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1.
Cell ; 178(5): 1145-1158.e20, 2019 08 22.
Artigo em Inglês | MEDLINE | ID: mdl-31402173

RESUMO

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.


Assuntos
Complexo Mediador/metabolismo , RNA Polimerase II/metabolismo , Animais , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/metabolismo , Sistemas CRISPR-Cas/genética , Proteínas de Ciclo Celular/metabolismo , Células Cultivadas , Proteínas Cromossômicas não Histona/metabolismo , Microscopia Crioeletrônica , Elementos Facilitadores Genéticos , Edição de Genes , Humanos , Masculino , Complexo Mediador/química , Complexo Mediador/genética , Camundongos , Camundongos Endogâmicos C57BL , Células-Tronco Embrionárias Murinas/citologia , Células-Tronco Embrionárias Murinas/metabolismo , Regiões Promotoras Genéticas , Estrutura Quaternária de Proteína , RNA Polimerase II/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Coesinas
2.
Cancer Immunol Immunother ; 72(12): 4195-4207, 2023 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-37848682

RESUMO

T cells expressing a mesothelin (MSLN)-specific T cell receptor fusion construct (TRuC®), called TC-210, have demonstrated robust antitumor activity in preclinical models of mesothelioma, ovarian cancer, and lung cancer. However, they are susceptible to suppression by the programmed cell death protein 1 (PD-1)/programmed cell death protein ligand 1 (PD-L1) axis and lack intrinsic costimulatory signaling elements. To enhance the function of anti-MSLN TRuC-T cells, chimeric switch receptors (CSRs) have been designed to co-opt the immunosuppressive PD-1/PD-L1 axis and to deliver a CD28-mediated costimulatory signal. Here, we report that coexpression of the PD1-CD28 CSR in TRuC-T cells enhanced T cell receptor signaling, increased proinflammatory effector cytokines, decreased anti-inflammatory cytokines, and sustained effector function in the presence of PD-L1 when compared with TC-210. Anti-MSLN TRuC-T cells engineered to coexpress PD1-CD28 CSRs comprising the ectodomain of PD-1 and the intracellular domain of CD28 linked by the transmembrane domain of PD-1 were selected for integration into an anti-MSLN TRuC-T cell therapy product called TC-510. In vitro, TC-510 showed significant improvements in persistence and resistance to exhaustion upon chronic stimulation by tumor cells expressing MSLN and PD-L1 when compared with TC-210. In vivo, TC-510 showed a superior ability to provide durable protection following tumor rechallenge, versus TC-210. These data demonstrate that integration of a PD1-CD28 CSR into TRuC-T cells improves effector function, resistance to exhaustion, and prolongs persistence. Based on these findings, TC-510 is currently being evaluated in patients with MSLN-expressing solid tumors.


Assuntos
Antígenos CD28 , Mesotelioma , Humanos , Mesotelina , Receptor de Morte Celular Programada 1/metabolismo , Antígeno B7-H1/genética , Antígeno B7-H1/metabolismo , Linfócitos T/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Citocinas/metabolismo
3.
PLoS Pathog ; 9(9): e1003584, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24039573

RESUMO

Kaposi's sarcoma (KS) is caused by infection with Kaposi's sarcoma-associated herpesvirus (KSHV). The virus expresses unique microRNAs (miRNAs), but the targets and functions of these miRNAs are not completely understood. In order to identify human targets of viral miRNAs, we measured protein expression changes caused by multiple KSHV miRNAs using pulsed stable labeling with amino acids in cell culture (pSILAC) in primary endothelial cells. This led to the identification of multiple human genes that are repressed at the protein level, but not at the miRNA level. Further analysis also identified that KSHV miRNAs can modulate activity or expression of upstream regulatory factors, resulting in suppressed activation of a protein involved in leukocyte recruitment (ICAM1) following lysophosphatidic acid treatment, as well as up-regulation of a pro-angiogenic protein (HIF1α), and up-regulation of a protein involved in stimulating angiogenesis (HMOX1). This study aids in our understanding of miRNA mechanisms of repression and miRNA contributions to viral pathogenesis.


Assuntos
Herpesvirus Humano 8/metabolismo , MicroRNAs/metabolismo , Neovascularização Patológica/metabolismo , RNA Neoplásico/metabolismo , RNA Viral/metabolismo , Sarcoma de Kaposi/metabolismo , Evasão Tumoral , Células HEK293 , Herpesvirus Humano 8/genética , Células Endoteliais da Veia Umbilical Humana , Humanos , MicroRNAs/genética , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Neovascularização Patológica/genética , Neovascularização Patológica/patologia , Neovascularização Patológica/virologia , RNA Neoplásico/genética , RNA Viral/genética , Sarcoma de Kaposi/genética , Sarcoma de Kaposi/patologia , Sarcoma de Kaposi/virologia
4.
J Virol ; 86(21): 11663-74, 2012 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-22896623

RESUMO

Kaposi's sarcoma (KS)-associated herpesvirus (KSHV) is the causative agent of KS, an important AIDS-associated malignancy. KSHV expresses at least 18 different mature microRNAs (miRNAs). We identified interleukin-1 receptor (IL-1R)-associated kinase 1 (IRAK1) as a potential target of miR-K12-9 (miR-K9) in an array data set examining changes in cellular gene expression levels in the presence of KSHV miRNAs. Using 3'-untranslated region (3'UTR) luciferase reporter assays, we confirmed that miR-K9 and other miRNAs inhibit IRAK1 expression. In addition, IRAK1 expression is downregulated in cells transfected with miR-K9 and during de novo KSHV infection. IRAK1 is an important component of the Toll-like receptor (TLR)/IL-1R signaling cascade. The downregulation of IRAK1 by miR-K9 resulted in the decreased stimulation of NF-κB activity in endothelial cells treated with IL-1α and in B cells treated with a TLR7/8 agonist. Interestingly, miR-K9 had a greater effect on NF-κB activity than did a small interfering RNA (siRNA) targeting IRAK1 despite the more efficient downregulation of IRAK1 expression with the siRNA. We hypothesized that KSHV miRNAs may also be regulating a second component of the TLR/IL-1R signaling cascade, resulting in a stronger phenotype. Reanalysis of the array data set identified myeloid differentiation primary response protein 88 (MYD88) as an additional potential target. 3'UTR luciferase reporter assays and Western blot analysis confirmed the targeting of MYD88 by miR-K5. The presence of miR-K9 and miR-K5 inhibited the production of IL-6 and IL-8 upon the IL-1α stimulation of endothelial cells. These results demonstrate KSHV-encoded miRNAs regulating the TLR/IL-1R signaling cascade at two distinct points and suggest the importance of these pathways during viral infection.


Assuntos
Citocinas/antagonistas & inibidores , Herpesvirus Humano 8/imunologia , Herpesvirus Humano 8/patogenicidade , Quinases Associadas a Receptores de Interleucina-1/antagonistas & inibidores , MicroRNAs/metabolismo , Fator 88 de Diferenciação Mieloide/antagonistas & inibidores , Transdução de Sinais , Fusão Gênica Artificial , Western Blotting , Linhagem Celular , Perfilação da Expressão Gênica , Inativação Gênica , Genes Reporter , Humanos , Evasão da Resposta Imune , Quinases Associadas a Receptores de Interleucina-1/genética , Interleucinas/imunologia , Interleucinas/metabolismo , Luciferases/análise , Luciferases/genética , MicroRNAs/genética , Análise em Microsséries , Fator 88 de Diferenciação Mieloide/genética , Receptores Toll-Like/imunologia , Receptores Toll-Like/metabolismo
5.
Mol Biol Cell ; 15(10): 4356-68, 2004 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-15269281

RESUMO

TATA-binding protein (TBP)-related factor 2 (TRF2) is one of four closely related RNA polymerase II transcription factors. We compared the intracellular localizations of TBP and TRF2 during the cell cycle and mitosis in HeLa cells. We show that during interphase, endogenous or exogenously expressed TRF2 is located almost exclusively in the nucleolus in HeLa or Cos cells. TRF2 localization is not affected by stress or mitotic stimuli, but TRF2 is rapidly released from the nucleolus upon inhibition of pol I transcription or treatment by RNase. These results suggest that localization of HeLa TRF2 requires a nucleolar-associated RNA species. In contrast, in 3T3 fibroblast cells, exogenously expressed TRF2 localizes to the nucleoplasm. Constitutive expression of ectopic TRF2 in 3T3 cells leads to a prolonged S phase of the cell cycle and reduced proliferation. Together with previous data, our results highlight the cell-specific localization and functions of TRF2. Furthermore, we show that during cell division, HeLa TRF2 and TBP are localized in the mitotic cytoplasm and TRF2 relocalizes into the nascent nucleoli immediately after mitosis, whereas TBP reassociates with the chromatin. Although partially contradictory results have been reported, our data are consistent with a model where only small proportion of the cellular TBP remains associated with specific promoter loci during mitosis.


Assuntos
Ciclo Celular/fisiologia , Nucléolo Celular/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Semelhantes à Proteína de Ligação a TATA-Box/metabolismo , Células 3T3 , Animais , Células COS , Chlorocebus aethiops , DNA Polimerase I/metabolismo , Células HeLa , Humanos , Imuno-Histoquímica , Camundongos , Proteína de Ligação a TATA-Box/metabolismo , Proteína 2 de Ligação a Repetições Teloméricas , Transcrição Gênica
6.
PLoS One ; 10(8): e0135560, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26263384

RESUMO

Kaposi's sarcoma (KS) is characterized by highly vascularized spindle-cell tumors induced after infection of endothelial cells by Kaposi's sarcoma-associated herpesvirus (KSHV). In KS tumors, KSHV expresses only a few latent proteins together with 12 pre-microRNAs. Previous microarray and proteomic studies predicted that multiple splice variants of the tumor suppressor protein tropomyosin 1 (TPM1) were targets of KSHV microRNAs. Here we show that at least two microRNAs of KSHV, miR-K2 and miR-K5, repress protein levels of specific isoforms of TPM1. We identified a functional miR-K5 binding site in the 3' untranslated region (UTR) of one TPM1 isoform. Furthermore, the inhibition or loss of miR-K2 or miR-K5 restores expression of TPM1 in KSHV-infected cells. TPM1 protein levels were also repressed in KSHV-infected clinical samples compared to uninfected samples. Functionally, miR-K2 increases viability of unanchored human umbilical vein endothelial cells (HUVEC) by inhibiting anoikis (apoptosis after cell detachment), enhances tube formation of HUVECs, and enhances VEGFA expression. Taken together, KSHV miR-K2 and miR-K5 may facilitate KSHV pathogenesis.


Assuntos
Células Endoteliais/metabolismo , Herpesvirus Humano 8/genética , MicroRNAs/genética , Interferência de RNA , RNA Viral , Tropomiosina/genética , Regiões 3' não Traduzidas , Anoikis/genética , Linhagem Celular , Células Endoteliais/virologia , Regulação da Expressão Gênica , Ordem dos Genes , Células Endoteliais da Veia Umbilical Humana , Humanos , Peso Molecular , Isoformas de Proteínas , RNA Mensageiro/genética , Tropomiosina/química , Tropomiosina/metabolismo
7.
Viruses ; 4(9): 1687-710, 2012 09.
Artigo em Inglês | MEDLINE | ID: mdl-23170179

RESUMO

EBV and KSHV are both gamma-herpesviruses which express multiple viral microRNAs. Various methods have been used to investigate the functions of these microRNAs, largely through identification of microRNA target genes. Surprisingly, these related viruses do not share significant sequence homology in their microRNAs. A number of reports have described functions of EBV and KSHV microRNA targets, however only three experimentally validated target genes have been shown to be targeted by microRNAs from both viruses. More sensitive methods to identify microRNA targets have predicted approximately 60% of host targets could be shared by EBV and KSHV microRNAs, but by targeting different sequences in the host targets. In this review, we explore the similarities of microRNA functions and targets of these related viruses.


Assuntos
Herpesvirus Humano 4/patogenicidade , Herpesvirus Humano 8/patogenicidade , MicroRNAs/metabolismo , RNA Viral/metabolismo , Fatores de Virulência/metabolismo , Regulação da Expressão Gênica , Herpesvirus Humano 4/genética , Herpesvirus Humano 8/genética , Humanos , MicroRNAs/genética , RNA Viral/genética , Fatores de Virulência/genética
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