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1.
Annu Rev Immunol ; 38: 1-21, 2020 04 26.
Artigo em Inglês | MEDLINE | ID: mdl-31594433

RESUMO

It is difficult to believe that in about 1960 practically nothing was known about the thymus and some of its products, T cells bearing αß receptors for antigen. Thus I was lucky to join the field of T cell biology almost at its beginning, when knowledge about the cells was just getting off the ground and there was so much to discover. This article describes findings about these cells made by others and myself that led us all from ignorance, via complete confusion, to our current state of knowledge. I believe I was fortunate to practice science in very supportive institutions and with very collaborative colleagues in two countries that both encourage independent research by independent scientists, while simultaneously ignoring or somehow being able to avoid some of the difficulties of being a woman in what was, at the time, a male-dominated profession.


Assuntos
Suscetibilidade a Doenças , Transtorno Obsessivo-Compulsivo/etiologia , Transtorno Obsessivo-Compulsivo/metabolismo , Animais , Autoimunidade , Biomarcadores , Morte Celular , Citocinas/metabolismo , Suscetibilidade a Doenças/imunologia , Antígenos de Histocompatibilidade/genética , Antígenos de Histocompatibilidade/imunologia , Antígenos de Histocompatibilidade/metabolismo , Humanos , Imunidade Inata , Transtorno Obsessivo-Compulsivo/psicologia , Ligação Proteica , Receptores de Antígenos de Linfócitos T/metabolismo , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo , Superantígenos/imunologia , Linfócitos T/imunologia , Linfócitos T/metabolismo , Timo/imunologia , Timo/metabolismo
2.
Annu Rev Cell Dev Biol ; 33: 511-535, 2017 10 06.
Artigo em Inglês | MEDLINE | ID: mdl-28661722

RESUMO

A majority of T cells bearing the αß T cell receptor (TCR) are specific for peptides bound to polymorphic classical major histocompatibility complex (MHC) molecules. Smaller subsets of T cells are reactive toward various nonpeptidic ligands associated with nonpolymorphic MHC class-Ib (MHC-Ib) molecules. These cells have been termed unconventional for decades, even though only the composite antigen is different from the one seen by classical T cells. Herein, we discuss the identity of these particular T cells in light of the coevolution of their TCR and MHC-Ib restricting elements. We examine their original thymic development: selection on hematopoietic cells leading to the acquisition of an original differentiation program. Most of these cells acquire memory cell features during thymic maturation and exhibit unique patterns of migration into peripheral nonlymphoid tissues to become tissue resident. Thus, these cells are termed preset T cells, as they also display a variety of effector functions. They may act as microbial or danger sentinels, fight microbes, or regulate tissue homeostasis.


Assuntos
Peptídeos/metabolismo , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo , Linfócitos T/metabolismo , Animais , Antígenos de Histocompatibilidade/metabolismo , Humanos , Ligantes , Timo/citologia
4.
Nature ; 626(8000): 881-890, 2024 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-38297124

RESUMO

The pace of human brain development is highly protracted compared with most other species1-7. The maturation of cortical neurons is particularly slow, taking months to years to develop adult functions3-5. Remarkably, such protracted timing is retained in cortical neurons derived from human pluripotent stem cells (hPSCs) during in vitro differentiation or upon transplantation into the mouse brain4,8,9. Those findings suggest the presence of a cell-intrinsic clock setting the pace of neuronal maturation, although the molecular nature of this clock remains unknown. Here we identify an epigenetic developmental programme that sets the timing of human neuronal maturation. First, we developed a hPSC-based approach to synchronize the birth of cortical neurons in vitro which enabled us to define an atlas of morphological, functional and molecular maturation. We observed a slow unfolding of maturation programmes, limited by the retention of specific epigenetic factors. Loss of function of several of those factors in cortical neurons enables precocious maturation. Transient inhibition of EZH2, EHMT1 and EHMT2 or DOT1L, at progenitor stage primes newly born neurons to rapidly acquire mature properties upon differentiation. Thus our findings reveal that the rate at which human neurons mature is set well before neurogenesis through the establishment of an epigenetic barrier in progenitor cells. Mechanistically, this barrier holds transcriptional maturation programmes in a poised state that is gradually released to ensure the prolonged timeline of human cortical neuron maturation.


Assuntos
Epigênese Genética , Regulação da Expressão Gênica no Desenvolvimento , Células-Tronco Embrionárias Humanas , Células-Tronco Neurais , Neurogênese , Neurônios , Adulto , Animais , Humanos , Camundongos , Antígenos de Histocompatibilidade/metabolismo , Histona-Lisina N-Metiltransferase/antagonistas & inibidores , Histona-Lisina N-Metiltransferase/metabolismo , Células-Tronco Embrionárias Humanas/citologia , Células-Tronco Embrionárias Humanas/metabolismo , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Neurogênese/genética , Neurônios/citologia , Neurônios/metabolismo , Fatores de Tempo , Transcrição Gênica
5.
Immunity ; 53(6): 1182-1201.e8, 2020 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-33242395

RESUMO

αß lineage T cells, most of which are CD4+ or CD8+ and recognize MHC I- or MHC II-presented antigens, are essential for immune responses and develop from CD4+CD8+ thymocytes. The absence of in vitro models and the heterogeneity of αß thymocytes have hampered analyses of their intrathymic differentiation. Here, combining single-cell RNA and ATAC (chromatin accessibility) sequencing, we identified mouse and human αß thymocyte developmental trajectories. We demonstrated asymmetric emergence of CD4+ and CD8+ lineages, matched differentiation programs of agonist-signaled cells to their MHC specificity, and identified correspondences between mouse and human transcriptomic and epigenomic patterns. Through computational analysis of single-cell data and binding sites for the CD4+-lineage transcription factor Thpok, we inferred transcriptional networks associated with CD4+- or CD8+-lineage differentiation, and with expression of Thpok or of the CD8+-lineage factor Runx3. Our findings provide insight into the mechanisms of CD4+ and CD8+ T cell differentiation and a foundation for mechanistic investigations of αß T cell development.


Assuntos
Diferenciação Celular/imunologia , Linhagem da Célula/imunologia , Subpopulações de Linfócitos T/imunologia , Timócitos/imunologia , Animais , Apresentação de Antígeno/imunologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Diferenciação Celular/genética , Linhagem da Célula/genética , Epigenoma , Regulação da Expressão Gênica , Redes Reguladoras de Genes , Antígenos de Histocompatibilidade/genética , Antígenos de Histocompatibilidade/imunologia , Antígenos de Histocompatibilidade/metabolismo , Humanos , Camundongos , Subpopulações de Linfócitos T/metabolismo , Timócitos/metabolismo , Timo/imunologia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transcriptoma
6.
Genes Dev ; 35(13-14): 1035-1054, 2021 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-34168040

RESUMO

G9a is a histone methyltransferase responsible for the dimethylation of histone H3 at lysine 9 (H3K9me2). G9a plays key roles in transcriptional silencing of developmentally regulated genes, but its role in X-chromosome inactivation (XCI) has been under debate. Here, we uncover a female-specific function of G9a and demonstrate that deleting G9a has a disproportionate impact on the X chromosome relative to the rest of the genome. G9a deficiency causes a failure of XCI and female-specific hypersensitivity to drug inhibition of H3K9me2. We show that G9a interacts with Tsix and Xist RNAs, and that competitive inhibition of the G9a-RNA interaction recapitulates the XCI defect. During XCI, Xist recruits G9a to silence X-linked genes on the future inactive X. In parallel on the future Xa, Tsix recruits G9a to silence Xist in cis Thus, RNA tethers G9a for allele-specific targeting of the H3K9me2 modification and the G9a-RNA interaction is essential for XCI.


Assuntos
Cromossomos Humanos X , Antígenos de Histocompatibilidade/metabolismo , Histona-Lisina N-Metiltransferase/metabolismo , Metiltransferases , RNA Longo não Codificante , Feminino , Histonas/metabolismo , Humanos , Metiltransferases/genética , RNA Longo não Codificante/genética , Inativação do Cromossomo X/genética
7.
Cell ; 153(1): 178-92, 2013 Mar 28.
Artigo em Inglês | MEDLINE | ID: mdl-23523135

RESUMO

The nuclear lamina (NL) interacts with hundreds of large genomic regions termed lamina associated domains (LADs). The dynamics of these interactions and the relation to epigenetic modifications are poorly understood. We visualized the fate of LADs in single cells using a "molecular contact memory" approach. In each nucleus, only ~30% of LADs are positioned at the periphery; these LADs are in intermittent molecular contact with the NL but remain constrained to the periphery. Upon mitosis, LAD positioning is not detectably inherited but instead is stochastically reshuffled. Contact of individual LADs with the NL is linked to transcriptional repression and H3K9 dimethylation in single cells. Furthermore, we identify the H3K9 methyltransferase G9a as a regulator of NL contacts. Collectively, these results highlight principles of the dynamic spatial architecture of chromosomes in relation to gene regulation.


Assuntos
Cromossomos/metabolismo , Regulação da Expressão Gênica , Lâmina Nuclear/química , Análise de Célula Única/métodos , Adenina/metabolismo , Linhagem Celular Tumoral , Metilação de DNA , Genoma , Heterocromatina/metabolismo , Antígenos de Histocompatibilidade/metabolismo , Histona-Lisina N-Metiltransferase/metabolismo , Humanos , Mitose , Lâmina Nuclear/metabolismo
8.
Immunity ; 48(3): 453-473, 2018 03 20.
Artigo em Inglês | MEDLINE | ID: mdl-29562195

RESUMO

Most studies on the immunotherapeutic potential of T cells have focused on CD8 and CD4 T cells that recognize peptide antigens (Ag) presented by polymorphic major histocompatibility complex (MHC) class I and MHC class II molecules, respectively. However, unconventional T cells, which interact with MHC class Ib and MHC-I like molecules, are also implicated in tumor immunity, although their role therein is unclear. These include unconventional T cells targeting MHC class Ib molecules such as HLA-E and its murine ortholog Qa-1b, natural killer T (NKT) cells, mucosal associated invariant T (MAIT) cells, and γδ T cells. Here, we review the current understanding of the roles of these unconventional T cells in tumor immunity and discuss why further studies into the immunotherapeutic potential of these cells is warranted.


Assuntos
Imunoterapia , Terapia de Alvo Molecular , Neoplasias/imunologia , Neoplasias/terapia , Linfócitos T/imunologia , Linfócitos T/metabolismo , Animais , Biomarcadores , Estudos Clínicos como Assunto , Terapia Combinada , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Antígenos de Histocompatibilidade/imunologia , Antígenos de Histocompatibilidade/metabolismo , Humanos , Imunomodulação/efeitos dos fármacos , Neoplasias/metabolismo , Transdução de Sinais/efeitos dos fármacos , Subpopulações de Linfócitos T/efeitos dos fármacos , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Linfócitos T/efeitos dos fármacos , Resultado do Tratamento
9.
Proc Natl Acad Sci U S A ; 121(9): e2315985121, 2024 Feb 27.
Artigo em Inglês | MEDLINE | ID: mdl-38377192

RESUMO

Recurrent, ancient arms races between viruses and hosts have shaped both host immunological defense strategies as well as viral countermeasures. One such battle is waged by the glycoprotein US11 encoded by the persisting human cytomegalovirus. US11 mediates degradation of major histocompatibility class I (MHC-I) molecules to prevent CD8+ T-cell activation. Here, we studied the consequences of the arms race between US11 and primate MHC-A proteins, leading us to uncover a tit-for-tat coevolution and its impact on MHC-A diversification. We found that US11 spurred MHC-A adaptation to evade viral antagonism: In an ancestor of great apes, the MHC-A A2 lineage acquired a Pro184Ala mutation, which confers resistance against the ancestral US11 targeting strategy. In response, US11 deployed a unique low-complexity region (LCR), which exploits the MHC-I peptide loading complex to target the MHC-A2 peptide-binding groove. In addition, the global spread of the human HLA-A*02 allelic family prompted US11 to employ a superior LCR strategy with an optimally fitting peptide mimetic that specifically antagonizes HLA-A*02. Thus, despite cytomegaloviruses low pathogenic potential, the increasing commitment of US11 to MHC-A has significantly promoted diversification of MHC-A in hominids.


Assuntos
Antígenos de Histocompatibilidade Classe I , Hominidae , Animais , Humanos , Proteínas Virais/metabolismo , Citomegalovirus , Hominidae/genética , Hominidae/metabolismo , Linhagem Celular , Antígenos de Histocompatibilidade/metabolismo , Antígenos HLA-A/metabolismo , Peptídeos/metabolismo
10.
Proc Natl Acad Sci U S A ; 120(52): e2308366120, 2023 Dec 26.
Artigo em Inglês | MEDLINE | ID: mdl-38113261

RESUMO

Immune system threat detection hinges on T cells' ability to perceive varying peptide-major histocompatibility complex (pMHC) antigens. As the Erk and NFAT pathways link T cell receptor engagement to gene regulation, their signaling dynamics may convey information about pMHC inputs. To test this idea, we developed a dual reporter mouse strain and a quantitative imaging assay that, together, enable simultaneous monitoring of Erk and NFAT dynamics in live T cells over day-long timescales as they respond to varying pMHC inputs. Both pathways initially activate uniformly across various pMHC inputs but diverge only over longer (9+ h) timescales, enabling independent encoding of pMHC affinity and dose. These late signaling dynamics are decoded via multiple temporal and combinatorial mechanisms to generate pMHC-specific transcriptional responses. Our findings underscore the importance of long timescale signaling dynamics in antigen perception and establish a framework for understanding T cell responses under diverse contexts.


Assuntos
Ativação Linfocitária , Linfócitos T , Camundongos , Animais , Receptores de Antígenos de Linfócitos T , Antígenos/metabolismo , Antígenos de Histocompatibilidade/metabolismo , Peptídeos/metabolismo , Complexo Principal de Histocompatibilidade , Percepção , Ligação Proteica
11.
Proc Natl Acad Sci U S A ; 120(43): e2304689120, 2023 10 24.
Artigo em Inglês | MEDLINE | ID: mdl-37856544

RESUMO

The importance of classical CD8+ T cells in tumor eradication is well acknowledged. However, the anti-tumor activity of MHC (major histocompatibility complex) Ib-restricted CD8+ T (Ib-CD8+ T) cells remains obscure. Here, we show that CX3CR1-expressing Ib-CD8+ T cells (Ib-restricted CD8+ T cells) highly express cytotoxic factors, austerely resist exhaustion, and effectively eliminate various tumors. These Ib-CD8+ T cells can be primed by MHC Ia (MHC class Ia molecules) expressed on various cell types for optimal activation in a Tbet-dependent manner. Importantly, MHC Ia does not allogeneically activate Ib-CD8+ T cells, rather, sensitizes these cells for T cell receptor activation. Such effects were observed when MHC Ia+ cells were administered to tumor-bearing Kb-/-Db-/-mice. A similar population of tumoricidal CX3CR1+CD8+ T cells was identified in wild-type mice and melanoma patients. Adoptive transfer of Ib-CD8+ T cells to wild-type mice inhibited tumor progression without damaging normal tissues. Taken together, we demonstrate that MHC class Ia can prime Ib-CD8+ T cells for robust tumoricidal activities.


Assuntos
Linfócitos T CD8-Positivos , Antígenos de Histocompatibilidade Classe I , Humanos , Camundongos , Animais , Antígenos de Histocompatibilidade Classe I/genética , Antígenos de Histocompatibilidade Classe I/metabolismo , Antígenos H-2 , Antígenos de Histocompatibilidade/metabolismo , Camundongos Endogâmicos C57BL
12.
Nat Immunol ; 14(6): 619-32, 2013 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-23644507

RESUMO

The differentiation of αßT cells from thymic precursors is a complex process essential for adaptive immunity. Here we exploited the breadth of expression data sets from the Immunological Genome Project to analyze how the differentiation of thymic precursors gives rise to mature T cell transcriptomes. We found that early T cell commitment was driven by unexpectedly gradual changes. In contrast, transit through the CD4(+)CD8(+) stage involved a global shutdown of housekeeping genes that is rare among cells of the immune system and correlated tightly with expression of the transcription factor c-Myc. Selection driven by major histocompatibility complex (MHC) molecules promoted a large-scale transcriptional reactivation. We identified distinct signatures that marked cells destined for positive selection versus apoptotic deletion. Differences in the expression of unexpectedly few genes accompanied commitment to the CD4(+) or CD8(+) lineage, a similarity that carried through to peripheral T cells and their activation, demonstrated by mass cytometry phosphoproteomics. The transcripts newly identified as encoding candidate mediators of key transitions help define the 'known unknowns' of thymocyte differentiation.


Assuntos
Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Diferenciação Celular/imunologia , Receptores de Antígenos de Linfócitos T alfa-beta/imunologia , Animais , Antígenos CD/imunologia , Antígenos CD/metabolismo , Antígenos de Diferenciação de Linfócitos T/imunologia , Antígenos de Diferenciação de Linfócitos T/metabolismo , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD8-Positivos/metabolismo , Diferenciação Celular/genética , Linhagem da Célula/genética , Linhagem da Célula/imunologia , Proliferação de Células , Células Cultivadas , Análise por Conglomerados , Citometria de Fluxo , Antígenos de Histocompatibilidade/genética , Antígenos de Histocompatibilidade/imunologia , Antígenos de Histocompatibilidade/metabolismo , Lectinas Tipo C/imunologia , Lectinas Tipo C/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Análise de Sequência com Séries de Oligonucleotídeos , Fosforilação/imunologia , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo , Timócitos/citologia , Timócitos/imunologia , Timócitos/metabolismo , Transcriptoma/genética , Transcriptoma/imunologia
13.
Immunity ; 44(5): 1091-101, 2016 05 17.
Artigo em Inglês | MEDLINE | ID: mdl-27192576

RESUMO

Signaling through the T cell receptor (TCR) controls adaptive immune responses. Antigen binding to TCRαß transmits signals through the plasma membrane to induce phosphorylation of the CD3 cytoplasmic tails by incompletely understood mechanisms. Here we show that cholesterol bound to the TCRß transmembrane region keeps the TCR in a resting, inactive conformation that cannot be phosphorylated by active kinases. Only TCRs that spontaneously detached from cholesterol could switch to the active conformation (termed primed TCRs) and then be phosphorylated. Indeed, by modulating cholesterol binding genetically or enzymatically, we could switch the TCR between the resting and primed states. The active conformation was stabilized by binding to peptide-MHC, which thus controlled TCR signaling. These data are explained by a model of reciprocal allosteric regulation of TCR phosphorylation by cholesterol and ligand binding. Our results provide both a molecular mechanism and a conceptual framework for how lipid-receptor interactions regulate signal transduction.


Assuntos
Imunidade Adaptativa , Colesterol/metabolismo , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo , Linfócitos T/imunologia , Regulação Alostérica , Antígenos/imunologia , Antígenos/metabolismo , Antígenos de Histocompatibilidade/metabolismo , Humanos , Células Jurkat , Ativação Linfocitária , Modelos Imunológicos , Fragmentos de Peptídeos/imunologia , Fragmentos de Peptídeos/metabolismo , Fosforilação , Ligação Proteica , Conformação Proteica , Estabilidade Proteica , Transdução de Sinais
14.
EMBO Rep ; 24(1): e54969, 2023 01 09.
Artigo em Inglês | MEDLINE | ID: mdl-36327141

RESUMO

T cell activation and effector functions are determined by the affinity of the interaction between T cell receptor (TCR) and its antigenic peptide MHC (pMHC) ligand. A better understanding of the quantitative aspects of TCR-pMHC affinity-dependent T cell activation is critical for the development of new immunotherapeutic strategies. However, the role of TCR-pMHC affinity in regulating the kinetics of CD8+ T cell commitment to proliferation and differentiation is unknown. Here, we show that the stronger the TCR-pMHC affinity, the shorter the time of T cell-APC co-culture required to commit CD8+ T cells to proliferation. The time threshold for T cell cytokine production is much lower than that for cell proliferation. There is a strong correlation between affinity-dependent differences in AKT phosphorylation and T cell proliferation. The cytokine IL-15 increases the poor proliferation of T cells stimulated with low affinity pMHC, suggesting that pro-inflammatory cytokines can override the affinity-dependent features of T cell proliferation.


Assuntos
Linfócitos T CD8-Positivos , Citocinas , Receptores de Antígenos de Linfócitos T/metabolismo , Antígenos de Histocompatibilidade/metabolismo , Ativação Linfocitária , Ligação Proteica , Proliferação de Células
15.
J Immunol ; 210(8): 1156-1165, 2023 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-36883850

RESUMO

The study of Ag-specific lymphocytes has been a key advancement in immunology over the past few decades. The development of multimerized probes containing Ags, peptide:MHC complexes, or other ligands was one innovation allowing the direct study of Ag-specific lymphocytes by flow cytometry. Although these types of study are now common and performed by thousands of laboratories, quality control and assessment of probe quality are often minimal. In fact, many of these types of probe are made in-house, and protocols vary between laboratories. Although peptide:MHC multimers can often be obtained from commercial sources or core facilities, few such services exist for Ag multimers. To ensure high quality and consistency with ligand probes, we have developed an easy and robust multiplexed approach using commercially available beads able to bind Abs specific for the ligand of interest. Using this assay, we have sensitively assessed the performance of peptide:MHC and Ag tetramers and have found considerable batch-to-batch variability in performance and stability over time more easily than using murine or human cell-based assays. This bead-based assay can also reveal common production errors such as miscalculation of Ag concentration. This work could set the stage for the development of standardized assays for all commonly used ligand probes to limit laboratory-to-laboratory technical variation and experimental failure caused by probe underperformance.


Assuntos
Peptídeos , Linfócitos T Citotóxicos , Humanos , Animais , Camundongos , Ligantes , Antígenos de Histocompatibilidade Classe I/metabolismo , Antígeno HLA-A2 , Antígenos de Histocompatibilidade/metabolismo
16.
Mol Ther ; 32(8): 2662-2675, 2024 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-38796700

RESUMO

Prader-Willi syndrome (PWS) is the prototypic genomic disorder resulting from deficiency of paternally expressed genes in the human chromosome 15q11-q13 region. The unique molecular mechanism involving epigenetic modifications renders PWS as the most attractive candidate to explore a proof-of-concept of epigenetic therapy in humans. The premise is that epigenetic modulations could reactivate the repressed PWS candidate genes from the maternal chromosome and offer therapeutic benefit. Our prior study identifies an EHMT2/G9a inhibitor, UNC0642, that reactivates the expression of PWS genes via reduction of H3K9me2. However, low brain permeability and poor oral bioavailability of UNC0642 preclude its advancement into translational studies in humans. In this study, a newly developed inhibitor, MS152, modified from the structure of UNC0642, has better brain penetration and greater potency and selectivity against EHMT2/G9a. MS152 reactivated maternally silenced PWS genes in PWS patient fibroblasts and in brain and liver tissues of PWS mouse models. Importantly, the molecular efficacy of oral administration is comparable with the intraperitoneal route. MS152 treatment in newborns ameliorates the perinatal lethality and poor growth, maintaining reactivation in a PWS mouse model at postnatal 90 days. Our findings provide strong support for MS152 as a first-in-class inhibitor to advance the epigenetic therapy of PWS in humans.


Assuntos
Modelos Animais de Doenças , Epigênese Genética , Síndrome de Prader-Willi , Humanos , Animais , Síndrome de Prader-Willi/tratamento farmacológico , Síndrome de Prader-Willi/genética , Camundongos , Epigênese Genética/efeitos dos fármacos , Administração Oral , Antígenos de Histocompatibilidade/genética , Antígenos de Histocompatibilidade/metabolismo , Encéfalo/metabolismo , Encéfalo/efeitos dos fármacos , Histona-Lisina N-Metiltransferase
17.
Mol Cell ; 67(4): 550-565.e5, 2017 Aug 17.
Artigo em Inglês | MEDLINE | ID: mdl-28803780

RESUMO

DNA methylation is an essential epigenetic mark in mammals that has to be re-established after each round of DNA replication. The protein UHRF1 is essential for this process; it has been proposed that the protein targets newly replicated DNA by cooperatively binding hemi-methylated DNA and H3K9me2/3, but this model leaves a number of questions unanswered. Here, we present evidence for a direct recruitment of UHRF1 by the replication machinery via DNA ligase 1 (LIG1). A histone H3K9-like mimic within LIG1 is methylated by G9a and GLP and, compared with H3K9me2/3, more avidly binds UHRF1. Interaction with methylated LIG1 promotes the recruitment of UHRF1 to DNA replication sites and is required for DNA methylation maintenance. These results further elucidate the function of UHRF1, identify a non-histone target of G9a and GLP, and provide an example of a histone mimic that coordinates DNA replication and DNA methylation maintenance.


Assuntos
Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , DNA Ligase Dependente de ATP/metabolismo , Metilação de DNA , Replicação do DNA , DNA/biossíntese , Epigênese Genética , Antígenos de Histocompatibilidade/metabolismo , Histona-Lisina N-Metiltransferase/metabolismo , Processamento de Proteína Pós-Traducional , Animais , Proteínas Estimuladoras de Ligação a CCAAT/química , Proteínas Estimuladoras de Ligação a CCAAT/genética , DNA/genética , DNA Ligase Dependente de ATP/química , DNA Ligase Dependente de ATP/genética , Células-Tronco Embrionárias/enzimologia , Células HEK293 , Células HeLa , Antígenos de Histocompatibilidade/química , Antígenos de Histocompatibilidade/genética , Histona-Lisina N-Metiltransferase/química , Histona-Lisina N-Metiltransferase/genética , Histonas/metabolismo , Humanos , Lisina , Metilação , Camundongos , Modelos Moleculares , Mimetismo Molecular , Mutação , Ligação Proteica , Conformação Proteica , Relação Estrutura-Atividade , Transfecção , Domínio Tudor , Ubiquitina-Proteína Ligases
18.
Mol Cell ; 66(4): 517-532.e9, 2017 May 18.
Artigo em Inglês | MEDLINE | ID: mdl-28525743

RESUMO

Autophagy is a membrane-trafficking process that directs degradation of cytoplasmic material in lysosomes. The process promotes cellular fidelity, and while the core machinery of autophagy is known, the mechanisms that promote and sustain autophagy are less well defined. Here we report that the epigenetic reader BRD4 and the methyltransferase G9a repress a TFEB/TFE3/MITF-independent transcriptional program that promotes autophagy and lysosome biogenesis. We show that BRD4 knockdown induces autophagy in vitro and in vivo in response to some, but not all, situations. In the case of starvation, a signaling cascade involving AMPK and histone deacetylase SIRT1 displaces chromatin-bound BRD4, instigating autophagy gene activation and cell survival. Importantly, this program is directed independently and also reciprocally to the growth-promoting properties of BRD4 and is potently repressed by BRD4-NUT, a driver of NUT midline carcinoma. These findings therefore identify a distinct and selective mechanism of autophagy regulation.


Assuntos
Autofagia , Carcinoma Ductal Pancreático/metabolismo , Lisossomos/metabolismo , Proteínas Nucleares/metabolismo , Neoplasias Pancreáticas/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Gênica , Proteínas Quinases Ativadas por AMP/metabolismo , Animais , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/patologia , Proteínas de Ciclo Celular , Linhagem Celular Tumoral , Proliferação de Células , Cromatina/genética , Cromatina/metabolismo , Regulação para Baixo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Metabolismo Energético , Regulação Neoplásica da Expressão Gênica , Células HEK293 , Antígenos de Histocompatibilidade/genética , Antígenos de Histocompatibilidade/metabolismo , Histona-Lisina N-Metiltransferase/genética , Histona-Lisina N-Metiltransferase/metabolismo , Humanos , Lisossomos/patologia , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas Nucleares/genética , Proteínas de Fusão Oncogênica/genética , Proteínas de Fusão Oncogênica/metabolismo , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Agregados Proteicos , Ligação Proteica , Proteólise , Interferência de RNA , Transdução de Sinais , Sirtuína 1/genética , Sirtuína 1/metabolismo , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismo , Fatores de Tempo , Fatores de Transcrição/genética , Transfecção
19.
Proc Natl Acad Sci U S A ; 119(9)2022 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-35149556

RESUMO

Development of the messenger RNA (mRNA) vaccine has emerged as an effective and speedy strategy to control the spread of new pathogens. After vaccination, the mRNA is translated into the real protein vaccine, and there is no need to manufacture the protein in vitro. However, the fate of mRNA and its posttranslational modification inside the cell may affect immune response. Here, we showed that the mRNA vaccine of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein with deletion of glycosites in the receptor-binding domain (RBD) or especially the subunit 2 (S2) domain to expose more conserved epitopes elicited stronger antibody and CD8+ T cell responses with broader protection against the alpha, beta, gamma, delta, and omicron variants, compared to the unmodified mRNA. Immunization of such mRNA resulted in accumulation of misfolded spike protein in the endoplasmic reticulum, causing the up-regulation of BiP/GRP78, XBP1, and p-eIF2α to induce cell apoptosis and strong CD8+ T cell response. In addition, dendritic cells (DCs) incubated with S2-glysosite deleted mRNA vaccine increased class I major histocompatibility complex (MHC I) expression. This study provides a direction for the development of broad-spectrum mRNA vaccines which may not be achieved with the use of expressed proteins as antigens.


Assuntos
Vacinas contra COVID-19/imunologia , Glicoproteína da Espícula de Coronavírus/genética , Animais , Anticorpos Antivirais/imunologia , Formação de Anticorpos , Linfócitos T CD8-Positivos/imunologia , Citocinas/metabolismo , Glicosilação , Células HEK293 , Antígenos de Histocompatibilidade/metabolismo , Humanos , Imunidade , Camundongos Endogâmicos BALB C , Resposta a Proteínas não Dobradas , Vacinas Sintéticas/imunologia , Vacinas de mRNA/imunologia
20.
Proc Natl Acad Sci U S A ; 119(27): e2118529119, 2022 07 05.
Artigo em Inglês | MEDLINE | ID: mdl-35767640

RESUMO

During normal T cell development in mouse and human, a low-frequency population of immature CD4-CD8- double-negative (DN) thymocytes expresses early, mature αß T cell antigen receptor (TCR). We report that these early αß TCR+ DN (EADN) cells are DN3b-DN4 stage and require CD3δ but not major histocompatibility complex (MHC) for their generation/detection. When MHC - is present, however, EADN cells can respond to it, displaying a degree of coreceptor-independent MHC reactivity not typical of mature, conventional αß T cells. We found these data to be connected with observations that EADN cells were susceptible to T cell acute lymphoblastic leukemia (T-ALL) transformation in both humans and mice. Using the OT-1 TCR transgenic system to model EADN-stage αß TCR expression, we found that EADN leukemogenesis required MHC to induce development of T-ALL bearing NOTCH1 mutations. This leukemia-driving MHC requirement could be lost, however, upon passaging the tumors in vivo, even when matching MHC was continuously present in recipient animals and on the tumor cells themselves. These data demonstrate that MHC:TCR signaling can be required to initiate a cancer phenotype from an understudied developmental state that appears to be represented in the mouse and human disease spectrum.


Assuntos
Linfócitos T CD8-Positivos , Leucemia-Linfoma Linfoblástico de Células T Precursoras , Receptor Notch1 , Receptores de Antígenos de Linfócitos T alfa-beta , Animais , Linfócitos T CD8-Positivos/metabolismo , Diferenciação Celular , Antígenos de Histocompatibilidade/metabolismo , Humanos , Complexo Principal de Histocompatibilidade , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mutação , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Receptor Notch1/genética , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/metabolismo , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo , Timo/metabolismo
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