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The mild hypothermia response (MHR) maintains organismal homeostasis during cold exposure and is thought to be critical for the neuroprotection documented with therapeutic hypothermia. To date, little is known about the transcriptional regulation of the MHR. We utilize a forward CRISPR-Cas9 mutagenesis screen to identify the histone lysine methyltransferase SMYD5 as a regulator of the MHR. SMYD5 represses the key MHR gene SP1 at euthermia. This repression correlates with temperature-dependent levels of histone H3 lysine 26 trimethylation (H3K36me3) at the SP1 locus and globally, indicating that the mammalian MHR is regulated at the level of histone modifications. We have identified 37 additional SMYD5-regulated temperature-dependent genes, suggesting a broader MHR-related role for SMYD5. Our study provides an example of how histone modifications integrate environmental cues into the genetic circuitry of mammalian cells and provides insights that may yield therapeutic avenues for neuroprotection after catastrophic events.
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Introduction: The microphthalmia transcription factor Mitf has been shown to regulate B cell activation and tolerance. However, the underlying B cell-specific mechanisms responsible, and those that distinguish Mitf from closely related Mitf/TFE (MiT) transcription factors Tfe3, Tfeb, and Tfec, remain obscure. Methods: Two complementary mouse models of Mitf and MiT deficiency were used: the Mitfmi-vga9/mi-vga9 systemic loss-of-function mutation, and B-cell specific MiT family inactivation via transgenic expression of a trans-dominant negative (TDN) protein (TDN-B). These models were employed to identify MiT family candidate target genes and pathways. Results: Both models displayed spontaneous splenomegaly coincident with elevated plasma cell numbers, autoantibody titers, and proteinuria. These abnormalities appeared dependent on T helper cells, but independent of other non-B cell intrinsic effects of systemic Mitf inactivation. MiT inactivation in B cells augmented aspects of lupus-like autoimmune disease on the C57BL/6-Faslpr/lpr background. In both models, RNAseq of ex vivo resting B cells showed transcriptional upregulation of genes that control cell cycle, germinal center responses, and plasma cell differentiation. Among the genes strongly upregulated in both models were Socs6, Isp53 (Baiap1), S1pR2, and IgG2b/c. Mitf null B cells, but not TDN-B cells, showed evidence of type I interferon dysregulation. Discussion: These studies clarify Mitf's role as 1) a key regulator of a B cell intrinsic germinal center program that influences self-tolerance through novel target genes, and 2) a regulator of systemic inflammatory processes that can impact the B cell microenvironment. This distinction of Mitf's function from that of related MiT transcription factors advances our understanding of B cell regulation and autoimmunity.
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Linfócitos B , Centro Germinativo , Animais , Camundongos , Expressão Gênica , Homeostase , Camundongos Endogâmicos C57BLRESUMO
BACKGROUND: Increasing evidence shows that lifestyle interventions can improve the symptoms, quality of life (QoL), and even overall survival of patients with cancer. Digital therapeutics (DTx) can help implement behavioral modifications and empower patients through education, lifestyle support, and remote symptom monitoring. OBJECTIVE: We aimed to test the feasibility of a DTx program for patients with cancer, as measured by engagement, retention, and acceptability. In addition, we explored the effects of the program on cancer-related QoL. METHODS: We conducted a 4-week single-arm trial in Iceland, where DTx was delivered through a smartphone app. The intervention consisted of patient education about mindfulness, sleep, stress, and nutrition; lifestyle coaching; and the completion of daily missions for tracking physical activity and exercise, reporting patient-reported outcomes (PROs), practicing mindfulness, and logging healthy food intake. Information on program engagement and retention, step goal attainment, as well as PROs were collected throughout the study. QoL was measured using the European Organization for Research and Treatment of Cancer Quality of Life Questionnaire C30 at baseline and follow-up. RESULTS: In total, 30 patients with cancer undergoing active therapy were enrolled, and 29 registered in the app (23 female, 18 with breast cancer; mean age 52.6, SD 11.5 years). Overall, 97% (28/29) of participants were active in 3 of the 4 weeks and completed the pre- and postprogram questionnaires. The weekly active days (median) were 6.8 (IQR 5.8-6.8), and 72% (21/29) of participants were active at least 5 days a week. Users interacted with the app on average 7.7 (SD 1.9) times per day. On week 1, all 29 participants used the step counter and logged an average of 20,306 steps; 21 (72%) participants reached their step goals of at least 3000 steps per day. On week 4, of the 28 active users, 27 (96%) were still logging their steps, with 19 (68%) reaching their step goals. Of the 28 participants who completed the satisfaction questionnaire, 25 (89%) were likely to recommend the program, 23 (82%) said the program helped them deal with the disease, and 24 (86%) said it helped them remember their medication. QoL assessment showed that the average global health status, functioning, and symptom burden remained stable from baseline to follow-up. In all, 50% (14/28) of participants reported less pain, and the average pain score decreased from 31 (SD 20.1) to 22.6 (SD 23.2; P=.16). There was no significant change in PROs on the quality of sleep, energy, and stress levels from the first to the last week. CONCLUSIONS: The high retention, engagement, and acceptability found in this study demonstrate that multidisciplinary DTx is feasible for patients with cancer. A longer, full-scale randomized controlled trial is currently being planned to evaluate the efficacy of the intervention.
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Despite the important role that monozygotic twins have played in genetics research, little is known about their genomic differences. Here we show that monozygotic twins differ on average by 5.2 early developmental mutations and that approximately 15% of monozygotic twins have a substantial number of these early developmental mutations specific to one of them. Using the parents and offspring of twins, we identified pre-twinning mutations. We observed instances where a twin was formed from a single cell lineage in the pre-twinning cell mass and instances where a twin was formed from several cell lineages. CpG>TpG mutations increased in frequency with embryonic development, coinciding with an increase in DNA methylation. Our results indicate that allocations of cells during development shapes genomic differences between monozygotic twins.
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Genoma Humano , Células Germinativas/metabolismo , Gêmeos Monozigóticos/genética , Desenvolvimento Embrionário/genética , Feminino , Frequência do Gene/genética , Humanos , Masculino , Mosaicismo , Mutação/genética , Zigoto/metabolismoRESUMO
The microphthalmia-associated transcription factor (MITF) is a critical regulator of melanocyte development and differentiation. It also plays an important role in melanoma where it has been described as a molecular rheostat that, depending on activity levels, allows reversible switching between different cellular states. Here, we show that MITF directly represses the expression of genes associated with the extracellular matrix (ECM) and focal adhesion pathways in human melanoma cells as well as of regulators of epithelial-to-mesenchymal transition (EMT) such as CDH2, thus affecting cell morphology and cell-matrix interactions. Importantly, we show that these effects of MITF are reversible, as expected from the rheostat model. The number of focal adhesion points increased upon MITF knockdown, a feature observed in drug-resistant melanomas. Cells lacking MITF are similar to the cells of minimal residual disease observed in both human and zebrafish melanomas. Our results suggest that MITF plays a critical role as a repressor of gene expression and is actively involved in shaping the microenvironment of melanoma cells in a cell-autonomous manner.
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Transição Epitelial-Mesenquimal , Matriz Extracelular/metabolismo , Adesões Focais/metabolismo , Fator de Transcrição Associado à Microftalmia/genética , Linhagem Celular Tumoral , Humanos , Melanoma/metabolismo , Fator de Transcrição Associado à Microftalmia/metabolismoRESUMO
Waldenström's macroglobulinemia (WM) is a non-Hodgkin lymphoma, resulting in antibody-secreting lymphoplasmacytic cells in the bone marrow and pathologies resulting from high levels of monoclonal immunoglobulin M (IgM) in the blood. Despite the key role for BLIMP1 in plasma cell maturation and antibody secretion, its potential effect on WM cell biology has not yet been explored. Here we provide evidence of a crucial role for BLIMP1 in the survival of cells from WM cell line models and further demonstrate that BLIMP1 is necessary for the expression of the histone methyltransferase EZH2 in both WM and multiple myeloma cell lines. The effect of BLIMP1 on EZH2 levels is post-translational, at least partially through the regulation of proteasomal targeting of EZH2. Chromatin immunoprecipitation analysis and transcriptome profiling suggest that the two factors co-operate in regulating genes involved in cancer cell immune evasion. Co-cultures of natural killer cells and cells from a WM cell line further suggest that both factors participate in immune evasion by promoting escape from natural killer cell-mediated cytotoxicity. Together, the interplay of BLIMP1 and EZH2 plays a vital role in promoting the survival of WM cell lines, suggesting a role for the two factors in Waldenström's macroglobulinaemia.
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Proteína Potenciadora do Homólogo 2 de Zeste/genética , Perfilação da Expressão Gênica/métodos , Regulação Neoplásica da Expressão Gênica , Linfoma não Hodgkin/genética , Fator 1 de Ligação ao Domínio I Regulador Positivo/genética , Linhagem Celular Tumoral , Sobrevivência Celular/genética , Proteína Potenciadora do Homólogo 2 de Zeste/metabolismo , Células HEK293 , Humanos , Linfoma não Hodgkin/metabolismo , Linfoma não Hodgkin/patologia , Mieloma Múltiplo/genética , Mieloma Múltiplo/metabolismo , Mieloma Múltiplo/patologia , Fator 1 de Ligação ao Domínio I Regulador Positivo/metabolismo , Ligação Proteica , Macroglobulinemia de Waldenstrom/genética , Macroglobulinemia de Waldenstrom/metabolismo , Macroglobulinemia de Waldenstrom/patologiaRESUMO
Germ cells are the precursors to the gametes that carry genetic and epigenetic information between human generations and generate a new individual. Because germ cells are specified early during embryogenesis, at the time of embryo implantation, they are inaccessible for research. Our understanding of their biology has therefore developed slowly since their identification over one hundred years ago. As a result of research into the properties of human and mouse embryonic stem cells and primordial germ cells, scientists have now succeeded in efficiently generating human primordial germ cells in culture by embryonic stem cell and induced pluripotent stem cell culture. In this review we will discuss the state of our knowledge of human primordial germ cells and how research into the pluripotent properties of human and mouse embryonic germ cells has led to this breakthrough. In addition we will discuss the possible utilization of a cell culture system of human primordial germ cells for research into and treatment of germ cell related abnormalities.
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Técnicas de Cultura de Células , Diferenciação Celular , Linhagem da Célula , Células-Tronco Embrionárias/fisiologia , Células Germinativas/fisiologia , Células-Tronco Pluripotentes/fisiologia , Animais , Células Cultivadas , Células-Tronco Embrionárias/transplante , Células Germinativas/transplante , Humanos , Células-Tronco Pluripotentes/transplante , Transplante de Células-TroncoRESUMO
During embryonic development, the foundation of the germline is laid by the specification of primordial germ cells (PGCs) from the postimplantation epiblast via bone morphogenetic protein (BMP) and WNT signalling. While the majority of epiblast cells undergo differentiation towards somatic cell lineages, PGCs initiate a unique cellular programme driven by the cooperation of the transcription factors BLIMP1, PRDM14 and AP2γ. These factors synergistically suppress the ongoing somatic differentiation and drive the re-expression of pluripotency and germ cell-specific genes accompanied by global epigenetic changes. However, an unresolved question is how postimplantation epiblast cells acquire the developmental competence for the PGC fate downstream of BMP/WNT signalling. One emerging concept is that transcriptional enhancers might play a central role in the establishment of developmental competence and the execution of cell fate determination. Here, we discuss recent advances on the specification and reprogramming of PGCs thereby highlighting the concept of enhancer function.
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Diferenciação Celular/fisiologia , Desenvolvimento Embrionário/fisiologia , Epigênese Genética/fisiologia , Redes Reguladoras de Genes/genética , Camadas Germinativas/embriologia , Transdução de Sinais/fisiologia , Animais , Camundongos , Modelos Biológicos , Transdução de Sinais/genéticaRESUMO
Primordial germ cells (PGCs) are the precursors of sperm and eggs, which generate a new organism that is capable of creating endless new generations through germ cells. PGCs are specified during early mammalian postimplantation development, and are uniquely programmed for transmission of genetic and epigenetic information to subsequent generations. In this Primer, we summarise the establishment of the fundamental principles of PGC specification during early development and discuss how it is now possible to make mouse PGCs from pluripotent embryonic stem cells, and indeed somatic cells if they are first rendered pluripotent in culture.
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Células Germinativas/citologia , Células-Tronco/citologia , Animais , Desdiferenciação Celular , Linhagem da Célula , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Epigênese Genética , Feminino , Gametogênese/genética , Gametogênese/fisiologia , Redes Reguladoras de Genes , Células Germinativas/metabolismo , Humanos , Masculino , Camundongos , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo , Células-Tronco/metabolismoRESUMO
Meiotic recombination contributes to genetic diversity by yielding new combinations of alleles. Individuals vary with respect to the genome-wide recombination counts in their gametes. Exploiting data resources in Iceland, we compiled a data set consisting of 35,927 distinct parents and 71,929 parent-offspring pairs. Within this data set, we called over 2.2 million recombination events and imputed variants with sequence-level resolution from 2,261 whole genome-sequenced individuals into the parents to search for variants influencing recombination rate. We identified 13 variants in 8 regions that are associated with genome-wide recombination rate, 8 of which were previously unknown. Three of these variants associate with male recombination rate only, seven variants associate with female recombination rate only and three variants affect both. Two are low-frequency variants with large effects, one of which is estimated to increase the male and female genetic maps by 111 and 416 cM, respectively. This variant, located in an intron, would not be found by exome sequencing.
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Variação Genética , Genoma Humano , Polimorfismo de Nucleotídeo Único , Recombinação Genética , Inversão Cromossômica , Cromossomos Humanos Par 17 , Cromossomos Humanos Par 4 , Feminino , Frequência do Gene , Histona-Lisina N-Metiltransferase/genética , Humanos , Islândia , Masculino , Telômero/genéticaRESUMO
Sequence polymorphisms linked to human diseases and phenotypes in genome-wide association studies often affect noncoding regions. A SNP within an intron of the gene encoding Interferon Regulatory Factor 4 (IRF4), a transcription factor with no known role in melanocyte biology, is strongly associated with sensitivity of skin to sun exposure, freckles, blue eyes, and brown hair color. Here, we demonstrate that this SNP lies within an enhancer of IRF4 transcription in melanocytes. The allele associated with this pigmentation phenotype impairs binding of the TFAP2A transcription factor that, together with the melanocyte master regulator MITF, regulates activity of the enhancer. Assays in zebrafish and mice reveal that IRF4 cooperates with MITF to activate expression of Tyrosinase (TYR), an essential enzyme in melanin synthesis. Our findings provide a clear example of a noncoding polymorphism that affects a phenotype by modulating a developmental gene regulatory network.
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Fatores Reguladores de Interferon/metabolismo , Polimorfismo de Nucleotídeo Único , Animais , Sequência de Bases , Elementos Facilitadores Genéticos , Humanos , Fatores Reguladores de Interferon/química , Fatores Reguladores de Interferon/genética , Melanócitos/metabolismo , Camundongos , Dados de Sequência Molecular , Pigmentação , Transdução de Sinais , Fator de Transcrição AP-2/química , Fator de Transcrição AP-2/metabolismo , Peixe-ZebraRESUMO
Transitions in cell states are controlled by combinatorial actions of transcription factors. BLIMP1, the key regulator of primordial germ cell (PGC) specification, apparently acts together with PRDM14 and AP2γ. To investigate their individual and combinatorial functions, we first sought an in vitro system for transcriptional readouts and chromatin immunoprecipitation sequencing analysis. We then integrated this data with information from single-cell transcriptome analysis of normal and mutant PGCs. Here we show that BLIMP1 binds directly to repress somatic and cell proliferation genes. It also directly induces AP2γ, which together with PRDM14 initiates the PGC-specific fate. We determined the occupancy of critical genes by AP2γ-which, when computed altogether with those of BLIMP1 and PRDM14 (both individually and cooperatively), reveals a tripartite mutually interdependent transcriptional network for PGCs. We also demonstrate that, in principle, BLIMP1, AP2γ and PRDM14 are sufficient for PGC specification, and the unprecedented resetting of the epigenome towards a basal state.
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Diferenciação Celular , Células Germinativas/citologia , Células Germinativas/metabolismo , Fatores de Transcrição/metabolismo , Animais , Linhagem Celular Tumoral , Proliferação de Células , Células Cultivadas , Proteínas de Ligação a DNA , Regulação da Expressão Gênica no Desenvolvimento , Camundongos , Fator 1 de Ligação ao Domínio I Regulador Positivo , Ligação Proteica , Proteínas de Ligação a RNA , Proteínas Repressoras/metabolismo , Fator de Transcrição AP-2/metabolismoRESUMO
Primordial germ cells (PGCs) and somatic cells originate from postimplantation epiblast cells in mice. As pluripotency is lost upon differentiation of somatic lineages, a naive epigenome and the pluripotency network are re-established during PGC development. Here we demonstrate that Prdm14 contributes not only to PGC specification, but also to naive pluripotency in embryonic stem (ES) cells by repressing the DNA methylation machinery and fibroblast growth factor (FGF) signalling. This indicates a critical role for Prdm14 in programming PGCs and promoting pluripotency in ES cells.
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Células-Tronco Embrionárias/metabolismo , Fatores de Crescimento de Fibroblastos/genética , Regulação da Expressão Gênica no Desenvolvimento , Células Germinativas/metabolismo , Células-Tronco Pluripotentes/metabolismo , Fatores de Transcrição/genética , Animais , Diferenciação Celular , Metilação de DNA , Proteínas de Ligação a DNA , Células-Tronco Embrionárias/citologia , Fatores de Crescimento de Fibroblastos/metabolismo , Perfilação da Expressão Gênica , Células Germinativas/citologia , Camadas Germinativas/citologia , Camadas Germinativas/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas Quinases Ativadas por Mitógeno/genética , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Células-Tronco Pluripotentes/citologia , Proteínas de Ligação a RNA , Transdução de Sinais , Fatores de Transcrição/metabolismoRESUMO
Development of mammalian primordial germ cells (PGCs) presents a unique example of a cell fate specification event that is intimately linked with epigenetic reprogramming. Cell fate commitment is governed by transcription factors which, together with epigenetic regulators, instruct lineage choice in response to signalling cues. Similarly, the reversal of epigenetic silencing is driven by the combinatorial action of transcriptional regulators, resulting in an increase in cellular plasticity. PGCs constitute a paradox, since their development as a unipotent specialised lineage is coupled with extensive reprogramming, which eventually leads to an increase in cellular potency. In this review we discuss the role of key factors in the specification of the germ cell lineage that are also important for the comprehensive erasure of epigenetic modifications, which provides the foundation for regeneration of totipotency. We further discuss current concepts of transcriptional and epigenetic control of cell fate decisions, with a particular focus on emerging principles of enhancer activity and their potential implications for the transcriptional control of PGC specification.
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Diferenciação Celular , Reprogramação Celular , Células-Tronco Embrionárias/citologia , Células Germinativas/metabolismo , Animais , Linhagem da Célula , Metilação de DNA , Células-Tronco Embrionárias/metabolismo , Epigênese Genética , Epigenômica , Inativação Gênica , Humanos , Mamíferos , Transdução de Sinais , Fatores de TranscriçãoRESUMO
Epigenetic reprogramming in early germ cells is critical toward the establishment of totipotency, but investigations of the germline events are intractable. An objective cell culture-based system could provide mechanistic insight on how the key determinants of primordial germ cells (PGCs), including Prdm14, induce reprogramming in germ cells to an epigenetic ground state. Here we show a Prdm14-Klf2 synergistic effect that can accelerate and enhance reversion of mouse epiblast stem cells (epiSCs) to a naive pluripotent state, including X reactivation and DNA demethylation. Notably, Prdm14 alone has little effect on epiSC reversion, but it enhances the competence for reprogramming and potentially PGC specification. Reprogramming of epiSCs by the combinatorial effect of Prdm14-Klf2 involves key epigenetic changes, which might have an analogous role in PGCs. Our study provides a paradigm toward a systematic analysis of how other key genes contribute to complex and dynamic events of reprogramming in the germline.
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Desdiferenciação Celular , Epigênese Genética , Camadas Germinativas/metabolismo , Fatores de Transcrição Kruppel-Like/metabolismo , Proteínas Repressoras/metabolismo , Células-Tronco/metabolismo , Fatores de Transcrição/metabolismo , Animais , Metilação de DNA , Proteínas de Ligação a DNA , Células Germinativas/citologia , Camadas Germinativas/citologia , Humanos , Camundongos , Proteínas de Ligação a RNA , Células-Tronco/citologiaRESUMO
The zinc-finger PR domain transcriptional repressor Blimp-1/Prdm1 plays essential roles in primordial germ cell specification, placental, heart, and forelimb development, plasma cell differentiation, and T-cell homeostasis. The present experiments demonstrate that the mouse Prdm1 gene has three alternative promoter regions. All three alternative first exons splice directly to exon 3, containing the translational start codon. To examine possible cell-type-specific functional activities in vivo, we generated targeted deletions that selectively eliminate two of these transcriptional start sites. Remarkably, mice lacking the previously described first exon develop normally and are fertile. However, this region contains NF-kappaB binding sites, and as shown here, NF-kappaB signaling is required for Prdm1 induction. Thus, mutant B cells fail to express Prdm1 in response to lipopolysaccharide stimulation and lack the ability to become antibody-secreting cells. An alternative distal promoter located approximately 70 kb upstream, giving rise to transcripts strongly expressed in the yolk sac, is dispensable. Thus, the deletion of exon 1B has no noticeable effect on expression levels in the embryo or adult tissues. Collectively, these experiments provide insight into the organization of the Prdm1 gene and demonstrate that NF-kappaB is a key mediator of Prdm1 expression.
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Diferenciação Celular , Desenvolvimento Embrionário/genética , Plasmócitos/citologia , Plasmócitos/metabolismo , Regiões Promotoras Genéticas , Fatores de Transcrição/genética , Animais , Pareamento de Bases/genética , Sequência de Bases , Células Dendríticas/metabolismo , Éxons/genética , Deleção de Genes , Dosagem de Genes , Regulação da Expressão Gênica no Desenvolvimento , Marcação de Genes , Heterozigoto , Íntrons/genética , Camundongos , Dados de Sequência Molecular , NF-kappa B/metabolismo , Fator 1 de Ligação ao Domínio I Regulador Positivo , Ligação Proteica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transdução de Sinais , Linfócitos T/metabolismo , Fatores de Transcrição/metabolismo , Sítio de Iniciação de Transcrição , Ativação TranscricionalRESUMO
Mice with a T cell-specific deletion of Prdm1, encoding Blimp-1, have aberrant T cell homeostasis and develop fatal colitis. In this study, we show that one critical activity of Blimp-1 in T cells is to repress IL-2, and that it does so by direct repression of Il2 transcription, and also by repression of Fos transcription. Using these mechanisms Blimp-1 participates in an autoregulatory loop by which IL-2 induces Prdm1 expression and thus represses its own expression after T cell activation, ensuring that the immune response is appropriately controlled. This activity of Blimp-1 is important for cytokine deprivation-induced T cell death and for attenuating T cell proliferation in antigen-specific responses both in vitro and in vivo.
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Interleucina-2/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-fos/metabolismo , Linfócitos T/citologia , Fatores de Transcrição/fisiologia , Animais , Apresentação de Antígeno , Diferenciação Celular , Proliferação de Células , Sobrevivência Celular , Homeostase , Sistema Imunitário , Interleucina-2/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Fator 1 de Ligação ao Domínio I Regulador Positivo , Estrutura Terciária de Proteína , Fatores de Transcrição/metabolismo , Transcrição GênicaRESUMO
T cell-specific deletion of Blimp-1 causes abnormal T cell homeostasis and function, leading to spontaneous, fatal colitis in mice. Herein we explore the role of Blimp-1 in Th1/Th2 differentiation. Blimp-1 mRNA and protein are more highly expressed in Th2 cells compared with Th1 cells, and Blimp-1 attenuates IFN-gamma production in CD4 cells activated under nonpolarizing conditions. Although Blimp-1-deficient T cells differentiate normally to Th2 cytokines in vitro, Blimp-1 is required in vivo for normal Th2 humoral responses to NP-KLH (4-hydroxy-3-nitrophenylacetyl/keyhole lymphocyte hemocyanin) immunization. Lack of Blimp-1 in CD4 T cells causes increased IFN-gamma, T-bet, and Bcl-6 mRNA. By chromatin immunoprecipitation we show that Blimp-1 binds directly to a distal regulatory region in the ifng gene and at multiple sites in tbx21 and bcl6 genes. Our data provide evidence that Blimp-1 functions in Th2 cells to reinforce Th2 differentiation by repressing critical Th1 genes.
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Diferenciação Celular/imunologia , Proteínas de Ligação a DNA/antagonistas & inibidores , Interferon gama/antagonistas & inibidores , Proteínas Repressoras/fisiologia , Proteínas com Domínio T/antagonistas & inibidores , Células Th1/imunologia , Células Th1/metabolismo , Fatores de Transcrição/fisiologia , Animais , Diferenciação Celular/genética , Células Cultivadas , Proteínas de Ligação a DNA/biossíntese , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/fisiologia , Regulação da Expressão Gênica/imunologia , Inibidores do Crescimento/biossíntese , Inibidores do Crescimento/deficiência , Inibidores do Crescimento/fisiologia , Interferon gama/biossíntese , Interferon gama/genética , Depleção Linfocítica , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Fator 1 de Ligação ao Domínio I Regulador Positivo , Proteínas Proto-Oncogênicas c-bcl-6 , Proteínas Repressoras/biossíntese , Proteínas Repressoras/genética , Proteínas com Domínio T/biossíntese , Proteínas com Domínio T/genética , Células Th1/citologia , Células Th2/citologia , Células Th2/imunologia , Células Th2/metabolismo , Fatores de Transcrição/biossíntese , Fatores de Transcrição/deficiência , Fatores de Transcrição/genéticaRESUMO
The cornified layer is a compacted lattice of lipid-embedded corneocytes that provides an organism's barrier to the external environment. Cornification is the final differentiative step for epidermal keratinocytes and involves dramatic cell condensation before death. Using conditional gene deletion in mice, we identified the transcriptional repressor Blimp-1 (B lymphocyte-induced maturation protein-1) as an important regulator of keratinocyte transition from the granular to the cornified layer. More than 250 genes are misregulated in conditional knockout epidermis, including those encoding transcription factors, signal transduction components, proteinases, and enzymes involved in lipid metabolism. Steady-state mRNA and ChIP analyses of a subset of these genes provide evidence that nfat5, fos, prdm1, and dusp16 are novel direct targets of Blimp-1. Identifying nfat5 as a target of Blimp-1 repression indicates that cornification involves suppression of normal osmotic regulation in granular cells. Consistently, conditional knockout mice have delayed barrier formation as embryos, enlarged granular layer cells and corneocytes, and a morphologically abnormal cornified layer. These studies provide insight into cornification, identifying transcriptional regulatory circuitry and indicating the importance of blocking osmotic homeostasis.