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1.
Circ Res ; 135(4): e94-e113, 2024 Aug 02.
Artigo em Inglês | MEDLINE | ID: mdl-38957991

RESUMO

BACKGROUND: Cerebral vascular malformations (CCMs) are primarily found within the brain, where they result in increased risk for stroke, seizures, and focal neurological deficits. The unique feature of the brain vasculature is the blood-brain barrier formed by the brain neurovascular unit. Recent studies suggest that loss of CCM genes causes disruptions of blood-brain barrier integrity as the inciting events for CCM development. CCM lesions are proposed to be initially derived from a single clonal expansion of a subset of angiogenic venous capillary endothelial cells (ECs) and respective resident endothelial progenitor cells (EPCs). However, the critical signaling events in the subclass of brain ECs/EPCs for CCM lesion initiation and progression are unclear. METHODS: Brain EC-specific CCM3-deficient (Pdcd10BECKO) mice were generated by crossing Pdcd10fl/fl mice with Mfsd2a-CreERT2 mice. Single-cell RNA-sequencing analyses were performed by the chromium single-cell platform (10× genomics). Cell clusters were annotated into EC subtypes based on visual inspection and GO analyses. Cerebral vessels were visualized by 2-photon in vivo imaging and tissue immunofluorescence analyses. Regulation of mTOR (mechanistic target of rapamycin) signaling by CCM3 and Cav1 (caveolin-1) was performed by cell biology and biochemical approaches. RESULTS: Single-cell RNA-sequencing analyses from P10 Pdcd10BECKO mice harboring visible CCM lesions identified upregulated CCM lesion signature and mitotic EC clusters but decreased blood-brain barrier-associated EC clusters. However, a unique EPC cluster with high expression levels of stem cell markers enriched with mTOR signaling was identified from early stages of the P6 Pdcd10BECKO brain. Indeed, mTOR signaling was upregulated in both mouse and human CCM lesions. Genetic deficiency of Raptor (regulatory-associated protein of mTOR), but not of Rictor (rapamycin-insensitive companion of mTOR), prevented CCM lesion formation in the Pdcd10BECKO model. Importantly, the mTORC1 (mTOR complex 1) pharmacological inhibitor rapamycin suppressed EPC proliferation and ameliorated CCM pathogenesis in Pdcd10BECKO mice. Mechanistic studies suggested that Cav1/caveolae increased in CCM3-depleted EPC-mediated intracellular trafficking and complex formation of the mTORC1 signaling proteins. CONCLUSIONS: CCM3 is critical for maintaining blood-brain barrier integrity and CCM3 loss-induced mTORC1 signaling in brain EPCs initiates and facilitates CCM pathogenesis.


Assuntos
Células Progenitoras Endoteliais , Hemangioma Cavernoso do Sistema Nervoso Central , Alvo Mecanístico do Complexo 1 de Rapamicina , Transdução de Sinais , Animais , Hemangioma Cavernoso do Sistema Nervoso Central/metabolismo , Hemangioma Cavernoso do Sistema Nervoso Central/genética , Hemangioma Cavernoso do Sistema Nervoso Central/patologia , Camundongos , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Células Progenitoras Endoteliais/metabolismo , Células Progenitoras Endoteliais/patologia , Encéfalo/metabolismo , Encéfalo/patologia , Encéfalo/irrigação sanguínea , Camundongos Knockout , Barreira Hematoencefálica/metabolismo , Barreira Hematoencefálica/patologia , Proteínas Reguladoras de Apoptose/metabolismo , Proteínas Reguladoras de Apoptose/genética , Camundongos Endogâmicos C57BL , Proteínas de Membrana/metabolismo , Proteínas de Membrana/genética
2.
J Immunol ; 213(9): 1338-1348, 2024 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-39302113

RESUMO

Expression of IL-15 on the surface of human graft endothelial cells (ECs) bound to the IL-15Rα subunit can increase the activation of CTLs, potentiating allograft rejection. Our previous work showed that surface expression of this protein complex could be induced by alloantibody-mediated complement activation through increased IL-1ß synthesis, secretion, and autocrine/paracrine IL-1-mediated activation of NF-κB. In this article, we report that cultured human ECs express eight differently spliced IL-15 transcripts. Remarkably, IL-1ß does not alter the expression level of any IL-15 transcript but induces surface expression independently of RNA polymerase II-mediated transcription while requiring new protein translation. Mechanistically, IL-1ß causes an NF-κB-mediated reduction in the level of microRNA Let-7c-3p, thereby relieving a block of translation of IL-15 surface protein. Let7c-3p anti-miR can induce EC surface expression of IL-15/IL-15Rα in the absence of complement activation or of IL-1, enabling IL-15 transpresentation to boost CD8 T cell activation. Because of the complexity we have uncovered in IL-15 regulation, we recommend caution in interpreting increased total IL-15 mRNA or protein levels as a surrogate for transpresentation.


Assuntos
Células Endoteliais , Interleucina-15 , Interleucina-1beta , MicroRNAs , Biossíntese de Proteínas , Humanos , Interleucina-15/metabolismo , MicroRNAs/genética , Interleucina-1beta/metabolismo , Células Endoteliais/metabolismo , Células Endoteliais/imunologia , NF-kappa B/metabolismo , Células Cultivadas , Rejeição de Enxerto/imunologia , Regulação da Expressão Gênica , Ativação Linfocitária/imunologia , Subunidade alfa de Receptor de Interleucina-15/metabolismo , Subunidade alfa de Receptor de Interleucina-15/genética
3.
PLoS Genet ; 19(11): e1011019, 2023 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-37934795

RESUMO

Lineage-specific genes (LSGs) have long been postulated to play roles in the establishment of genetic barriers to intercrossing and speciation. In the genome of Neurospora crassa, most of the 670 Neurospora LSGs that are aggregated adjacent to the telomeres are clustered with 61% of the HET-domain genes, some of which regulate self-recognition and define vegetative incompatibility groups. In contrast, the LSG-encoding proteins possess few to no domains that would help to identify potential functional roles. Possible functional roles of LSGs were further assessed by performing transcriptomic profiling in genetic mutants and in response to environmental alterations, as well as examining gene knockouts for phenotypes. Among the 342 LSGs that are dynamically expressed during both asexual and sexual phases, 64% were detectable on unusual carbon sources such as furfural, a wildfire-produced chemical that is a strong inducer of sexual development, and the structurally-related furan 5-hydroxymethyl furfural (HMF). Expression of a significant portion of the LSGs was sensitive to light and temperature, factors that also regulate the switch from asexual to sexual reproduction. Furthermore, expression of the LSGs was significantly affected in the knockouts of adv-1 and pp-1 that regulate hyphal communication, and expression of more than one quarter of the LSGs was affected by perturbation of the mating locus. These observations encouraged further investigation of the roles of clustered lineage-specific and HET-domain genes in ecology and reproduction regulation in Neurospora, especially the regulation of the switch from the asexual growth to sexual reproduction, in response to dramatic environmental conditions changes.


Assuntos
Neurospora crassa , Neurospora , Neurospora/genética , Genes Fúngicos , Neurospora crassa/genética , Fenótipo , Perfilação da Expressão Gênica , Reprodução/genética , Proteínas Fúngicas/genética
4.
J Cell Sci ; 135(6)2022 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-35178554

RESUMO

Mutations in SPAG1, a dynein axonemal assembly factor (DNAAF) that facilitates the assembly of dynein arms in the cytoplasm before their transport into the cilium, result in primary ciliary dyskinesia (PCD), a genetically heterogenous disorder characterized by chronic oto-sino-pulmonary disease, infertility and laterality defects. To further elucidate the role of SPAG1 in dynein assembly, we examined its expression, interactions and ciliary defects in control and PCD human airway epithelia. Immunoprecipitations showed that SPAG1 interacts with multiple DNAAFs, dynein chains and canonical components of the R2TP complex. Protein levels of dynein heavy chains (DHCs) and interactions between DHCs and dynein intermediate chains (DICs) were reduced in SPAG1 mutants. We also identified a previously uncharacterized 60 kDa SPAG1 isoform, through examination of PCD subjects with an atypical ultrastructural defect for SPAG1 variants, that can partially compensate for the absence of full-length SPAG1 to assemble a reduced number of outer dynein arms. In summary, our data show that SPAG1 is necessary for axonemal dynein arm assembly by scaffolding R2TP-like complexes composed of several DNAAFs that facilitate the folding and/or binding of the DHCs to the DIC complex.


Assuntos
Dineínas do Axonema , Axonema , Antígenos de Superfície/metabolismo , Dineínas do Axonema/genética , Dineínas do Axonema/metabolismo , Axonema/metabolismo , Cílios/metabolismo , Dineínas/genética , Dineínas/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Humanos , Mutação/genética , Sistema Respiratório/metabolismo
5.
EMBO J ; 38(16): e102003, 2019 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-31313851

RESUMO

Many eukaryotic proteins are regulated by modification with the ubiquitin-like protein small ubiquitin-like modifier (SUMO). This linkage is reversed by SUMO proteases, of which there are two in Saccharomyces cerevisiae, Ulp1 and Ulp2. SUMO-protein conjugation regulates transcription, but the roles of SUMO proteases in transcription remain unclear. We report that Ulp2 is recruited to transcriptionally active genes to control local polysumoylation. Mutant ulp2 cells show impaired association of RNA polymerase II (RNAPII) with, and diminished expression of, constitutively active genes and the inducible CUP1 gene. Ulp2 loss sensitizes cells to 6-azauracil, a hallmark of transcriptional elongation defects. We also describe a novel chromatin regulatory mechanism whereby histone-H2B ubiquitylation stimulates histone sumoylation, which in turn appears to inhibit nucleosome association of the Ctk1 kinase. Ctk1 phosphorylates serine-2 (S2) in the RNAPII C-terminal domain (CTD) and promotes transcript elongation. Removal of both ubiquitin and SUMO from histones is needed to overcome the impediment to S2 phosphorylation. These results suggest sequential ubiquitin-histone and SUMO-histone modifications recruit Ulp2, which removes polySUMO chains and promotes RNAPII transcription elongation.


Assuntos
Endopeptidases/metabolismo , Histonas/metabolismo , Mutação , Proteínas de Saccharomyces cerevisiae/metabolismo , Elongação da Transcrição Genética , Endopeptidases/genética , Regulação Fúngica da Expressão Gênica , Metalotioneína/genética , RNA Polimerase II/metabolismo , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/genética , Sumoilação , Elongação da Transcrição Genética/efeitos dos fármacos , Ativação Transcricional/efeitos dos fármacos , Uracila/análogos & derivados , Uracila/farmacologia
6.
Mol Ecol ; 2023 Oct 16.
Artigo em Inglês | MEDLINE | ID: mdl-37843462

RESUMO

The origin of new genes has long been a central interest of evolutionary biologists. However, their novelty means that they evade reconstruction by the classical tools of evolutionary modelling. This evasion of deep ancestral investigation necessitates intensive study of model species within well-sampled, recently diversified, clades. One such clade is the model genus Neurospora, members of which lack recent gene duplications. Several Neurospora species are comprehensively characterized organisms apt for studying the evolution of lineage-specific genes (LSGs). Using gene synteny, we documented that 78% of Neurospora LSG clusters are located adjacent to the telomeres featuring extensive tracts of non-coding DNA and duplicated genes. Here, we report several instances of LSGs that are likely from regional rearrangements and potentially from gene rebirth. To broadly investigate the functions of LSGs, we assembled transcriptomics data from 68 experimental data points and identified co-regulatory modules using Weighted Gene Correlation Network Analysis, revealing that LSGs are widely but peripherally involved in known regulatory machinery for diverse functions. The ancestral status of the LSG mas-1, a gene with roles in cell-wall integrity and cellular sensitivity to antifungal toxins, was investigated in detail alongside its genomic neighbours, indicating that it arose from an ancient lysophospholipase precursor that is ubiquitous in lineages of the Sordariomycetes. Our discoveries illuminate a "rummage region" in the N. crassa genome that enables the formation of new genes and functions to arise via gene duplication and relocation, followed by fast mutation and recombination facilitated by sequence repeats and unconstrained non-coding sequences.

7.
FASEB J ; 36(4): e22254, 2022 04.
Artigo em Inglês | MEDLINE | ID: mdl-35294066

RESUMO

Overwhelming inflammation in the setting of acute critical illness induces capillary leak resulting in hypovolemia, edema, tissue dysoxia, organ failure and even death. The tight junction (TJ)-dependent capillary barrier is regulated by small GTPases, but the specific regulatory molecules most active in this vascular segment under such circumstances are not well described. We set out to identify GTPase regulatory molecules specific to endothelial cells (EC) that form TJs. Transcriptional profiling of confluent monolayers of TJ-forming human dermal microvascular ECs (HDMECs) and adherens junction only forming-human umbilical vein EC (HUVECs) demonstrate ARHGEF12 is basally expressed at higher levels and is only downregulated in HDMECs by junction-disrupting tumor necrosis factor (TNF). HDMECs depleted of ArhGEF12 by siRNA demonstrate a significantly exacerbated TNF-induced decrease in trans-endothelial electrical resistance and disruption of TJ continuous staining. ArhGEF12 is established as a RhoA-GEF in HUVECs and its knock down would be expected to reduce RhoA activity and barrier disruption. Pulldown of active GEFs from HDMECs depleted of ArhGEF12 and treated with TNF show decreased GTP-bound Rap1A after four hours but increased GTP-bound RhoA after 12 h. In cell-free assays, ArhGEF12 immunoprecipitated from HDMECs is able to activate both Rap1A and RhoA, but not act on Rap2A-C, RhoB-C, or even Rap1B which shares 95% sequence identity with Rap1A. We conclude that in TJ-forming HDMECs, ArhGEF12 selectively activates Rap1A to limit capillary barrier disruption in a mechanism independent of cAMP-mediated Epac1 activation.


Assuntos
Fatores de Troca do Nucleotídeo Guanina , Proteína rhoA de Ligação ao GTP , Fatores de Troca do Nucleotídeo Guanina/genética , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Guanosina Trifosfato , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Fator de Necrose Tumoral alfa/farmacologia , Proteínas rap1 de Ligação ao GTP/genética , Proteína rhoA de Ligação ao GTP/genética , Proteína rhoA de Ligação ao GTP/metabolismo , Proteína rhoB de Ligação ao GTP/metabolismo
8.
J Lipid Res ; 63(6): 100209, 2022 06.
Artigo em Inglês | MEDLINE | ID: mdl-35460704

RESUMO

Low levels of high density lipoprotein-cholesterol (HDL-C) are associated with an elevated risk of arteriosclerotic coronary heart disease. Heritability of HDL-C levels is high. In this research discovery study, we used whole-exome sequencing to identify damaging gene variants that may play significant roles in determining HDL-C levels. We studied 204 individuals with a mean HDL-C level of 27.8 ± 6.4 mg/dl (range: 4-36 mg/dl). Data were analyzed by statistical gene burden testing and by filtering against candidate gene lists. We found 120 occurrences of probably damaging variants (116 heterozygous; four homozygous) among 45 of 104 recognized HDL candidate genes. Those with the highest prevalence of damaging variants were ABCA1 (n = 20), STAB1 (n = 9), OSBPL1A (n = 8), CPS1 (n = 8), CD36 (n = 7), LRP1 (n = 6), ABCA8 (n = 6), GOT2 (n = 5), AMPD3 (n = 5), WWOX (n = 4), and IRS1 (n = 4). Binomial analysis for damaging missense or loss-of-function variants identified the ABCA1 and LDLR genes at genome-wide significance. In conclusion, whole-exome sequencing of individuals with low HDL-C showed the burden of damaging rare variants in the ABCA1 and LDLR genes is particularly high and revealed numerous occurrences in HDL candidate genes, including many genes identified in genome-wide association study reports. Many of these genes are involved in cancer biology, which accords with epidemiologic findings of the association of HDL deficiency with increased risk of cancer, thus presenting a new area of interest in HDL genomics.


Assuntos
Estudo de Associação Genômica Ampla , Hipoalfalipoproteinemias , HDL-Colesterol/genética , Heterozigoto , Humanos , Sequenciamento do Exoma
9.
FASEB J ; 35(6): e21627, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33948992

RESUMO

Capillary endothelial cells (ECs) maintain a semi-permeable barrier between the blood and tissue by forming inter-EC tight junctions (TJs), regulating selective transport of fluid and solutes. Overwhelming inflammation, as occurs in sepsis, disrupts these TJs, leading to leakage of fluid, proteins, and small molecules into the tissues. Mechanistically, disruption of capillary barrier function is mediated by small Rho-GTPases, such as RhoA, -B, and -C, which are activated by guanine nucleotide exchange factors (GEFs) and disrupted by GTPase-activating factors (GAPs). We previously reported that a mutation in a specific RhoB GAP (p190BRhoGAP) underlays a hereditary capillary leak syndrome. Tumor necrosis factor (TNF) treatment disrupts TJs in cultured human microvascular ECs, a model of capillary leak. This response requires new gene transcription and involves increased RhoB activation. However, the specific GEF that activates RhoB in capillary ECs remains unknown. Transcriptional profiling of cultured tight junction-forming human dermal microvascular endothelial cells (HDMECs) revealed that 17 GEFs were significantly induced by TNF. The function of each candidate GEF was assessed by short interfering RNA depletion and trans-endothelial electrical resistance screening. Knockown of ArhGEF10 reduced the TNF-induced loss of barrier which was phenocopied by RhoB or dual ArhGEF10/RhoB knockdown. ArhGEF10 knockdown also reduced the extent of TNF-induced RhoB activation and disruption at tight junctions. In a cell-free assay, immunoisolated ArhGEF10 selectively catalyzed nucleotide exchange to activate RhoB, but not RhoA or RhoC. We conclude ArhGEF10 is a TNF-induced RhoB-selective GEF that mediates TJ disruption and barrier loss in human capillary endothelial cells.


Assuntos
Derme/metabolismo , Endotélio Vascular/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Fatores de Troca de Nucleotídeo Guanina Rho/metabolismo , Junções Íntimas/fisiologia , Proteína rhoB de Ligação ao GTP/metabolismo , Permeabilidade Capilar , Derme/citologia , Derme/efeitos dos fármacos , Endotélio Vascular/citologia , Endotélio Vascular/efeitos dos fármacos , Fatores de Troca do Nucleotídeo Guanina/genética , Humanos , Fatores de Troca de Nucleotídeo Guanina Rho/genética , Transdução de Sinais , Fator de Necrose Tumoral alfa/farmacologia , Proteína rhoB de Ligação ao GTP/genética
10.
PLoS Biol ; 17(9): e3000421, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31513564

RESUMO

Decidua is a transient uterine tissue shared by mammals with hemochorial placenta and is essential for pregnancy. The decidua is infiltrated by many immune cells promoting pregnancy. Adult bone marrow (BM)-derived cells (BMDCs) differentiate into rare populations of nonhematopoietic endometrial cells in the uterus. However, whether adult BMDCs become nonhematopoietic decidual cells and contribute functionally to pregnancy is unknown. Here, we show that pregnancy mobilizes mesenchymal stem cells (MSCs) to the circulation and that pregnancy induces considerable adult BMDCs recruitment to decidua, where some differentiate into nonhematopoietic prolactin-expressing decidual cells. To explore the functional importance of nonhematopoietic BMDCs to pregnancy, we used Homeobox a11 (Hoxa11)-deficient mice, having endometrial stromal-specific defects precluding decidualization and successful pregnancy. Hoxa11 expression in BM is restricted to nonhematopoietic cells. BM transplant (BMT) from wild-type (WT) to Hoxa11-/- mice results in stromal expansion, gland formation, and marked decidualization otherwise absent in Hoxa11-/- mice. Moreover, in Hoxa11+/- mice, which have increased pregnancy losses, BMT from WT donors leads to normalized uterine expression of numerous decidualization-related genes and rescue of pregnancy loss. Collectively, these findings reveal that adult BMDCs have a previously unrecognized nonhematopoietic physiologic contribution to decidual stroma, thereby playing important roles in decidualization and pregnancy.


Assuntos
Células da Medula Óssea/fisiologia , Decídua/citologia , Implantação do Embrião , Células-Tronco Mesenquimais/fisiologia , Gravidez/fisiologia , Animais , Feminino , Proteínas de Homeodomínio/genética , Masculino , Camundongos Knockout
11.
Am J Med Genet A ; 185(1): 119-133, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33098347

RESUMO

Dubowitz syndrome (DubS) is considered a recognizable syndrome characterized by a distinctive facial appearance and deficits in growth and development. There have been over 200 individuals reported with Dubowitz or a "Dubowitz-like" condition, although no single gene has been implicated as responsible for its cause. We have performed exome (ES) or genome sequencing (GS) for 31 individuals clinically diagnosed with DubS. After genome-wide sequencing, rare variant filtering and computational and Mendelian genomic analyses, a presumptive molecular diagnosis was made in 13/27 (48%) families. The molecular diagnoses included biallelic variants in SKIV2L, SLC35C1, BRCA1, NSUN2; de novo variants in ARID1B, ARID1A, CREBBP, POGZ, TAF1, HDAC8, and copy-number variation at1p36.11(ARID1A), 8q22.2(VPS13B), Xp22, and Xq13(HDAC8). Variants of unknown significance in known disease genes, and also in genes of uncertain significance, were observed in 7/27 (26%) additional families. Only one gene, HDAC8, could explain the phenotype in more than one family (N = 2). All but two of the genomic diagnoses were for genes discovered, or for conditions recognized, since the introduction of next-generation sequencing. Overall, the DubS-like clinical phenotype is associated with extensive locus heterogeneity and the molecular diagnoses made are for emerging clinical conditions sharing characteristic features that overlap the DubS phenotype.


Assuntos
Eczema/diagnóstico , Eczema/genética , Predisposição Genética para Doença , Transtornos do Crescimento/diagnóstico , Transtornos do Crescimento/genética , Histona Desacetilases/genética , Deficiência Intelectual/diagnóstico , Deficiência Intelectual/genética , Microcefalia/diagnóstico , Microcefalia/genética , Proteínas Repressoras/genética , Adolescente , Criança , Pré-Escolar , Variações do Número de Cópias de DNA/genética , Eczema/patologia , Exoma/genética , Fácies , Feminino , Genoma Humano/genética , Genômica/métodos , Transtornos do Crescimento/patologia , Humanos , Lactente , Deficiência Intelectual/patologia , Masculino , Microcefalia/patologia , Fenótipo , Sequenciamento do Exoma
12.
Am J Hum Genet ; 97(2): 199-215, 2015 Aug 06.
Artigo em Inglês | MEDLINE | ID: mdl-26166479

RESUMO

Discovering the genetic basis of a Mendelian phenotype establishes a causal link between genotype and phenotype, making possible carrier and population screening and direct diagnosis. Such discoveries also contribute to our knowledge of gene function, gene regulation, development, and biological mechanisms that can be used for developing new therapeutics. As of February 2015, 2,937 genes underlying 4,163 Mendelian phenotypes have been discovered, but the genes underlying ∼50% (i.e., 3,152) of all known Mendelian phenotypes are still unknown, and many more Mendelian conditions have yet to be recognized. This is a formidable gap in biomedical knowledge. Accordingly, in December 2011, the NIH established the Centers for Mendelian Genomics (CMGs) to provide the collaborative framework and infrastructure necessary for undertaking large-scale whole-exome sequencing and discovery of the genetic variants responsible for Mendelian phenotypes. In partnership with 529 investigators from 261 institutions in 36 countries, the CMGs assessed 18,863 samples from 8,838 families representing 579 known and 470 novel Mendelian phenotypes as of January 2015. This collaborative effort has identified 956 genes, including 375 not previously associated with human health, that underlie a Mendelian phenotype. These results provide insight into study design and analytical strategies, identify novel mechanisms of disease, and reveal the extensive clinical variability of Mendelian phenotypes. Discovering the gene underlying every Mendelian phenotype will require tackling challenges such as worldwide ascertainment and phenotypic characterization of families affected by Mendelian conditions, improvement in sequencing and analytical techniques, and pervasive sharing of phenotypic and genomic data among researchers, clinicians, and families.


Assuntos
Doenças Genéticas Inatas/genética , Genética Médica/métodos , Genética Médica/tendências , Fenótipo , Proteínas/genética , Humanos
13.
PLoS Pathog ; 12(11): e1006008, 2016 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-27855219

RESUMO

Subversion of host immune surveillance is a crucial step in viral pathogenesis. Epstein-Barr virus (EBV) encodes two immune evasion gene products, BCRF1 (viral IL-10) and BPLF1 (deubiquitinase/deneddylase); both proteins suppress antiviral immune responses during primary infection. The BCRF1 and BPLF1 genes are expressed during the late phase of the lytic cycle, an essential but poorly understood phase of viral gene expression. Several late gene regulators recently identified in beta and gamma herpesviruses form a viral pre-initiation complex for transcription. Whether each of these late gene regulators is necessary for transcription of all late genes is not known. Here, studying viral gene expression in the absence and presence of siRNAs to individual components of the viral pre-initiation complex, we identified two distinct groups of late genes. One group includes late genes encoding the two immunoevasins, BCRF1 and BPLF1, and is transcribed independently of the viral pre-initiation complex. The second group primarily encodes viral structural proteins and is dependent on the viral pre-initiation complex. The protein kinase BGLF4 is the only known late gene regulator necessary for expression of both groups of late genes. ChIP-seq analysis showed that the transcription activator Rta associates with the promoters of eight late genes including genes encoding the viral immunoevasins. Our results demonstrate that late genes encoding immunomodulatory proteins are transcribed by a mechanism distinct from late genes encoding viral structural proteins. Understanding the mechanisms that specifically regulate expression of the late immunomodulatory proteins could aid the development of antiviral drugs that impair immune evasion by the oncogenic EB virus.


Assuntos
Infecções por Vírus Epstein-Barr/genética , Regulação Viral da Expressão Gênica/fisiologia , Herpesvirus Humano 4/genética , Evasão da Resposta Imune/genética , Proteínas Virais/biossíntese , Proteínas Virais Reguladoras e Acessórias/biossíntese , Western Blotting , Imunoprecipitação da Cromatina , Infecções por Vírus Epstein-Barr/imunologia , Técnicas de Silenciamento de Genes , Células HEK293 , Herpesvirus Humano 4/imunologia , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Evasão da Resposta Imune/imunologia , Reação em Cadeia da Polimerase em Tempo Real , Proteínas Virais/genética , Proteínas Virais Reguladoras e Acessórias/genética , Proteínas Estruturais Virais/biossíntese
14.
PLoS Pathog ; 10(8): e1004307, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-25166506

RESUMO

The mechanism regulating expression of late genes, encoding viral structural components, is an unresolved problem in the biology of DNA tumor viruses. Here we show that BGLF4, the only protein kinase encoded by Epstein-Barr virus (EBV), controls expression of late genes independent of its effect on viral DNA replication. Ectopic expression of BGLF4 in cells lacking the kinase gene stimulated the transcript levels of six late genes by 8- to 10-fold. Introduction of a BGLF4 mutant that eliminated its kinase activity did not stimulate late gene expression. In cells infected with wild-type EBV, siRNA to BGLF4 (siG4) markedly reduced late gene expression without compromising viral DNA replication. Synthesis of late products was restored upon expression of a form of BGLF4 resistant to the siRNA. Studying the EBV transcriptome using mRNA-seq during the late phase of the lytic cycle in the absence and presence of siG4 showed that BGLF4 controlled expression of 31 late genes. Analysis of the EBV transcriptome identified BGLF3 as a gene whose expression was reduced as a result of silencing BGLF4. Knockdown of BGLF3 markedly reduced late gene expression but had no effect on viral DNA replication or expression of BGLF4. Our findings reveal the presence of a late control locus encompassing BGLF3 and BGLF4 in the EBV genome, and provide evidence for the importance of both proteins in post-replication events that are necessary for expression of late genes.


Assuntos
Regulação Viral da Expressão Gênica/genética , Proteínas Serina-Treonina Quinases/biossíntese , Proteínas Serina-Treonina Quinases/genética , Proteínas Virais/biossíntese , Proteínas Virais/genética , Proteínas Estruturais Virais/biossíntese , Proteínas Estruturais Virais/genética , Northern Blotting , Loci Gênicos , Células HEK293 , Humanos , Reação em Cadeia da Polimerase em Tempo Real , Transfecção , Replicação Viral/genética
15.
Proc Natl Acad Sci U S A ; 110(14): 5428-33, 2013 Apr 02.
Artigo em Inglês | MEDLINE | ID: mdl-23509252

RESUMO

Mutant human Cu/Zn superoxide dismutase 1 (SOD1) is associated with motor neuron toxicity and death in an inherited form of amyotrophic lateral sclerosis (ALS; Lou Gehrig disease). One aspect of toxicity in motor neurons involves diminished fast axonal transport, observed both in transgenic mice and, more recently, in axoplasm isolated from squid giant axons. The latter effect appears to be directly mediated by misfolded SOD1, whose addition activates phosphorylation of p38 MAPK and phosphorylation of kinesin. Here, we observe that several different oligomeric states of a fusion protein, comprising ALS-associated human G85R SOD1 joined with yellow fluorescent protein (G85R SOD1YFP), which produces ALS in transgenic mice, inhibited anterograde transport when added to squid axoplasm. Inhibition was blocked both by an apoptosis signal-regulating kinase 1 (ASK1; MAPKKK) inhibitor and by a p38 inhibitor, indicating the transport defect is mediated through the MAPK cascade. In further incubations, we observed that addition of the mammalian molecular chaperone Hsc70, abundantly associated with G85R SOD1YFP in spinal cord of transgenic mice, exerted partial correction of the transport defect, associated with diminished phosphorylation of p38. Most striking, the addition of the molecular chaperone Hsp110, in a concentration substoichiometric to the mutant SOD1 protein, completely rescued both the transport defect and the phosphorylation of p38. Hsp110 has been demonstrated to act as a nucleotide exchange factor for Hsc70 and, more recently, to be able to cooperate with it to mediate protein disaggregation. We speculate that it can cooperate with endogenous squid Hsp(c)70 to mediate binding and/or disaggregation of mutant SOD1 protein, abrogating toxicity.


Assuntos
Transporte Axonal/fisiologia , Proteínas de Choque Térmico HSP110/farmacologia , Proteínas Recombinantes de Fusão/metabolismo , Superóxido Dismutase/metabolismo , Vesículas Transportadoras/metabolismo , Animais , Proteínas de Bactérias/metabolismo , Decapodiformes , Perfilação da Expressão Gênica , Proteínas de Choque Térmico HSP110/metabolismo , Humanos , Proteínas Luminescentes/metabolismo , MAP Quinase Quinase Quinase 5/antagonistas & inibidores , Camundongos , Camundongos Transgênicos , Mutação de Sentido Incorreto/genética , Fosforilação/efeitos dos fármacos , Dobramento de Proteína , Proteômica , Medula Espinal/citologia , Medula Espinal/metabolismo , Superóxido Dismutase/química , Superóxido Dismutase/genética , Superóxido Dismutase-1 , Vesículas Transportadoras/efeitos dos fármacos , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores
16.
Development ; 139(12): 2161-9, 2012 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-22573623

RESUMO

The establishment and maintenance of organ boundaries are vital for animal and plant development. In the Arabidopsis flower, three microRNA164 genes (MIR164a, b and c) regulate the expression of CUP-SHAPED COTYLEDON1 (CUC1) and CUC2, which encode key transcriptional regulators involved in organ boundary specification. These three miR164 genes are expressed in distinct spatial and temporal domains that are crucial for their function. Here, we show that the C2H2 zinc finger transcriptional repressor encoded by RABBIT EARS (RBE) regulates the expression of all three miR164 genes. Furthermore, we demonstrate that RBE directly interacts with the promoter of MIR164c and negatively regulates its expression. We also show that the role of RBE in sepal and petal development is mediated in part through the concomitant regulation of the CUC1 and CUC2 gene products. These results indicate that one role of RBE is to fine-tune miR164 expression to regulate the CUC1 and CUC2 effector genes, which, in turn, regulate developmental events required for sepal and petal organogenesis.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/embriologia , Arabidopsis/genética , Flores/embriologia , Regulação da Expressão Gênica de Plantas , MicroRNAs/genética , Organogênese/genética , Proteínas Repressoras/metabolismo , Arabidopsis/citologia , Proteínas de Arabidopsis/genética , Flores/citologia , Flores/genética , Regulação da Expressão Gênica no Desenvolvimento , Genes de Plantas/genética , MicroRNAs/metabolismo , Mutação/genética , Especificidade de Órgãos/genética , Regiões Promotoras Genéticas/genética , Ligação Proteica/genética , Proteínas Repressoras/genética , Fatores de Tempo
17.
Eukaryot Cell ; 13(1): 154-69, 2014 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-24243796

RESUMO

Fungi can serve as highly tractable models for understanding genetic basis of sexual development in multicellular organisms. Applying a reverse-genetic approach to advance such a model, we used random and multitargeted primers to assay gene expression across perithecial development in Neurospora crassa. We found that functionally unclassified proteins accounted for most upregulated genes, whereas downregulated genes were enriched for diverse functions. Moreover, genes associated with developmental traits exhibited stage-specific peaks of expression. Expression increased significantly across sexual development for mating type gene mat a-1 and for mat A-1 specific pheromone precursor ccg-4. In addition, expression of a gene encoding a protein similar to zinc finger, stc1, was highly upregulated early in perithecial development, and a strain with a knockout of this gene exhibited arrest at the same developmental stage. A similar expression pattern was observed for genes in RNA silencing and signaling pathways, and strains with knockouts of these genes were also arrested at stages of perithecial development that paralleled their peak in expression. The observed stage specificity allowed us to correlate expression upregulation and developmental progression and to identify regulators of sexual development. Bayesian networks inferred from our expression data revealed previously known and new putative interactions between RNA silencing genes and pathways. Overall, our analysis provides a fine-scale transcriptomic landscape and novel inferences regarding the control of the multistage development process of sexual crossing and fruiting body development in N. crassa.


Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Regulação Fúngica da Expressão Gênica , Genoma Fúngico , Neurospora crassa/genética , Esporos Fúngicos/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Deleção de Genes , Genes Fúngicos Tipo Acasalamento , Neurospora crassa/crescimento & desenvolvimento , Neurospora crassa/metabolismo , Esporos Fúngicos/crescimento & desenvolvimento , Regulação para Cima
18.
bioRxiv ; 2024 Sep 08.
Artigo em Inglês | MEDLINE | ID: mdl-39282330

RESUMO

In utero gene editing has the potential to modify disease causing genes in multiple developing tissues before birth, possibly allowing for normal organ development, disease improvement, and conceivably, cure. In cystic fibrosis (CF), a disease that arises from mutations in the cystic fibrosis transmembrane conductance regulator ( CFTR ) gene, there are signs of multiorgan disease affecting the function of the respiratory, gastrointestinal, and reproductive systems already present at birth. Thus, treating CF patients early is crucial for preventing or delaying irreversible organ damage. Here we demonstrate proof-of-concept of multiorgan mutation correction in CF using peptide nucleic acids (PNAs) encapsulated in polymeric nanoparticles and delivered systemically in utero. In utero editing was associated with sustained postnatal CFTR activity, at a level similar to that of wild-type mice, in both respiratory and gastrointestinal tissue, without detection of off-target mutations in partially homologous loci. This work suggests that systemic in utero gene editing represents a viable strategy for treating monogenic diseases before birth that impact multiple tissue types.

19.
Cancer Res ; 84(8): 1303-1319, 2024 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-38359163

RESUMO

The majority of EGFR mutant lung adenocarcinomas respond well to EGFR tyrosine kinase inhibitors (TKI). However, most of these responses are partial, with drug-tolerant residual disease remaining even at the time of maximal response. This residual disease can ultimately lead to relapses, which eventually develop in most patients. To investigate the cellular and molecular properties of residual tumor cells in vivo, we leveraged patient-derived xenograft (PDX) models of EGFR mutant lung cancer. Subcutaneous EGFR mutant PDXs were treated with the third-generation TKI osimertinib until maximal tumor regression. Residual tissue inevitably harbored tumor cells that were transcriptionally distinct from bulk pretreatment tumor. Single-cell transcriptional profiling provided evidence of cells matching the profiles of drug-tolerant cells present in the pretreatment tumor. In one of the PDXs analyzed, osimertinib treatment caused dramatic transcriptomic changes that featured upregulation of the neuroendocrine lineage transcription factor ASCL1. Mechanistically, ASCL1 conferred drug tolerance by initiating an epithelial-to-mesenchymal gene-expression program in permissive cellular contexts. This study reveals fundamental insights into the biology of drug tolerance, the plasticity of cells through TKI treatment, and why specific phenotypes are observed only in certain tumors. SIGNIFICANCE: Analysis of residual disease following tyrosine kinase inhibitor treatment identified heterogeneous and context-specific mechanisms of drug tolerance in lung cancer that could lead to the development of strategies to forestall drug resistance. See related commentary by Rumde and Burns, p. 1188.


Assuntos
Acrilamidas , Carcinoma Pulmonar de Células não Pequenas , Indóis , Neoplasias Pulmonares , Pirimidinas , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , Receptores ErbB/genética , Resistencia a Medicamentos Antineoplásicos/genética , Recidiva Local de Neoplasia/tratamento farmacológico , Compostos de Anilina/farmacologia , Compostos de Anilina/uso terapêutico , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Mutação , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética
20.
Mycologia ; 105(6): 1350-73, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23935031

RESUMO

We present a phylogenetic and phylogenomic overview of the Polyporales. The newly sequenced genomes of Bjerkandera adusta, Ganoderma sp., and Phlebia brevispora are introduced and an overview of 10 currently available Polyporales genomes is provided. The new genomes are 39 500 000-49 900 00 bp and encode for 12 910-16 170 genes. We searched available genomes for single-copy genes and performed phylogenetic informativeness analyses to evaluate their potential for phylogenetic systematics of the Polyporales. Phylogenomic datasets (25, 71, 356 genes) were assembled for the 10 Polyporales species with genome data and compared with the most comprehensive dataset of Polyporales to date (six-gene dataset for 373 taxa, including taxa with missing data). Maximum likelihood and Bayesian phylogenetic analyses of genomic datasets yielded identical topologies, and the corresponding clades also were recovered in the 373-taxa dataset although with different support values in some datasets. Three previously recognized lineages of Polyporales, antrodia, core polyporoid and phlebioid clades, are supported in most datasets, while the status of the residual polyporoid clade remains uncertain and certain taxa (e.g. Gelatoporia, Grifola, Tyromyces) apparently do not belong to any of the major lineages of Polyporales. The most promising candidate single-copy genes are presented, and nodes in the Polyporales phylogeny critical for the suprageneric taxonomy of the order are identified and discussed.


Assuntos
Filogenia , Polyporales/classificação , Polyporales/genética , Evolução Molecular , Proteínas Fúngicas/genética , Genômica
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