSPCs and provide a delivery system for in vivo application. Here, we developed CRISPR/Cas9-PLGA-NPs efficiently encapsulating Cas9 protein, single gRNA and a fluorescent probe. The initial 'burst' of Cas9 and gRNA release was followed by a sustained release pattern. CRISPR/Cas9-PLGA-NPs were taken up and processed by human HSPCs, without inducing cellular cytotoxicity. Upon escape from the lysosomal compartment, CRISPR/Cas9-PLGA-NPs-mediated gene editing of the γ-globin gene locus resulted in elevated expression of fetal hemoglobin (HbF) in primary erythroid cells. The development of CRISPR/Cas9-PLGA-NPs provides an attractive tool for the delivery of the CRISPR components to target HSPCs, and could provide the basis for in vivo treatment of hemoglobinopathies and other genetic diseases.


Assuntos
Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Nanopartículas , Sistemas CRISPR-Cas/genética , Células Eritroides , Edição de Genes , Humanos
7.
Hum Genomics ; 14(1): 39, 2020 10 16.
Artigo em Inglês | MEDLINE | ID: mdl-33066815

RESUMO

The expression of the human ß-like globin genes follows a well-orchestrated developmental pattern, undergoing two essential switches, the first one during the first weeks of gestation (ε to γ), and the second one during the perinatal period (γ to ß). The γ- to ß-globin gene switching mechanism includes suppression of fetal (γ-globin, HbF) and activation of adult (ß-globin, HbA) globin gene transcription. In hereditary persistence of fetal hemoglobin (HPFH), the γ-globin suppression mechanism is impaired leaving these individuals with unusual elevated levels of fetal hemoglobin (HbF) in adulthood. Recently, the transcription factors KLF1 and BCL11A have been established as master regulators of the γ- to ß-globin switch. Previously, a genomic variant in the KLF1 gene, identified by linkage analysis performed on twenty-seven members of a Maltese family, was found to be associated with HPFH. However, variation in the levels of HbF among family members, and those from other reported families carrying genetic variants in KLF1, suggests additional contributors to globin switching. ASF1B was downregulated in the family members with HPFH. Here, we investigate the role of ASF1B in γ- to ß-globin switching and erythropoiesis in vivo. Mouse-human interspecies ASF1B protein identity is 91.6%. By means of knockdown functional assays in human primary erythroid cultures and analysis of the erythroid lineage in Asf1b knockout mice, we provide evidence that ASF1B is a novel contributor to steady-state erythroid differentiation, and while its loss affects the balance of globin expression, it has no major role in hemoglobin switching.

8.
Hum Mol Genet ; 29(15): 2535-2550, 2020 08 29.
Artigo em Inglês | MEDLINE | ID: mdl-32628253

RESUMO

The transcription factor zinc finger E-box binding protein 2 (ZEB2) controls embryonic and adult cell fate decisions and cellular maturation in many stem/progenitor cell types. Defects in these processes in specific cell types underlie several aspects of Mowat-Wilson syndrome (MOWS), which is caused by ZEB2 haplo-insufficiency. Human ZEB2, like mouse Zeb2, is located on chromosome 2 downstream of a ±3.5 Mb-long gene-desert, lacking any protein-coding gene. Using temporal targeted chromatin capture (T2C), we show major chromatin structural changes based on mapping in-cis proximities between the ZEB2 promoter and this gene desert during neural differentiation of human-induced pluripotent stem cells, including at early neuroprogenitor cell (NPC)/rosette state, where ZEB2 mRNA levels increase significantly. Combining T2C with histone-3 acetylation mapping, we identified three novel candidate enhancers about 500 kb upstream of the ZEB2 transcription start site. Functional luciferase-based assays in heterologous cells and NPCs reveal co-operation between these three enhancers. This study is the first to document in-cis Regulatory Elements located in ZEB2's gene desert. The results further show the usability of T2C for future studies of ZEB2 REs in differentiation and maturation of multiple cell types and the molecular characterization of newly identified MOWS patients that lack mutations in ZEB2 protein-coding exons.

9.
Nat Commun ; 11(1): 2511, 2020 05 14.
Artigo em Inglês | MEDLINE | ID: mdl-32409714

RESUMO

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

10.
Blood ; 136(3): 269-278, 2020 07 16.
Artigo em Inglês | MEDLINE | ID: mdl-32396940

RESUMO

The oxygen transport function of hemoglobin (HB) is thought to have arisen ∼500 million years ago, roughly coinciding with the divergence between jawless (Agnatha) and jawed (Gnathostomata) vertebrates. Intriguingly, extant HBs of jawless and jawed vertebrates were shown to have evolved twice, and independently, from different ancestral globin proteins. This raises the question of whether erythroid-specific expression of HB also evolved twice independently. In all jawed vertebrates studied to date, one of the HB gene clusters is linked to the widely expressed NPRL3 gene. Here we show that the nprl3-linked hb locus of a jawless vertebrate, the river lamprey (Lampetra fluviatilis), shares a range of structural and functional properties with the equivalent jawed vertebrate HB locus. Functional analysis demonstrates that an erythroid-specific enhancer is located in intron 7 of lamprey nprl3, which corresponds to the NPRL3 intron 7 MCS-R1 enhancer of jawed vertebrates. Collectively, our findings signify the presence of an nprl3-linked multiglobin gene locus, which contains a remote enhancer that drives globin expression in erythroid cells, before the divergence of jawless and jawed vertebrates. Different globin genes from this ancestral cluster evolved in the current NPRL3-linked HB genes in jawless and jawed vertebrates. This provides an explanation of the enigma of how, in different species, globin genes linked to the same adjacent gene could undergo convergent evolution.


Assuntos
Eritrócitos/metabolismo , Evolução Molecular , Proteínas de Peixes , Regulação da Expressão Gênica/fisiologia , Hemoglobinas , Lampreias , Animais , Proteínas de Peixes/biossíntese , Proteínas de Peixes/genética , Hemoglobinas/biossíntese , Hemoglobinas/genética , Lampreias/genética , Lampreias/metabolismo , Família Multigênica
11.
Nat Commun ; 11(1): 2251, 2020 05 04.
Artigo em Inglês | MEDLINE | ID: mdl-32366817

RESUMO

The emergence of the novel human coronavirus SARS-CoV-2 in Wuhan, China has caused a worldwide epidemic of respiratory disease (COVID-19). Vaccines and targeted therapeutics for treatment of this disease are currently lacking. Here we report a human monoclonal antibody that neutralizes SARS-CoV-2 (and SARS-CoV) in cell culture. This cross-neutralizing antibody targets a communal epitope on these viruses and may offer potential for prevention and treatment of COVID-19.


Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Betacoronavirus/imunologia , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/prevenção & controle , Pandemias/prevenção & controle , Pneumonia Viral/imunologia , Pneumonia Viral/prevenção & controle , Enzima de Conversão de Angiotensina 2 , Animais , Anticorpos Monoclonais/farmacologia , Anticorpos Neutralizantes/farmacologia , Anticorpos Antivirais/farmacologia , Afinidade de Anticorpos/imunologia , Betacoronavirus/química , Betacoronavirus/efeitos dos fármacos , COVID-19 , Chlorocebus aethiops , Sequência Conservada , Infecções por Coronavirus/tratamento farmacológico , Infecções por Coronavirus/virologia , Reações Cruzadas/imunologia , Epitopos de Linfócito B/química , Epitopos de Linfócito B/imunologia , Humanos , Técnicas In Vitro , Concentração Inibidora 50 , Modelos Moleculares , Peptidil Dipeptidase A/química , Peptidil Dipeptidase A/metabolismo , Pneumonia Viral/tratamento farmacológico , Pneumonia Viral/virologia , Ligação Proteica/efeitos dos fármacos , Domínios Proteicos/imunologia , Receptores Virais/química , Receptores Virais/metabolismo , Vírus da SARS/química , Vírus da SARS/efeitos dos fármacos , Vírus da SARS/imunologia , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/imunologia , Células Vero
12.
Development ; 147(10)2020 05 26.
Artigo em Inglês | MEDLINE | ID: mdl-32253238

RESUMO

The transcription factor Zeb2 controls fate specification and subsequent differentiation and maturation of multiple cell types in various embryonic tissues. It binds many protein partners, including activated Smad proteins and the NuRD co-repressor complex. How Zeb2 subdomains support cell differentiation in various contexts has remained elusive. Here, we studied the role of Zeb2 and its domains in neurogenesis and neural differentiation in the young postnatal ventricular-subventricular zone (V-SVZ), in which neural stem cells generate olfactory bulb-destined interneurons. Conditional Zeb2 knockouts and separate acute loss- and gain-of-function approaches indicated that Zeb2 is essential for controlling apoptosis and neuronal differentiation of V-SVZ progenitors before and after birth, and we identified Sox6 as a potential downstream target gene of Zeb2. Zeb2 genetic inactivation impaired the differentiation potential of the V-SVZ niche in a cell-autonomous fashion. We also provide evidence that its normal function in the V-SVZ also involves non-autonomous mechanisms. Additionally, we demonstrate distinct roles for Zeb2 protein-binding domains, suggesting that Zeb2 partners co-determine neuronal output from the mouse V-SVZ in both quantitative and qualitative ways in early postnatal life.


Assuntos
Ventrículos Laterais/embriologia , Ventrículos Laterais/crescimento & desenvolvimento , Neurogênese/genética , Bulbo Olfatório/embriologia , Bulbo Olfatório/crescimento & desenvolvimento , Homeobox 2 de Ligação a E-box com Dedos de Zinco/metabolismo , Animais , Apoptose/genética , Movimento Celular/genética , Proliferação de Células/genética , Técnicas de Inativação de Genes , Interneurônios/metabolismo , Ventrículos Laterais/metabolismo , Camundongos , Camundongos Knockout , Células-Tronco Neurais/metabolismo , Bulbo Olfatório/metabolismo , Fatores de Transcrição SOXD/metabolismo , Transdução de Sinais/imunologia , Homeobox 2 de Ligação a E-box com Dedos de Zinco/genética
13.
Elife ; 92020 04 21.
Artigo em Inglês | MEDLINE | ID: mdl-32314955

RESUMO

The World Health Organization has included three bunyaviruses posing an increasing threat to human health on the Blueprint list of viruses likely to cause major epidemics and for which no, or insufficient countermeasures exist. Here, we describe a broadly applicable strategy, based on llama-derived single-domain antibodies (VHHs), for the development of bunyavirus biotherapeutics. The method was validated using the zoonotic Rift Valley fever virus (RVFV) and Schmallenberg virus (SBV), an emerging pathogen of ruminants, as model pathogens. VHH building blocks were assembled into highly potent neutralizing complexes using bacterial superglue technology. The multimeric complexes were shown to reduce and prevent virus-induced morbidity and mortality in mice upon prophylactic administration. Bispecific molecules engineered to present two different VHHs fused to an Fc domain were further shown to be effective upon therapeutic administration. The presented VHH-based technology holds great promise for the development of bunyavirus antiviral therapies.


Assuntos
Antivirais/farmacologia , Infecções por Bunyaviridae , Anticorpos de Domínio Único/farmacologia , Animais , Anticorpos Neutralizantes/farmacologia , Camelídeos Americanos , Feminino , Humanos , Masculino , Camundongos
14.
Haematologica ; 105(7): 1802-1812, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-31582556

RESUMO

GATA1 is an essential transcriptional regulator of myeloid hematopoietic differentiation towards red blood cells. During erythroid differentiation, GATA1 forms different complexes with other transcription factors such as LDB1, TAL1, E2A and LMO2 ("the LDB1 complex") or with FOG1. The functions of GATA1 complexes have been studied extensively in definitive erythroid differentiation; however, the temporal and spatial formation of these complexes during erythroid development is unknown. We applied proximity ligation assay (PLA) to detect, localize and quantify individual interactions during embryonic stem cell differentiation and in mouse fetal liver (FL) tissue. We show that GATA1/LDB1 interactions appear before the proerythroblast stage and increase in a subset of the CD71+/TER119- cells to activate the terminal erythroid differentiation program in 12.5 day FL. Using Ldb1 and Gata1 knockdown FL cells, we studied the functional contribution of the GATA1/LDB1 complex during differentiation. This shows that the active LDB1 complex appears quite late at the proerythroblast stage of differentiation and confirms the power of PLA in studying the dynamic interaction of proteins in cell differentiation at the single cell level. We provide dynamic insight into the temporal and spatial formation of the GATA1 and LDB1 transcription factor complexes during hematopoietic development and differentiation.


Assuntos
Células-Tronco Embrionárias/citologia , Fator de Transcrição GATA1 , Proteínas com Domínio LIM , Animais , Diferenciação Celular , Proteínas de Ligação a DNA , Fator de Transcrição GATA1/genética , Fígado , Camundongos , Fatores de Transcrição
15.
Stem Cells ; 38(2): 202-217, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31675135

RESUMO

Cooperative actions of extrinsic signals and cell-intrinsic transcription factors alter gene regulatory networks enabling cells to respond appropriately to environmental cues. Signaling by transforming growth factor type ß (TGFß) family ligands (eg, bone morphogenetic proteins [BMPs] and Activin/Nodal) exerts cell-type specific and context-dependent transcriptional changes, thereby steering cellular transitions throughout embryogenesis. Little is known about coordinated regulation and transcriptional interplay of the TGFß system. To understand intrafamily transcriptional regulation as part of this system's actions during development, we selected 95 of its components and investigated their mRNA-expression dynamics, gene-gene interactions, and single-cell expression heterogeneity in mouse embryonic stem cells transiting to neural progenitors. Interrogation at 24 hour intervals identified four types of temporal gene transcription profiles that capture all stages, that is, pluripotency, epiblast formation, and neural commitment. Then, between each stage we performed esiRNA-based perturbation of each individual component and documented the effect on steady-state mRNA levels of the remaining 94 components. This exposed an intricate system of multilevel regulation whereby the majority of gene-gene interactions display a marked cell-stage specific behavior. Furthermore, single-cell RNA-profiling at individual stages demonstrated the presence of detailed co-expression modules and subpopulations showing stable co-expression modules such as that of the core pluripotency genes at all stages. Our combinatorial experimental approach demonstrates how intrinsically complex transcriptional regulation within a given pathway is during cell fate/state transitions.


Assuntos
Proteínas Morfogenéticas Ósseas/metabolismo , Células-Tronco Embrionárias/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Diferenciação Celular , Humanos
16.
J Am Soc Nephrol ; 30(9): 1641-1658, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31405951

RESUMO

BACKGROUND: GATA3 is a dual-zinc finger transcription factor that regulates gene expression in many developing tissues. In the kidney, GATA3 is essential for ureteric bud branching, and mice without it fail to develop kidneys. In humans, autosomal dominant GATA3 mutations can cause renal aplasia as part of the hypoparathyroidism, renal dysplasia, deafness (HDR) syndrome that includes mesangioproliferative GN. This suggests that GATA3 may have a previously unrecognized role in glomerular development or injury. METHODS: To determine GATA3's role in glomerular development or injury, we assessed GATA3 expression in developing and mature kidneys from Gata3 heterozygous (+/-) knockout mice, as well as injured human and rodent kidneys. RESULTS: We show that GATA3 is expressed by FOXD1 lineage stromal progenitor cells, and a subset of these cells mature into mesangial cells (MCs) that continue to express GATA3 in adult kidneys. In mice, we uncover that GATA3 is essential for normal glomerular development, and mice with haploinsufficiency of Gata3 have too few MC precursors and glomerular abnormalities. Expression of GATA3 is maintained in MCs of adult kidneys and is markedly increased in rodent models of mesangioproliferative GN and in IgA nephropathy, suggesting that GATA3 plays a critical role in the maintenance of glomerular homeostasis. CONCLUSIONS: These results provide new insights on the role GATA3 plays in MC development and response to injury. It also shows that GATA3 may be a novel and robust nuclear marker for identifying MCs in tissue sections.


Assuntos
Fator de Transcrição GATA3/metabolismo , Glomerulonefrite/metabolismo , Glomérulos Renais/metabolismo , Animais , Movimento Celular , Proliferação de Células , Modelos Animais de Doenças , Feminino , Fatores de Transcrição Forkhead/metabolismo , Fator de Transcrição GATA3/genética , Haploinsuficiência , Humanos , Glomérulos Renais/anormalidades , Glomérulos Renais/embriologia , Glomérulos Renais/patologia , Masculino , Células Mesangiais/metabolismo , Células-Tronco Mesenquimais/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Cultura Primária de Células , Ratos , Ratos Wistar
17.
Emerg Microbes Infect ; 8(1): 516-530, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30938227

RESUMO

The Middle-East respiratory syndrome coronavirus (MERS-CoV) is a zoonotic virus that causes severe and often fatal respiratory disease in humans. Efforts to develop antibody-based therapies have focused on neutralizing antibodies that target the receptor binding domain of the viral spike protein thereby blocking receptor binding. Here, we developed a set of human monoclonal antibodies that target functionally distinct domains of the MERS-CoV spike protein. These antibodies belong to six distinct epitope groups and interfere with the three critical entry functions of the MERS-CoV spike protein: sialic acid binding, receptor binding and membrane fusion. Passive immunization with potently as well as with poorly neutralizing antibodies protected mice from lethal MERS-CoV challenge. Collectively, these antibodies offer new ways to gain humoral protection in humans against the emerging MERS-CoV by targeting different spike protein epitopes and functions.


Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Antivirais/imunologia , Infecções por Coronavirus/prevenção & controle , Coronavírus da Síndrome Respiratória do Oriente Médio/imunologia , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/imunologia , Animais , Anticorpos Neutralizantes/imunologia , Infecções por Coronavirus/genética , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/virologia , Epitopos/química , Epitopos/genética , Epitopos/imunologia , Humanos , Imunização Passiva , Camundongos , Coronavírus da Síndrome Respiratória do Oriente Médio/química , Coronavírus da Síndrome Respiratória do Oriente Médio/genética , Domínios Proteicos , Receptores Virais/genética , Receptores Virais/imunologia , Glicoproteína da Espícula de Coronavírus/genética
18.
PLoS One ; 14(3): e0208659, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30921348

RESUMO

Krüppel-like factor 1 (KLF1) is an essential transcription factor for erythroid development, as demonstrated by Klf1 knockout mice which die around E14 due to severe anemia. In humans, >140 KLF1 variants, causing different erythroid phenotypes, have been described. The KLF1 Nan variant, a single amino acid substitution (p.E339D) in the DNA binding domain, causes hemolytic anemia and is dominant over wildtype KLF1. Here we describe the effects of the KLF1 Nan variant during fetal development. We show that Nan embryos have defects in erythroid maturation. RNA-sequencing of the KLF1 Nan fetal liver cells revealed that Exportin 7 (Xpo7) was among the 782 deregulated genes. This nuclear exportin is implicated in terminal erythroid differentiation; in particular it is involved in nuclear condensation. Indeed, KLF1 Nan fetal liver cells had larger nuclei and reduced chromatin condensation. Knockdown of XPO7 in wildtype erythroid cells caused a similar phenotype. We propose that reduced expression of XPO7 is partially responsible for the erythroid defects observed in KLF1 Nan erythroid cells.


Assuntos
Anemia Hemolítica/genética , Células Eritroides/citologia , Fatores de Transcrição Kruppel-Like/genética , Proteína ran de Ligação ao GTP/genética , Substituição de Aminoácidos , Animais , Diferenciação Celular , Células Cultivadas , Cromatina/metabolismo , Modelos Animais de Doenças , Embrião não Mamífero/metabolismo , Células Eritroides/metabolismo , Eritropoese , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Masculino , Camundongos , Análise de Sequência de RNA/métodos , Proteína ran de Ligação ao GTP/metabolismo
19.
Mol Syst Biol ; 14(6): e8214, 2018 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-29858282

RESUMO

The last decade has radically renewed our understanding of higher order chromatin folding in the eukaryotic nucleus. As a result, most current models are in support of a mostly hierarchical and relatively stable folding of chromosomes dividing chromosomal territories into A- (active) and B- (inactive) compartments, which are then further partitioned into topologically associating domains (TADs), each of which is made up from multiple loops stabilized mainly by the CTCF and cohesin chromatin-binding complexes. Nonetheless, the structure-to-function relationship of eukaryotic genomes is still not well understood. Here, we focus on recent work highlighting the biophysical and regulatory forces that contribute to the spatial organization of genomes, and we propose that the various conformations that chromatin assumes are not so much the result of a linear hierarchy, but rather of both converging and conflicting dynamic forces that act on it.


Assuntos
Montagem e Desmontagem da Cromatina , Cromatina/química , Eucariotos/genética , Genoma , Animais , Humanos , Conformação de Ácido Nucleico , Dobramento de Proteína , Transcrição Genética
20.
Nat Protoc ; 13(3): 459-477, 2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-29419817

RESUMO

Chromosome conformation capture (3C) and its derivatives (e.g., 4C, 5C and Hi-C) are used to analyze the 3D organization of genomes. We recently developed targeted chromatin capture (T2C), an inexpensive method for studying the 3D organization of genomes, interactomes and structural changes associated with gene regulation, the cell cycle, and cell survival and development. Here, we present the protocol for T2C based on capture, describing all experimental steps and bio-informatic tools in full detail. T2C offers high resolution, a large dynamic interaction frequency range and a high signal-to-noise ratio. Its resolution is determined by the resulting fragment size of the chosen restriction enzyme, which can lead to sub-kilobase-pair resolution. T2C's high coverage allows the identification of the interactome of each individual DNA fragment, which makes binning of reads (often used in other methods) basically unnecessary. Notably, T2C requires low sequencing efforts. T2C also allows multiplexing of samples for the direct comparison of multiple samples. It can be used to study topologically associating domains (TADs), determining their position, shape, boundaries, and intra- and inter-domain interactions, as well as the composition of aggregated loops, interactions between nucleosomes, individual transcription factor binding sites, and promoters and enhancers. T2C can be performed by any investigator with basic skills in molecular biology techniques in ∼7-8 d. Data analysis requires basic expertise in bioinformatics and in Linux and Python environments.


Assuntos
Biologia Computacional/métodos , Mapeamento Físico do Cromossomo/métodos , Análise de Sequência de DNA/métodos , Animais , Cromatina/ultraestrutura , Montagem e Desmontagem da Cromatina/fisiologia , Mapeamento Cromossômico/métodos , DNA , Regulação da Expressão Gênica , Genoma/genética , Genoma Humano/genética , Genoma Humano/fisiologia , Genômica , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Camundongos , Nucleossomos , Software
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