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1.
Cell ; 186(13): 2802-2822.e22, 2023 06 22.
Artículo en Inglés | MEDLINE | ID: mdl-37220746

RESUMEN

Systemic candidiasis is a common, high-mortality, nosocomial fungal infection. Unexpectedly, it has emerged as a complication of anti-complement C5-targeted monoclonal antibody treatment, indicating a critical niche for C5 in antifungal immunity. We identified transcription of complement system genes as the top biological pathway induced in candidemic patients and as predictive of candidemia. Mechanistically, C5a-C5aR1 promoted fungal clearance and host survival in a mouse model of systemic candidiasis by stimulating phagocyte effector function and ERK- and AKT-dependent survival in infected tissues. C5ar1 ablation rewired macrophage metabolism downstream of mTOR, promoting their apoptosis and enhancing mortality through kidney injury. Besides hepatocyte-derived C5, local C5 produced intrinsically by phagocytes provided a key substrate for antifungal protection. Lower serum C5a concentrations or a C5 polymorphism that decreases leukocyte C5 expression correlated independently with poor patient outcomes. Thus, local, phagocyte-derived C5 production licenses phagocyte antimicrobial function and confers innate protection during systemic fungal infection.


Asunto(s)
Antifúngicos , Candidiasis , Animales , Ratones , Complemento C5/metabolismo , Fagocitos/metabolismo
2.
Nat Immunol ; 24(8): 1331-1344, 2023 08.
Artículo en Inglés | MEDLINE | ID: mdl-37443284

RESUMEN

CD4+ T helper 17 (TH17) cells protect barrier tissues but also trigger autoimmunity. The mechanisms behind these opposing processes remain unclear. Here, we found that the transcription factor EGR2 controlled the transcriptional program of pathogenic TH17 cells in the central nervous system (CNS) but not that of protective TH17 cells at barrier sites. EGR2 was significantly elevated in myelin-reactive CD4+ T cells from patients with multiple sclerosis and mice with autoimmune neuroinflammation. The EGR2 transcriptional program was intricately woven within the TH17 cell transcriptional regulatory network and showed high interconnectivity with core TH17 cell-specific transcription factors. Mechanistically, EGR2 enhanced TH17 cell differentiation and myeloid cell recruitment to the CNS by upregulating pathogenesis-associated genes and myelomonocytic chemokines. T cell-specific deletion of Egr2 attenuated neuroinflammation without compromising the host's ability to control infections. Our study shows that EGR2 regulates tissue-specific and disease-specific functions in pathogenic TH17 cells in the CNS.


Asunto(s)
Encefalomielitis Autoinmune Experimental , Esclerosis Múltiple , Animales , Ratones , Diferenciación Celular , Sistema Nervioso Central , Ratones Endogámicos C57BL , Enfermedades Neuroinflamatorias , Células TH1 , Células Th17 , Factores de Transcripción , Virulencia , Humanos
3.
Nat Immunol ; 23(1): 62-74, 2022 01.
Artículo en Inglés | MEDLINE | ID: mdl-34764490

RESUMEN

The molecular mechanisms governing orderly shutdown and retraction of CD4+ type 1 helper T (TH1) cell responses remain poorly understood. Here we show that complement triggers contraction of TH1 responses by inducing intrinsic expression of the vitamin D (VitD) receptor and the VitD-activating enzyme CYP27B1, permitting T cells to both activate and respond to VitD. VitD then initiated the transition from pro-inflammatory interferon-γ+ TH1 cells to suppressive interleukin-10+ cells. This process was primed by dynamic changes in the epigenetic landscape of CD4+ T cells, generating super-enhancers and recruiting several transcription factors, notably c-JUN, STAT3 and BACH2, which together with VitD receptor shaped the transcriptional response to VitD. Accordingly, VitD did not induce interleukin-10 expression in cells with dysfunctional BACH2 or STAT3. Bronchoalveolar lavage fluid CD4+ T cells of patients with COVID-19 were TH1-skewed and showed de-repression of genes downregulated by VitD, from either lack of substrate (VitD deficiency) and/or abnormal regulation of this system.


Asunto(s)
Interferón gamma/inmunología , Interleucina-10/inmunología , SARS-CoV-2/inmunología , Células TH1/inmunología , Vitamina D/metabolismo , 25-Hidroxivitamina D3 1-alfa-Hidroxilasa/metabolismo , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/metabolismo , Líquido del Lavado Bronquioalveolar/citología , COVID-19/inmunología , COVID-19/patología , Complemento C3a/inmunología , Complemento C3b/inmunología , Humanos , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Activación de Linfocitos/inmunología , Receptores de Calcitriol/metabolismo , Síndrome de Dificultad Respiratoria/inmunología , Síndrome de Dificultad Respiratoria/patología , Síndrome de Dificultad Respiratoria/virología , Factor de Transcripción STAT3/metabolismo , Transducción de Señal/inmunología , Transcripción Genética/genética
4.
Cell ; 178(5): 1145-1158.e20, 2019 08 22.
Artículo en Inglés | MEDLINE | ID: mdl-31402173

RESUMEN

While Mediator plays a key role in eukaryotic transcription, little is known about its mechanism of action. This study combines CRISPR-Cas9 genetic screens, degron assays, Hi-C, and cryoelectron microscopy (cryo-EM) to dissect the function and structure of mammalian Mediator (mMED). Deletion analyses in B, T, and embryonic stem cells (ESC) identified a core of essential subunits required for Pol II recruitment genome-wide. Conversely, loss of non-essential subunits mostly affects promoters linked to multiple enhancers. Contrary to current models, however, mMED and Pol II are dispensable to physically tether regulatory DNA, a topological activity requiring architectural proteins. Cryo-EM analysis revealed a conserved core, with non-essential subunits increasing structural complexity of the tail module, a primary transcription factor target. Changes in tail structure markedly increase Pol II and kinase module interactions. We propose that Mediator's structural pliability enables it to integrate and transmit regulatory signals and act as a functional, rather than an architectural bridge, between promoters and enhancers.


Asunto(s)
Complejo Mediador/metabolismo , ARN Polimerasa II/metabolismo , Animales , Linfocitos T CD4-Positivos/citología , Linfocitos T CD4-Positivos/metabolismo , Sistemas CRISPR-Cas/genética , Proteínas de Ciclo Celular/metabolismo , Células Cultivadas , Proteínas Cromosómicas no Histona/metabolismo , Microscopía por Crioelectrón , Elementos de Facilitación Genéticos , Edición Génica , Humanos , Masculino , Complejo Mediador/química , Complejo Mediador/genética , Ratones , Ratones Endogámicos C57BL , Células Madre Embrionarias de Ratones/citología , Células Madre Embrionarias de Ratones/metabolismo , Regiones Promotoras Genéticas , Estructura Cuaternaria de Proteína , ARN Polimerasa II/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Cohesinas
5.
Nat Immunol ; 22(3): 370-380, 2021 03.
Artículo en Inglés | MEDLINE | ID: mdl-33574619

RESUMEN

During chronic infection and cancer, a self-renewing CD8+ T cell subset maintains long-term immunity and is critical to the effectiveness of immunotherapy. These stem-like CD8+ T cells diverge from other CD8+ subsets early after chronic viral infection. However, pathways guarding stem-like CD8+ T cells against terminal exhaustion remain unclear. Here, we show that the gene encoding transcriptional repressor BACH2 is transcriptionally and epigenetically active in stem-like CD8+ T cells but not terminally exhausted cells early after infection. BACH2 overexpression enforced stem-like cell fate, whereas BACH2 deficiency impaired stem-like CD8+ T cell differentiation. Single-cell transcriptomic and epigenomic approaches revealed that BACH2 established the transcriptional and epigenetic programs of stem-like CD8+ T cells. In addition, BACH2 suppressed the molecular program driving terminal exhaustion through transcriptional repression and epigenetic silencing. Thus, our study reveals a new pathway that enforces commitment to stem-like CD8+ lineage and prevents an alternative terminally exhausted cell fate.


Asunto(s)
Infecciones por Arenaviridae/metabolismo , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/metabolismo , Linfocitos T CD8-positivos/metabolismo , Diferenciación Celular , Epigénesis Genética , Células Precursoras de Linfocitos T/metabolismo , Transcripción Genética , Animales , Infecciones por Arenaviridae/genética , Infecciones por Arenaviridae/inmunología , Infecciones por Arenaviridae/virología , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/deficiencia , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/genética , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/virología , Linaje de la Célula , Células Cultivadas , Enfermedad Crónica , Modelos Animales de Enfermedad , Interacciones Huésped-Patógeno , Virus de la Coriomeningitis Linfocítica/inmunología , Virus de la Coriomeningitis Linfocítica/patogenicidad , Ratones Endogámicos C57BL , Ratones Noqueados , Fenotipo , Células Precursoras de Linfocitos T/inmunología , Células Precursoras de Linfocitos T/virología , Transducción de Señal
6.
Nat Immunol ; 19(12): 1403-1414, 2018 12.
Artículo en Inglés | MEDLINE | ID: mdl-30397350

RESUMEN

Repair of tissue damaged during inflammatory processes is key to the return of local homeostasis and restoration of epithelial integrity. Here we describe CD161+ regulatory T (Treg) cells as a distinct, highly suppressive population of Treg cells that mediate wound healing. These Treg cells were enriched in intestinal lamina propria, particularly in Crohn's disease. CD161+ Treg cells had an all-trans retinoic acid (ATRA)-regulated gene signature, and CD161 expression on Treg cells was induced by ATRA, which directly regulated the CD161 gene. CD161 was co-stimulatory, and ligation with the T cell antigen receptor induced cytokines that accelerated the wound healing of intestinal epithelial cells. We identified a transcription-factor network, including BACH2, RORγt, FOSL2, AP-1 and RUNX1, that controlled expression of the wound-healing program, and found a CD161+ Treg cell signature in Crohn's disease mucosa associated with reduced inflammation. These findings identify CD161+ Treg cells as a population involved in controlling the balance between inflammation and epithelial barrier healing in the gut.


Asunto(s)
Mucosa Intestinal/inmunología , Subfamilia B de Receptores Similares a Lectina de Células NK/inmunología , Subgrupos de Linfocitos T/inmunología , Linfocitos T Reguladores/inmunología , Tretinoina/inmunología , Cicatrización de Heridas/inmunología , Enfermedad de Crohn/inmunología , Humanos
7.
Immunity ; 52(3): 513-527.e8, 2020 03 17.
Artículo en Inglés | MEDLINE | ID: mdl-32187519

RESUMEN

Intrinsic complement C3 activity is integral to human T helper type 1 (Th1) and cytotoxic T cell responses. Increased or decreased intracellular C3 results in autoimmunity and infections, respectively. The mechanisms regulating intracellular C3 expression remain undefined. We identified complement, including C3, as among the most significantly enriched biological pathway in tissue-occupying cells. We generated C3-reporter mice and confirmed that C3 expression was a defining feature of tissue-immune cells, including T cells and monocytes, occurred during transendothelial diapedesis, and depended on integrin lymphocyte-function-associated antigen 1 (LFA-1) signals. Immune cells from patients with leukocyte adhesion deficiency type 1 (LAD-1) had reduced C3 transcripts and diminished effector activities, which could be rescued proportionally by intracellular C3 provision. Conversely, increased C3 expression by T cells from arthritis patients correlated with disease severity. Our study defines integrins as key controllers of intracellular complement, demonstrates that perturbations in the LFA-1-C3-axis contribute to primary immunodeficiency, and identifies intracellular C3 as biomarker of severity in autoimmunity.


Asunto(s)
Complemento C3/inmunología , Integrinas/inmunología , Antígeno-1 Asociado a Función de Linfocito/inmunología , Linfocitos/inmunología , Monocitos/inmunología , Migración Transendotelial y Transepitelial/inmunología , Adulto , Anciano , Animales , Artritis Reumatoide/inmunología , Artritis Reumatoide/metabolismo , Artritis Reumatoide/patología , Niño , Preescolar , Complemento C3/genética , Complemento C3/metabolismo , Femenino , Humanos , Integrinas/metabolismo , Antígeno-1 Asociado a Función de Linfocito/metabolismo , Linfocitos/metabolismo , Masculino , Ratones Endogámicos C57BL , Persona de Mediana Edad , Monocitos/metabolismo , Transducción de Señal/inmunología
9.
J Autoimmun ; 134: 102982, 2023 01.
Artículo en Inglés | MEDLINE | ID: mdl-36592512

RESUMEN

Immune cell function is critically dependent on precise control over transcriptional output from the genome. In this respect, integration of environmental signals that regulate gene expression, specifically by transcription factors, enhancer DNA elements, genome topography and non-coding RNAs (ncRNAs), are key components. The first three have been extensively investigated. Even though non-coding RNAs represent the vast majority of cellular RNA species, this class of RNA remains historically understudied. This is partly because of a lag in technological and bioinformatic innovations specifically capable of identifying and accurately measuring their expression. Nevertheless, recent progress in this domain has enabled a profusion of publications identifying novel sub-types of ncRNAs and studies directly addressing the function of ncRNAs in human health and disease. Many ncRNAs, including circular and enhancer RNAs, have now been demonstrated to play key functions in the regulation of immune cells and to show associations with immune-mediated diseases. Some ncRNAs may function as biomarkers of disease, aiding in diagnostics and in estimating response to treatment, while others may play a direct role in the pathogenesis of disease. Importantly, some are relatively stable and are amenable to therapeutic targeting, for example through gene therapy. Here, we provide an overview of ncRNAs and review technological advances that enable their study and hold substantial promise for the future. We provide context-specific examples by examining the associations of ncRNAs with four prototypical human autoimmune diseases, specifically rheumatoid arthritis, psoriasis, inflammatory bowel disease and multiple sclerosis. We anticipate that the utility and mechanistic roles of these ncRNAs in autoimmunity will be further elucidated in the near future.


Asunto(s)
Artritis Reumatoide , Enfermedades Autoinmunes , Esclerosis Múltiple , Humanos , Autoinmunidad/genética , ARN no Traducido/genética , ARN no Traducido/metabolismo , Enfermedades Autoinmunes/diagnóstico , Enfermedades Autoinmunes/genética
10.
Exp Cell Res ; 412(2): 113043, 2022 03 15.
Artículo en Inglés | MEDLINE | ID: mdl-35101390

RESUMEN

The terminal steps of lens cell differentiation require elimination of all organelles to create a central Organelle Free Zone (OFZ) that is required for lens function of focusing images on the retina. Previous studies show that the spatiotemporal elimination of these organelles during development is autophagy-dependent. We now show that the inhibition of PI3K signaling in lens organ culture results in the premature induction of autophagy within 24 h, including a significant increase in LAMP1+ lysosomes, and the removal of lens organelles from the center of the lens. Specific inhibition of just the PI3K/Akt signaling axis was directly linked to the elimination of mitochondria and ER, while pan-PI3K inhibitors that block all PI3K downstream signaling removed all organelles, including nuclei. Therefore, blocking the PI3K/Akt pathway was alone insufficient to remove nuclei. RNAseq analysis revealed increased mRNA levels of the endogenous inhibitor of PI3K activation, PIK3IP1, in differentiating lens fiber cells preceding the induction of OFZ formation. Co-immunoprecipitation confirmed that PIK3IP1 associates with multiple PI3K p110 isoforms just prior to formation of the OFZ, providing a likely endogenous mechanism for blocking all PI3K signaling and activating the autophagy pathway required to form the OFZ during lens development.


Asunto(s)
Autofagia/fisiología , Cristalino/fisiología , Fosfatidilinositol 3-Quinasas/metabolismo , Transducción de Señal/fisiología , Animales , Diferenciación Celular/fisiología , Núcleo Celular/metabolismo , Núcleo Celular/fisiología , Embrión de Pollo , Células Epiteliales/metabolismo , Células Epiteliales/fisiología , Ojo/metabolismo , Ojo/fisiopatología , Cristalino/metabolismo , Mitocondrias/metabolismo , Mitocondrias/fisiología , Proteínas Proto-Oncogénicas c-akt/metabolismo
11.
Immunology ; 166(3): 299-309, 2022 07.
Artículo en Inglés | MEDLINE | ID: mdl-35322416

RESUMEN

In CD4+ T helper cells, the active form of vitamin D3 , 1,25-dihydroxyvitamin D3 (1,25D) suppresses production of inflammatory cytokines, including interferon-gamma (IFN-γ), but the mechanisms for this are not yet fully defined. In innate immune cells, response to 1,25D has been linked to metabolic reprogramming. It is unclear whether 1,25D has similar effects on CD4+ T cells, although it is known that antigen stimulation of these cells promotes an anabolic metabolic phenotype, characterized by high rates of aerobic glycolysis to support clonal expansion and effector cytokine expression. Here, we performed in-depth analysis of metabolic capacity and pathway usage, employing extracellular flux and stable isotope-based tracing approaches, in CD4+ T cells treated with 1,25D. We report that 1,25D significantly decreases rates of aerobic glycolysis in activated CD4+ T cells, whilst exerting a lesser effect on mitochondrial glucose oxidation. This is associated with transcriptional repression of Myc, but not repression of mTOR activity under these conditions. Consistent with the modest effect of 1,25D on mitochondrial activity, it also did not impact CD4+ T-cell mitochondrial mass or membrane potential. Finally, we demonstrate that inhibition of aerobic glycolysis by 1,25D substantially contributes to its immune-regulatory capacity in CD4+ T cells, since the suppression of IFN-γ expression was significantly blunted in the absence of aerobic glycolysis. 1,25-Dihydroxyvitamin D3 (1,25D) suppresses the production of inflammatory cytokines such as interferon-gamma (IFN-γ) by CD4+ T cells, but the underpinning mechanisms are not yet fully defined. Here, we identify that 1,25D inhibits aerobic glycolysis in activated CD4+ T cells, associated with decreased c-Myc expression. This mechanism appears to substantially contribute to the suppression of IFN-γ by 1,25D, since this is significantly blunted in the absence of aerobic glycolysis.


Asunto(s)
Calcitriol , Interferón gamma , Calcitriol/metabolismo , Calcitriol/farmacología , Glucólisis , Interferón gamma/metabolismo , Linfocitos T Colaboradores-Inductores/metabolismo , Vitamina D
12.
Am J Physiol Renal Physiol ; 322(6): F597-F610, 2022 06 01.
Artículo en Inglés | MEDLINE | ID: mdl-35379003

RESUMEN

We have previously reported that increased expression and activation of kidney cell complement components play an important role in the pathogenesis of renal scarring. Here, we used floxed green fluorescent protein (GFP)-C5a receptor 1 (C5aR1) knockin mice (GFP-C5ar1fl/fl) and the model of folic acid (FA)-induced kidney injury to define the cell types and potential mechanisms by which increased C5aR1 activation leads to fibrosis. Using flow cytometry and confocal microscopy, we identified macrophages as the major interstitial cell type showing increased expression of C5aR1 in FA-treated mice. C5ar1fl/fl.Lyz2Cre+/- mice, in which C5aR1 has been specifically deleted in lysozyme M-expressing myeloid cells, experienced reduced fibrosis compared with control C5ar1fl/fl mice. Examination of C5aR1-expressing macrophage transcriptomes by gene set enrichment analysis demonstrated that these cells were enriched in pathways corresponding to the complement cascade, collagen formation, and the NABA matrisome, strongly pointing to their critical roles in tissue repair/scarring. Since C5aR1 was also detected in a small population of platelet-derived growth factor receptor-ß+ GFP+ cells, we developed C5ar1fl/fl.Foxd1Cre+/- mice, in which C5aR1 is deleted specifically in pericytes, and found reduced FA-induced fibrosis. Primary cell cultures of platelet-derived growth factor receptor-ß+ pericytes isolated from FA-treated C5ar1fl/fl.Foxd1Cre+/- mice showed reduced secretion of several cytokines, including IL-6 and macrophage inflammatory protein-2, compared with pericytes isolated from FA-treated control GFP-C5ar1fl/fl mice. Collectively, these data imply that C5a/C5aR1 axis activation primarily in interstitial cells contributes to the development of renal fibrosis.NEW & NOTEWORTHY This study used novel green fluorescent protein C5a receptor 1 floxed mice and the model of folic acid-mediated kidney fibrosis to demonstrate the pathogenic role of increased expression of this complement receptor on macrophages.


Asunto(s)
Ácido Fólico , Receptor de Anafilatoxina C5a , Animales , Cicatriz , Fibrosis , Ácido Fólico/farmacología , Proteínas Fluorescentes Verdes , Riñón/patología , Ratones , Ratones Noqueados , Células Mieloides/patología , Receptor de Anafilatoxina C5a/genética , Receptores del Factor de Crecimiento Derivado de Plaquetas
13.
J Virol ; 95(15): e0029421, 2021 07 12.
Artículo en Inglés | MEDLINE | ID: mdl-33980601

RESUMEN

The pathogenic mechanisms underlying severe SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) infection remain largely unelucidated. High-throughput sequencing technologies that capture genome and transcriptome information are key approaches to gain detailed mechanistic insights from infected cells. These techniques readily detect both pathogen- and host-derived sequences, providing a means of studying host-pathogen interactions. Recent studies have reported the presence of host-virus chimeric (HVC) RNA in transcriptome sequencing (RNA-seq) data from SARS-CoV-2-infected cells and interpreted these findings as evidence of viral integration in the human genome as a potential pathogenic mechanism. Since SARS-CoV-2 is a positive-sense RNA virus that replicates in the cytoplasm, it does not have a nuclear phase in its life cycle. Thus, it is biologically unlikely to be in a location where splicing events could result in genome integration. Therefore, we investigated the biological authenticity of HVC events. In contrast to true biological events like mRNA splicing and genome rearrangement events, which generate reproducible chimeric sequencing fragments across different biological isolates, we found that HVC events across >100 RNA-seq libraries from patients with coronavirus disease 2019 (COVID-19) and infected cell lines were highly irreproducible. RNA-seq library preparation is inherently error prone due to random template switching during reverse transcription of RNA to cDNA. By counting chimeric events observed when constructing an RNA-seq library from human RNA and spiked-in RNA from an unrelated species, such as the fruit fly, we estimated that ∼1% of RNA-seq reads are artifactually chimeric. In SARS-CoV-2 RNA-seq, we found that the frequency of HVC events was, in fact, not greater than this background "noise." Finally, we developed a novel experimental approach to enrich SARS-CoV-2 sequences from bulk RNA of infected cells. This method enriched viral sequences but did not enrich HVC events, suggesting that the majority of HVC events are, in all likelihood, artifacts of library construction. In conclusion, our findings indicate that HVC events observed in RNA-sequencing libraries from SARS-CoV-2-infected cells are extremely rare and are likely artifacts arising from random template switching of reverse transcriptase and/or sequence alignment errors. Therefore, the observed HVC events do not support SARS-CoV-2 fusion to cellular genes and/or integration into human genomes. IMPORTANCE The pathogenic mechanisms underlying SARS-CoV-2, the virus responsible for COVID-19, are not fully understood. In particular, relatively little is known about the reasons some individuals develop life-threatening or persistent COVID-19. Recent studies identified host-virus chimeric (HVC) reads in RNA-sequencing data from SARS-CoV-2-infected cells and suggested that HVC events support potential "human genome invasion" and "integration" by SARS-CoV-2. This suggestion has fueled concerns about the long-term effects of current mRNA vaccines that incorporate elements of the viral genome. SARS-CoV-2 is a positive-sense, single-stranded RNA virus that does not encode a reverse transcriptase and does not include a nuclear phase in its life cycle, so some doubts have rightfully been expressed regarding the authenticity of HVCs and the role played by endogenous retrotransposons in this phenomenon. Thus, it is important to independently authenticate these HVC events. Here, we provide several lines of evidence suggesting that the observed HVC events are likely artifactual.


Asunto(s)
COVID-19/metabolismo , Interacciones Huésped-Patógeno , ARN Viral/metabolismo , RNA-Seq , SARS-CoV-2/fisiología , Replicación Viral , COVID-19/genética , COVID-19/patología , Línea Celular Tumoral , Humanos , ARN Viral/genética
14.
BMC Genomics ; 22(1): 497, 2021 Jul 03.
Artículo en Inglés | MEDLINE | ID: mdl-34215186

RESUMEN

BACKGROUND: During eye lens development the embryonic vasculature regresses leaving the lens without a direct oxygen source. Both embryonically and throughout adult life, the lens contains a decreasing oxygen gradient from the surface to the core that parallels the natural differentiation of immature surface epithelial cells into mature core transparent fiber cells. These properties of the lens suggest a potential role for hypoxia and the master regulator of the hypoxic response, hypoxia-inducible transcription factor 1 (HIF1), in the regulation of genes required for lens fiber cell differentiation, structure and transparency. Here, we employed a multiomics approach combining CUT&RUN, RNA-seq and ATACseq analysis to establish the genomic complement of lens HIF1α binding sites, genes activated or repressed by HIF1α and the chromatin states of HIF1α-regulated genes. RESULTS: CUT&RUN analysis revealed 8375 HIF1α-DNA binding complexes in the chick lens genome. One thousand one hundred ninety HIF1α-DNA binding complexes were significantly clustered within chromatin accessible regions (χ2 test p < 1 × 10- 55) identified by ATACseq. Formation of the identified HIF1α-DNA complexes paralleled the activation or repression of 526 genes, 116 of which contained HIF1α binding sites within 10kB of the transcription start sites. Some of the identified HIF1α genes have previously established lens functions while others have novel functions never before examined in the lens. GO and pathway analysis of these genes implicate HIF1α in the control of a wide-variety of cellular pathways potentially critical for lens fiber cell formation, structure and function including glycolysis, cell cycle regulation, chromatin remodeling, Notch and Wnt signaling, differentiation, development, and transparency. CONCLUSIONS: These data establish the first functional map of genomic HIF1α-DNA complexes in the eye lens. They identify HIF1α as an important regulator of a wide-variety of genes previously shown to be critical for lens formation and function and they reveal a requirement for HIF1α in the regulation of a wide-variety of genes not yet examined for lens function. They support a requirement for HIF1α in lens fiber cell formation, structure and function and they provide a basis for understanding the potential roles and requirements for HIF1α in the development, structure and function of more complex tissues.


Asunto(s)
Cristalino , Diferenciación Celular , Cromatina , ADN , Genómica , Subunidad alfa del Factor 1 Inducible por Hipoxia
15.
Dev Biol ; 453(1): 86-104, 2019 09 01.
Artículo en Inglés | MEDLINE | ID: mdl-31136738

RESUMEN

Changes in chromatin accessibility regulate the expression of multiple genes by controlling transcription factor access to key gene regulatory sequences. Here, we sought to establish a potential function for altered chromatin accessibility in control of key gene expression events during lens cell differentiation by establishing genome-wide chromatin accessibility maps specific for four distinct stages of lens cell differentiation and correlating specific changes in chromatin accessibility with genome-wide changes in gene expression. ATAC sequencing was employed to generate chromatin accessibility profiles that were correlated with the expression profiles of over 10,000 lens genes obtained by high-throughput RNA sequencing at the same stages of lens cell differentiation. Approximately 90,000 regions of the lens genome exhibited distinct changes in chromatin accessibility at one or more stages of lens differentiation. Over 1000 genes exhibited high Pearson correlation coefficients (r â€‹> â€‹0.7) between altered expression levels at specific stages of lens cell differentiation and changes in chromatin accessibility in potential promoter (-7.5kbp/+2.5kbp of the transcriptional start site) and/or other potential cis-regulatory regions ( ±10 â€‹kb of the gene body). Analysis of these regions identified consensus binding sequences for multiple transcription factors including members of the TEAD, FOX, and NFAT families of transcription factors as well as HIF1a, RBPJ and IRF1. Functional mapping of genes with high correlations between altered chromatin accessibility and differentiation state-specific gene expression changes identified multiple families of proteins whose expression could be regulated through changes in chromatin accessibility including those governing lens structure (BFSP1,BFSP2), gene expression (Pax-6, Sox 2), translation (TDRD7), cell-cell communication (GJA1), autophagy (FYCO1), signal transduction (SMAD3, EPHA2), and lens transparency (CRYBB1, CRYBA4). These data provide a novel relationship between altered chromatin accessibility and lens differentiation and they identify a wide-variety of lens genes and functions that could be regulated through altered chromatin accessibility. The data also point to a large number of potential DNA regulatory sequences and transcription factors whose functional analysis is likely to provide insight into novel regulatory mechanisms governing the lens differentiation program.


Asunto(s)
Diferenciación Celular/genética , Cromatina/metabolismo , Regulación del Desarrollo de la Expresión Génica , Cristalino/citología , Animales , Secuencia de Bases , Sitios de Unión , Biomarcadores/metabolismo , Pollos/genética , Secuencia de Consenso/genética , ADN/metabolismo , Genoma , Cristalino/metabolismo , Unión Proteica , ARN Mensajero/genética , ARN Mensajero/metabolismo , Factores de Transcripción/metabolismo
17.
J Biol Chem ; 290(51): 30253-66, 2015 Dec 18.
Artículo en Inglés | MEDLINE | ID: mdl-26527683

RESUMEN

Accumulation of apoptotic material is toxic and associated with cataract and other disease states. Identification of mechanisms that prevent accumulation of apoptotic debris is important for establishing the etiology of these diseases. The ocular lens is routinely assaulted by UV light that causes lens cell apoptosis and is associated with cataract formation. To date, no molecular mechanism for removal of toxic apoptotic debris has been identified in the lens. Vesicular debris within lens cells exposed to UV light has been observed raising speculation that lens cells themselves could act as phagocytes to remove toxic apoptotic debris. However, phagocytosis has not been confirmed as a function of the intact eye lens, and no mechanism for lens phagocytosis has been established. Here, we demonstrate that the eye lens is capable of phagocytizing extracellular lens cell debris. Using high throughput RNA sequencing and bioinformatics analysis, we establish that lens epithelial cells express members of the integrin αVß5-mediated phagocytosis pathway and that internalized cell debris co-localizes with αVß5 and with RAB7 and Rab-interacting lysosomal protein that are required for phagosome maturation and fusion with lysosomes. We demonstrate that the αVß5 receptor is required for lens epithelial cell phagocytosis and that UV light treatment of lens epithelial cells results in damage to the αVß5 receptor with concomitant loss of phagocytosis. These data suggest that loss of αVß5-mediated phagocytosis by the eye lens could result in accumulation of toxic cell debris that could contribute to UV light-induced cataract formation.


Asunto(s)
Apoptosis/efectos de la radiación , Proteínas Aviares/metabolismo , Células Epiteliales/metabolismo , Proteínas del Ojo/metabolismo , Cristalino/metabolismo , Receptores de Vitronectina/metabolismo , Rayos Ultravioleta/efectos adversos , Animales , Línea Celular , Embrión de Pollo , Pollos , Humanos , Lisosomas/metabolismo , Fagocitosis/efectos de la radiación , Proteínas de Unión al GTP rab/metabolismo , Proteínas de Unión a GTP rab7
18.
Mol Vis ; 21: 955-73, 2015.
Artículo en Inglés | MEDLINE | ID: mdl-26330747

RESUMEN

PURPOSE: Gene expression correlates with local chromatin structure. Our studies have mapped histone post-translational modifications, RNA polymerase II (pol II), and transcription factor Pax6 in lens chromatin. These data represent the first genome-wide insights into the relationship between lens chromatin structure and lens transcriptomes and serve as an excellent source for additional data analysis and refinement. The principal lens proteins, the crystallins, are encoded by predominantly expressed mRNAs; however, the regulatory mechanisms underlying their high expression in the lens remain poorly understood. METHODS: The formaldehyde-assisted identification of regulatory regions (FAIRE-Seq) was employed to analyze newborn lens chromatin. ChIP-seq and RNA-seq data published earlier (GSE66961) have been used to assist in FAIRE-seq data interpretation. RNA transcriptomes from murine lens epithelium, lens fibers, erythrocytes, forebrain, liver, neurons, and pancreas were compared to establish the gene expression levels of the most abundant mRNAs versus median gene expression across other differentiated cells. RESULTS: Normalized RNA expression data from multiple tissues show that crystallins rank among the most highly expressed genes in mammalian cells. These findings correlate with the extremely high abundance of pol II all across the crystallin loci, including crystallin genes clustered on chromosomes 1 and 5, as well as within regions of "open" chromatin, as identified by FAIRE-seq. The expression levels of mRNAs encoding DNA-binding transcription factors (e.g., Foxe3, Hsf4, Maf, Pax6, Prox1, Sox1, and Tfap2a) revealed that their transcripts form "clusters" of abundant mRNAs in either lens fibers or lens epithelium. The expression of three autophagy regulatory mRNAs, encoding Tfeb, FoxO1, and Hif1α, was found within a group of lens preferentially expressed transcription factors compared to the E12.5 forebrain. CONCLUSIONS: This study reveals novel features of lens chromatin, including the remarkably high abundance of pol II at the crystallin loci that exhibit features of "open" chromatin. Hsf4 ranks among the most abundant fiber cell-preferred DNA-binding transcription factors. Notable transcripts, including Atf4, Ctcf, E2F4, Hey1, Hmgb1, Mycn, RXRß, Smad4, Sp1, and Taf1 (transcription factors) and Ctsd, Gabarapl1, and Park7 (autophagy regulators) have been identified with high levels of expression in lens fibers, which suggests specific roles in lens fiber cell terminal differentiation.


Asunto(s)
Autofagia/genética , Cristalinas/genética , Cristalino/metabolismo , ARN Polimerasa II/metabolismo , Factores de Transcripción/genética , Animales , Canales de Calcio/genética , Diferenciación Celular/genética , Cromatina/genética , Cromatina/metabolismo , Expresión Génica , Hemoglobinas/genética , Cristalino/citología , Ratones , ARN Mensajero/genética , ARN Mensajero/metabolismo
19.
Sci Transl Med ; 16(765): eadk0845, 2024 09 18.
Artículo en Inglés | MEDLINE | ID: mdl-39292801

RESUMEN

Autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED) is a life-threatening monogenic autoimmune disorder primarily caused by biallelic deleterious variants in the autoimmune regulator (AIRE) gene. We prospectively evaluated 104 patients with clinically diagnosed APECED syndrome and identified 17 patients (16%) from 14 kindreds lacking biallelic AIRE variants in exons or flanking intronic regions; 15 had Puerto Rican ancestry. Through whole-genome sequencing, we identified a deep intronic AIRE variant (c.1504-818 G>A) cosegregating with the disease in all 17 patients. We developed a culture system of AIRE-expressing primary patient monocyte-derived dendritic cells and demonstrated that c.1504-818 G>A creates a cryptic splice site and activates inclusion of a 109-base pair frame-shifting pseudoexon. We also found low-level AIRE expression in patient-derived lymphoblastoid cell lines (LCLs) and confirmed pseudoexon inclusion in independent extrathymic AIRE-expressing cell lines. Through protein modeling and transcriptomic analyses of AIRE-transfected human embryonic kidney 293 and thymic epithelial cell 4D6 cells, we showed that this variant alters the carboxyl terminus of the AIRE protein, abrogating its function. Last, we developed an antisense oligonucleotide (ASO) that reversed pseudoexon inclusion and restored the normal AIRE transcript sequence in LCLs. Thus, our findings revealed c.1504-818 G>A as a founder APECED-causing AIRE variant in the Puerto Rican population and uncovered pseudoexon inclusion as an ASO-reversible genetic mechanism underlying APECED.


Asunto(s)
Proteína AIRE , Exones , Intrones , Oligonucleótidos Antisentido , Poliendocrinopatías Autoinmunes , Factores de Transcripción , Adolescente , Adulto , Niño , Femenino , Humanos , Masculino , Secuencia de Bases , Línea Celular , Exones/genética , Intrones/genética , Mutación/genética , Linaje , Poliendocrinopatías Autoinmunes/genética , Empalme del ARN/genética , Factores de Transcripción/genética , Factores de Transcripción/metabolismo
20.
Biochim Biophys Acta ; 1820(7): 921-30, 2012 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-22521365

RESUMEN

BACKGROUND: αB-crystallin/sHSP protects cells against oxidative stress damage. Here, we mechanistically examined its ability to preserve mitochondrial function in lens and retinal cells and protect cytochrome c under oxidative stress conditions. METHODS: αB-crystallin/sHSP was localized in human lens (HLE-B3) and retinal (ARPE-19) cells. αB-crystallin/sHSP was stably over-expressed and its ability to preserve mitochondrial membrane potential under oxidative stress conditions was monitored. Interactions between αB-crystallin/sHSP and cytochrome c were examined by fluorescent resonance energy transfer (FRET) and by co-immune precipitation. The ability of αB-crystallin/sHSP to protect cytochrome c against methionine-80 oxidation was monitored. RESULTS: αB-crystallin/sHSP is present in the mitochondria of lens and retinal cells and is translocated to the mitochondria under oxidative conditions. αB-crystallin/sHSP specifically interacts with cytochrome c in vitro and in vivo and its overexpression preserves mitochondrial membrane potential under oxidative stress conditions. αB-crystallin/sHSP directly protects cytochrome c against oxidation. GENERAL SIGNIFICANCE: These data demonstrate that αB-crystallin/sHSP maintains lens and retinal cells under oxidative stress conditions at least in part by preserving mitochondrial function and by protecting cytochrome c against oxidation. Since oxidative stress and loss of mitochondrial function are associated with eye lens cataract and age-related macular degeneration, loss of these αB-crystallin/sHSP functions likely plays a key role in the development of these diseases. αB-crystallin/sHSP is expressed throughout the body and its ability to maintain mitochondrial function is likely important for the prevention of multiple degenerative diseases.


Asunto(s)
Citocromos c/metabolismo , Cristalino/metabolismo , Mitocondrias/metabolismo , Estrés Oxidativo , Epitelio Pigmentado de la Retina/metabolismo , Cadena B de alfa-Cristalina/metabolismo , Western Blotting , Células Cultivadas , Humanos , Inmunoprecipitación , Cristalino/citología , Potencial de la Membrana Mitocondrial , Oxidación-Reducción , Epitelio Pigmentado de la Retina/citología , Transducción de Señal/efectos de los fármacos
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