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1.
PLoS Pathog ; 17(11): e1010074, 2021 11.
Artículo en Inglés | MEDLINE | ID: mdl-34793580

RESUMEN

Various pathogens systematically reprogram gene expression in macrophages, but the underlying mechanisms are largely unknown. We investigated whether the enteropathogen Yersinia enterocolitica alters chromatin states to reprogram gene expression in primary human macrophages. Genome-wide chromatin immunoprecipitation (ChIP) seq analyses showed that pathogen-associated molecular patterns (PAMPs) induced up- or down-regulation of histone modifications (HMod) at approximately 14500 loci in promoters and enhancers. Effectors of Y. enterocolitica reorganized about half of these dynamic HMod, with the effector YopP being responsible for about half of these modulatory activities. The reorganized HMod were associated with genes involved in immune response and metabolism. Remarkably, the altered HMod also associated with 61% of all 534 known Rho GTPase pathway genes, revealing a new level in Rho GTPase regulation and a new aspect of bacterial pathogenicity. Changes in HMod were associated to varying degrees with corresponding gene expression, e. g. depending on chromatin localization and cooperation of the HMod. In summary, infection with Y. enterocolitica remodels HMod in human macrophages to modulate key gene expression programs of the innate immune response.


Asunto(s)
Epigénesis Genética , Código de Histonas , Inmunidad Innata , Macrófagos/microbiología , Yersiniosis/microbiología , Yersinia enterocolitica/patogenicidad , Proteínas de Unión al GTP rho/metabolismo , Humanos , Macrófagos/inmunología , Macrófagos/metabolismo , Yersiniosis/genética , Yersiniosis/inmunología , Yersiniosis/metabolismo , Proteínas de Unión al GTP rho/genética
2.
PLoS Pathog ; 12(6): e1005660, 2016 06.
Artículo en Inglés | MEDLINE | ID: mdl-27300509

RESUMEN

Yersinia outer protein M (YopM) is a crucial immunosuppressive effector of the plaque agent Yersinia pestis and other pathogenic Yersinia species. YopM enters the nucleus of host cells but neither the mechanisms governing its nucleocytoplasmic shuttling nor its intranuclear activities are known. Here we identify the DEAD-box helicase 3 (DDX3) as a novel interaction partner of Y. enterocolitica YopM and present the three-dimensional structure of a YopM:DDX3 complex. Knockdown of DDX3 or inhibition of the exportin chromosomal maintenance 1 (CRM1) increased the nuclear level of YopM suggesting that YopM exploits DDX3 to exit the nucleus via the CRM1 export pathway. Increased nuclear YopM levels caused enhanced phosphorylation of Ribosomal S6 Kinase 1 (RSK1) in the nucleus. In Y. enterocolitica infected primary human macrophages YopM increased the level of Interleukin-10 (IL-10) mRNA and this effect required interaction of YopM with RSK and was enhanced by blocking YopM's nuclear export. We propose that the DDX3/CRM1 mediated nucleocytoplasmic shuttling of YopM determines the extent of phosphorylation of RSK in the nucleus to control transcription of immunosuppressive cytokines.


Asunto(s)
Proteínas de la Membrana Bacteriana Externa/metabolismo , ARN Helicasas DEAD-box/metabolismo , Regulación de la Expresión Génica/fisiología , Proteínas Quinasas S6 Ribosómicas 90-kDa/biosíntesis , Yersiniosis/inmunología , Proteínas de la Membrana Bacteriana Externa/química , Proteínas de la Membrana Bacteriana Externa/inmunología , Western Blotting , Línea Celular , Núcleo Celular/metabolismo , Cristalografía por Rayos X , ARN Helicasas DEAD-box/química , ARN Helicasas DEAD-box/inmunología , Técnica del Anticuerpo Fluorescente , Secuenciación de Nucleótidos de Alto Rendimiento , Interacciones Huésped-Parásitos/fisiología , Humanos , Tolerancia Inmunológica/fisiología , Inmunoprecipitación , Macrófagos/microbiología , Espectrometría de Masas , Microscopía Confocal , Reacción en Cadena de la Polimerasa , Transporte de Proteínas/fisiología , Factores de Virulencia/inmunología , Factores de Virulencia/metabolismo , Yersiniosis/metabolismo , Yersinia enterocolitica
3.
Eur J Cell Biol ; 102(4): 151364, 2023 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-37806297

RESUMEN

The multifunctional Yersinia effector YopM inhibits effector triggered immunity and increases production of the anti-inflammatory cytokine Interleukin-10 (IL-10) to suppress the host immune response. Previously it was shown that YopM induces IL-10 gene expression by elevating phosphorylation of the serine-threonine kinase RSK1 in the nucleus of human macrophages. Using transcriptomics, we found that YopM strongly affects expression of genes belonging to the JAK-STAT signaling pathway. Further analysis revealed that YopM mediates nuclear translocation of the transcription factor Stat3 in Y. enterocolitica infected macrophages and that knockdown of Stat3 inhibited YopM-induced IL-10 gene expression. YopM-induced Stat3 translocation did not depend on autocrine IL-10, activation of RSK1 or tyrosine phosphorylation of Stat3. Thus, besides activation of RSK1, stimulation of nuclear translocation of Stat3 is another mechanism by which YopM increases IL-10 gene expression in macrophages.


Asunto(s)
Proteínas Bacterianas , Interleucina-10 , Humanos , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Interleucina-10/genética , Interleucina-10/metabolismo , Proteínas de la Membrana Bacteriana Externa/genética , Proteínas de la Membrana Bacteriana Externa/metabolismo , Macrófagos/metabolismo , Regulación de la Expresión Génica , Factor de Transcripción STAT3/genética , Factor de Transcripción STAT3/metabolismo , Fosforilación
4.
Bio Protoc ; 9(12): e3277, 2019 Jun 20.
Artículo en Inglés | MEDLINE | ID: mdl-33654794

RESUMEN

Identification of protein-protein interactions of bacterial effectors and cellular targets during infection is at the core to understand how bacteria manipulate the infected host to overcome the immune response. Potential interacting proteins might be identified by genetic methods, i.e., two hybrid screens and could be verified by co-immunoprecipitation. The tandem affinity purification (TAP) method allows an unbiased screen of potential interaction partners of bacterial effectors in a physiological approach: target cells can be infected with a bacterial strain harboring the TAP-tagged bacterial effector protein which is translocated in the host similar as under physiological infection conditions. No transfection and overexpression of the bacterial protein in the eukaryotic host are needed. Therefore, also host target cells not easy to transfect can be analyzed by this method. Moreover, the two consecutive affinity tags Calmodulin-Binding-Peptide (CBP) and Streptavidin-Binding-Peptide (SBP) fused to the translocated bacterial protein allow an outstanding clear purification of protein complexes formed between the bacterial protein of interest and host cell proteins with less occurrence of contaminants. Mass spectrometry allows an unbiased identification of interacting eukaryotic proteins.

5.
Nat Genet ; 43(7): 630-8, 2011 Jun 19.
Artículo en Inglés | MEDLINE | ID: mdl-21685913

RESUMEN

Mammalian genomes are viewed as functional organizations that orchestrate spatial and temporal gene regulation. CTCF, the most characterized insulator-binding protein, has been implicated as a key genome organizer. However, little is known about CTCF-associated higher-order chromatin structures at a global scale. Here we applied chromatin interaction analysis by paired-end tag (ChIA-PET) sequencing to elucidate the CTCF-chromatin interactome in pluripotent cells. From this analysis, we identified 1,480 cis- and 336 trans-interacting loci with high reproducibility and precision. Associating these chromatin interaction loci with their underlying epigenetic states, promoter activities, enhancer binding and nuclear lamina occupancy, we uncovered five distinct chromatin domains that suggest potential new models of CTCF function in chromatin organization and transcriptional control. Specifically, CTCF interactions demarcate chromatin-nuclear membrane attachments and influence proper gene expression through extensive cross-talk between promoters and regulatory elements. This highly complex nuclear organization offers insights toward the unifying principles that govern genome plasticity and function.


Asunto(s)
Cromatina/genética , Cromatina/metabolismo , Proteínas de Unión al ADN/metabolismo , Embrión de Mamíferos/metabolismo , Genes Reguladores , Células Madre Pluripotentes/metabolismo , Proteínas Represoras/metabolismo , Animales , Factor de Unión a CCCTC , Células Cultivadas , Cromatina/química , Inmunoprecipitación de Cromatina , Proteínas de Unión al ADN/genética , Embrión de Mamíferos/citología , Epigenómica , Regulación de la Expresión Génica , Hibridación Fluorescente in Situ , Ratones , Regiones Promotoras Genéticas/genética , ARN Interferente Pequeño/genética , Proteínas Represoras/antagonistas & inhibidores , Proteínas Represoras/genética , Transcripción Genética
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