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1.
Immunogenetics ; 71(7): 489-499, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31297569

RESUMO

Epigenetic modifications have been shown to be important for immune cell differentiation by regulating gene transcription. However, the role and mechanism of histone methylation in the development and differentiation of iNKT cells in rheumatoid arthritis (RA) mice have yet to be deciphered. The DBA/1 mouse RA model was established by using a modified GPI mixed peptide. We demonstrated that total peripheral blood, thymus, and spleen iNKT cells in RA mice decreased significantly, while iNKT1 in the thymus and spleen was increased significantly. PLZF protein and PLZF mRNA levels were significantly decreased in thymus DP T cells, while T-bet protein and mRNA were significantly increased in thymus iNKT cells. We found a marked accumulation in H3K27me3 around the promoter regions of the signature gene Zbtb16 in RA mice thymus DP T cells, and an accumulation of H3K4me3 around the promoters of the Tbx21 gene in iNKT cells. The expression levels of UTX in the thymus of RA mice were significantly reduced. The changes in the above indicators were particularly significant in the progressive phase of inflammation (11 days after modeling) and the peak phase of inflammation (14 days after modeling) in RA mice. Developmental and differentiation defects of iNKT cells in RA mice were associated with abnormal methylation levels (H3K27me3 and H3K4me3) in the promoters of key genes Zbtb16 (encoding PLZF) and Tbx21 (encoding T-bet). Decreased UTX of thymus histone demethylase levels resulted in the accumulation of H3K27me3 modification.


Assuntos
Artrite Reumatoide/patologia , Lisina/metabolismo , Células T Matadoras Naturais/patologia , Regiões Promotoras Genéticas , Timo/fisiologia , Animais , Artrite Experimental/patologia , Diferenciação Celular , Epigênese Genética , Regulação da Expressão Gênica , Histona Desmetilases/genética , Histona Desmetilases/metabolismo , Metilação , Camundongos Endogâmicos DBA , Proteína com Dedos de Zinco da Leucemia Promielocítica/genética , Proteína com Dedos de Zinco da Leucemia Promielocítica/metabolismo , Baço/patologia , Proteínas com Domínio T/genética , Proteínas com Domínio T/metabolismo
2.
EMBO J ; 38(14): e101260, 2019 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-31304630

RESUMO

Tissue-resident iNKT cells maintain tissue homeostasis and peripheral surveillance against pathogens; however, studying these cells is challenging due to their low abundance and poor recovery from tissues. We here show that iNKT transnuclear mice, generated by somatic cell nuclear transfer, have increased tissue resident iNKT cells. We examined expression of PLZF, T-bet, and RORγt, as well as cytokine/chemokine profiles, and found that both monoclonal and polyclonal iNKT cells differentiated into functional subsets that faithfully replicated those seen in wild-type mice. We detected iNKT cells from tissues in which they are rare, including adipose, lung, skin-draining lymph nodes, and a previously undescribed population in Peyer's patches (PP). PP-NKT cells produce the majority of the IL-4 in Peyer's patches and provide indirect help for B-cell class switching to IgG1 in both transnuclear and wild-type mice. Oral vaccination with α-galactosylceramide shows enhanced fecal IgG1 titers in iNKT cell-sufficient mice. Transcriptional profiling reveals a unique signature of PP-NKT cells, characterized by tissue residency. We thus define PP-NKT as potentially important for surveillance for mucosal pathogens.


Assuntos
Perfilação da Expressão Gênica/métodos , Switching de Imunoglobulina , Imunoglobulina G/genética , Células T Matadoras Naturais/metabolismo , Nódulos Linfáticos Agregados/imunologia , Animais , Diferenciação Celular , Células Cultivadas , Feminino , Galactosilceramidas/administração & dosagem , Galactosilceramidas/imunologia , Interleucina-4/genética , Camundongos , Células T Matadoras Naturais/citologia , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/genética , Técnicas de Transferência Nuclear , Proteína com Dedos de Zinco da Leucemia Promielocítica/genética , Proteínas com Domínio T/genética , Vacinação
3.
Nat Commun ; 10(1): 2094, 2019 05 07.
Artigo em Inglês | MEDLINE | ID: mdl-31064978

RESUMO

Multiple sclerosis (MS) is an inflammatory, demyelinating disease of the central nervous system with a modest concordance rate in monozygotic twins, which strongly argues for involvement of epigenetic factors. We observe highly similar peripheral blood mononuclear cell-based methylomes in 45 MS-discordant monozygotic twins. Nevertheless, we identify seven MS-associated differentially methylated positions (DMPs) of which we validate two, including a region in the TMEM232 promoter and ZBTB16 enhancer. In CD4 + T cells we find an MS-associated differentially methylated region in FIRRE. Additionally, 45 regions show large methylation differences in individual pairs, but they do not clearly associate with MS. Furthermore, we present epigenetic biomarkers for current interferon-beta treatment, and extensive validation shows that the ZBTB16 DMP is a signature for prior glucocorticoid treatment. Taken together, this study represents an important reference for epigenomic MS studies, identifies new candidate epigenetic markers, and highlights treatment effects and genetic background as major confounders.


Assuntos
Metilação de DNA/genética , Doenças em Gêmeos/genética , Epigênese Genética , Predisposição Genética para Doença , Esclerose Múltipla/genética , Adulto , Idoso , Biomarcadores , Doenças em Gêmeos/sangue , Elementos Facilitadores Genéticos/genética , Epigenômica/métodos , Feminino , Humanos , Leucócitos Mononucleares , Masculino , Proteínas de Membrana/genética , Pessoa de Meia-Idade , Esclerose Múltipla/sangue , Regiões Promotoras Genéticas/genética , Proteína com Dedos de Zinco da Leucemia Promielocítica/genética , RNA Longo não Codificante/genética , Gêmeos Monozigóticos , Adulto Jovem
4.
J Biomed Sci ; 26(1): 30, 2019 Apr 26.
Artigo em Inglês | MEDLINE | ID: mdl-31027502

RESUMO

BACKGROUND: Promyelocytic leukemia zinc finger (Plzf), a transcriptional regulator involved in a lot of important biological processes during development, has been implied to maintain neural stem cells and inhibit their differentiation into neurons. However, the effects of Plzf on brain structures and functions are still not clarified. RESULTS: We showed that Plzf expression was detected as early as embryonic day (E) 9.5 in Pax6+ cells in the mouse brain, and was completely disappeared in telencephalon before the initiation of cortical neurogenesis. Loss of Plzf resulted in a smaller cerebral cortex with a decrease in the number of Tbr1+ deep layer neurons due to a decrease of mitotic cell number in the ventricular zone of forebrain at early developmental stage. Microarray, qRT-PCR, and flow cytometry analysis identified dysregulation of Mash1 proneural gene expression. We also observed an impairment of recognition memory in Plzf-deficient mice. CONCLUSIONS: Plzf is expressed at early stages of brain development and involved in the formation of deep layer cortical neurons. Loss of Plzf results in dysregulation of Mash1, microcephaly with reduced numbers of early-born neurons, and impairment of recognition memory.


Assuntos
Expressão Gênica/fisiologia , Neurogênese/genética , Neurônios/fisiologia , Proteína com Dedos de Zinco da Leucemia Promielocítica/genética , Animais , Córtex Cerebral/crescimento & desenvolvimento , Córtex Cerebral/fisiologia , Camundongos , Proteína com Dedos de Zinco da Leucemia Promielocítica/metabolismo
5.
Biochem Biophys Res Commun ; 511(4): 935-940, 2019 04 16.
Artigo em Inglês | MEDLINE | ID: mdl-30853180

RESUMO

BACKGROUND/AIMS: The expression of transcription factor Zbtb1 is essential for the maintenance and development of various blood cells in the hematopoietic system. In the current study, we found that the total number of thymocytes in PLZF deficient mice was reduced compared with thymocytes in wild-type mice, and the number of early T-cell progenitors decreased. However, the decrease of thymocytes in PLZF deficient mice was not cell intrinsic. This study adds new information regarding the regulation of the PLZF gene in the development and self-renewal of T cells. METHODS: The thymus was isolated from newborn mice, and the two lobes of each thymus were physically separated. Each host received a thymus, two lobes, each placed at one end of the kidney, as described in the literature. Tail vein blood was periodically collected from some of the recipients and analyzed for the presence of peripheral blood T cells. RESULTS: In PLZF-EGFP reporter mice and neonatal thymus transplantation to the kidney, we found that PLZF was highly expressed in DN1 (Lineage-CD44+CD25-) cells of thymic grafts of Rag2/γc-/- recipient mice. We found that the proportion of PLZF wild-type and mutant-derived cells in the thymocytes of recipient mice after bone marrow transplantation is approximately equal to the competitive bone marrow chimeric mouse model, and all mice contain a normal thymus. CONCLUSION: The development of T cells suggests that the effect of the PLZF gene on T cell differentiation and development is not cell intrinsic. However, in the neonatal mouse thymic transplant model in the Rag2/γc-/- recipient mouse, deletion of the PLZF gene results in a significant decrease in the proportion of DN1 cells from the donor in the thymic graft.


Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Proteína com Dedos de Zinco da Leucemia Promielocítica/genética , Linfócitos T/citologia , Animais , Contagem de Células , Autorrenovação Celular , Deleção de Genes , Camundongos , Camundongos Endogâmicos C57BL , Linfócitos T/metabolismo , Timócitos/citologia , Timócitos/metabolismo , Timo/citologia , Timo/crescimento & desenvolvimento , Timo/metabolismo
6.
Proc Natl Acad Sci U S A ; 116(15): 7439-7448, 2019 04 09.
Artigo em Inglês | MEDLINE | ID: mdl-30910955

RESUMO

Cellular metabolism and signaling pathways are key regulators to determine conventional T cell fate and function, but little is understood about the role of cell metabolism for natural killer T (NKT) cell survival, proliferation, and function. We found that NKT cells operate distinct metabolic programming from CD4 T cells. NKT cells are less efficient in glucose uptake than CD4 T cells with or without activation. Gene-expression data revealed that, in NKT cells, glucose is preferentially metabolized by the pentose phosphate pathway and mitochondria, as opposed to being converted into lactate. In fact, glucose is essential for the effector functions of NKT cells and a high lactate environment is detrimental for NKT cell survival and proliferation. Increased glucose uptake and IFN-γ expression in NKT cells is inversely correlated with bacterial loads in response to bacterial infection, further supporting the significance of glucose metabolism for NKT cell function. We also found that promyelocytic leukemia zinc finger seemed to play a role in regulating NKT cells' glucose metabolism. Overall, our study reveals that NKT cells use distinct arms of glucose metabolism for their survival and function.


Assuntos
Linfócitos T CD4-Positivos/imunologia , Proliferação de Células , Mitocôndrias/metabolismo , Células T Matadoras Naturais/imunologia , Fosforilação Oxidativa , Via de Pentose Fosfato/imunologia , Animais , Linfócitos T CD4-Positivos/citologia , Sobrevivência Celular/genética , Sobrevivência Celular/imunologia , Glucose/genética , Glucose/imunologia , Camundongos , Camundongos Knockout , Mitocôndrias/genética , Células T Matadoras Naturais/citologia , Via de Pentose Fosfato/genética , Proteína com Dedos de Zinco da Leucemia Promielocítica/genética , Proteína com Dedos de Zinco da Leucemia Promielocítica/imunologia
7.
Nucleic Acids Res ; 47(9): 4509-4520, 2019 05 21.
Artigo em Inglês | MEDLINE | ID: mdl-30892634

RESUMO

PLZF (promyelocytic leukemia zinc finger) is a transcription factor acting as a global regulator of hematopoietic commitment. PLZF displays an epigenetic specificity by recruiting chromatin-modifying factors but little is known about its role in remodeling chromatin of cells committed toward a given specific hematopoietic lineage. In murine myeloid progenitors, we decipher a new role for PLZF in restraining active genes and enhancers by targeting acetylated lysine 27 of Histone H3 (H3K27ac). Functional analyses reveal that active enhancers bound by PLZF are involved in biological processes related to metabolism and associated with hematopoietic aging. Comparing the epigenome of young and old myeloid progenitors, we reveal that H3K27ac variation at active enhancers is a hallmark of hematopoietic aging. Taken together, these data suggest that PLZF, associated with active enhancers, appears to restrain their activity as an epigenetic gatekeeper of hematopoietic aging.


Assuntos
Envelhecimento/genética , Células-Tronco Hematopoéticas/metabolismo , Proteína com Dedos de Zinco da Leucemia Promielocítica/genética , Transcrição Genética , Animais , Diferenciação Celular/genética , Elementos Facilitadores Genéticos , Epigênese Genética/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Histonas/genética , Humanos , Histona Desmetilases com o Domínio Jumonji/genética , Camundongos , Células Progenitoras Mieloides/metabolismo , Ligação Proteica , Sequências Reguladoras de Ácido Nucleico/genética
8.
Immunity ; 50(4): 1054-1068.e3, 2019 04 16.
Artigo em Inglês | MEDLINE | ID: mdl-30926235

RESUMO

Innate lymphoid cell (ILC) development proposes that ILC precursors (ILCPs) segregate along natural killer (NK) cell versus helper cell (ILC1, ILC2, ILC3) pathways, the latter depending on expression of Id2, Zbtb16, and Gata3. We have developed an Id2-reporter strain expressing red fluorescent protein (RFP) in the context of normal Id2 expression to re-examine ILCP phenotype and function. We show that bone-marrow ILCPs were heterogeneous and harbored extensive NK-cell potential in vivo and in vitro. By multiplexing Id2RFP with Zbtb16CreGFP and Bcl11btdTomato strains, we made a single-cell dissection of the ILCP compartment. In contrast with the current model, we have demonstrated that Id2+Zbtb16+ ILCPs included multi-potent ILCPs that retained NK-cell potential. Late-stage ILC2P and ILC3P compartments could be defined by differential Zbtb16 and Bcl11b expression. We suggest a revised model for ILC differentiation that redefines the cell-fate potential of helper-ILC-restricted Zbtb16+ ILCPs.


Assuntos
Regulação da Expressão Gênica/imunologia , Células-Tronco Hematopoéticas/citologia , Imunidade Inata , Proteína 2 Inibidora de Diferenciação/genética , Linfopoese/genética , Transferência Adotiva , Animais , Linhagem da Célula , Fator de Transcrição GATA3/biossíntese , Fator de Transcrição GATA3/genética , Fator de Transcrição GATA3/fisiologia , Genes Reporter , Células-Tronco Hematopoéticas/metabolismo , Proteína 2 Inibidora de Diferenciação/biossíntese , Células Matadoras Naturais/citologia , Proteínas Luminescentes/análise , Proteínas Luminescentes/genética , Camundongos , Camundongos Endogâmicos C57BL , Modelos Imunológicos , Proteína com Dedos de Zinco da Leucemia Promielocítica/biossíntese , Proteína com Dedos de Zinco da Leucemia Promielocítica/genética , Proteína com Dedos de Zinco da Leucemia Promielocítica/fisiologia , Análise de Célula Única , Linfócitos T Auxiliares-Indutores/citologia , Transcrição Genética
9.
Theriogenology ; 130: 120-124, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30884332

RESUMO

Microminipigs are one of the smallest miniature pigs characterized as sexually precocious; the males achieve sexual maturity at around 3-4.5 months of age. However, the physiology of this sexual precocity is still unclear. To understand sexual precocity in male microminipigs, we analyzed their testes at five developmental stages: neonatal (<7 days), 30-day-old, 45-day-old, 80-day-old, and adult (>24 months) stages. We used 4 pigs in each of the stages. To analyze testicular development histologically, the seminiferous tubule diameter (SD) was measured, and the presence or absence of the seminiferous lumen was confirmed. Changes in the expression of pluripotency markers, DBA, UCHL1, ZBTB16, and vimentin, were evaluated immunohistologically. For the analyses, cells positive for DBA, UCHL1, and ZBTB16 per 150 round seminiferous tubules in cross sections from each testis were counted to evaluate the total number of positive cells. The number of positive cells per 100 Sertoli cells (DBA+/Sertoli, UCHL1+/Sertoli, and ZBTB16+/Sertoli) was calculated to compare the five developmental stages. Histologically, SDs became larger with piglet growth, and precocity was confirmed; seminiferous lumens were observed from the 30-day-old stage. Immunohistologically, the number of DBA+/Sertoli, which indicates the number of gonocytes, decreased rapidly to an undetectable level by the 45-day-old stage. In the same period, the number of UCHL1+/Sertoli, which indicates total SSCs, increased significantly, suggesting that the proliferation of SSCs was accelerated before 30 days of age. Consequently, our study clarified that differentiation of SSCs in microminipigs started during the fetal period, the differentiation of gonocytes and proliferation of SSCs was then accelerated before 30 days of age, and the early phase of spermatogenesis was finally completed at around 45 days after birth. Consequently, sexual precocity in male microminipigs was characterized by a shorter duration of the early phase of spermatogenesis.


Assuntos
Células-Tronco Germinativas Adultas/metabolismo , Maturidade Sexual/fisiologia , Suínos/fisiologia , Animais , Biomarcadores , Diferenciação Celular , Regulação da Expressão Gênica no Desenvolvimento , Masculino , Proteína com Dedos de Zinco da Leucemia Promielocítica/genética , Proteína com Dedos de Zinco da Leucemia Promielocítica/metabolismo , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismo , Espermatogênese/fisiologia , Porco Miniatura , Ubiquitina Tiolesterase/genética , Ubiquitina Tiolesterase/metabolismo , Vimentina/genética , Vimentina/metabolismo
10.
Viruses ; 11(3)2019 03 09.
Artigo em Inglês | MEDLINE | ID: mdl-30857329

RESUMO

Expansion of natural killer (NK) cells expressing NKG2C occurs following human cytomegalovirus (HCMV) infection and is amplified by human immunodeficiency virus (HIV) co-infection. These NKG2C-expressing NK cells demonstrate enhanced CD16-dependent cytokine production and downregulate FcεRIγ and promyelocytic leukemia zinc finger protein (PLZF). Lacking NKG2C diminishes resistance to HIV infection, but whether this affects NK cell acquisition of superior antibody-dependent function is unclear. Therefore, our objective was to investigate whether HCMV-driven NK cell differentiation is impaired in NKG2Cnull HIV-infected individuals. Phenotypic (CD2, CD16, CD57, NKG2A, FcεRIγ, and PLZF expression) and functional (cytokine induction and cytotoxicity) properties were compared between HIV⁻infected NKG2Cnull and NKG2C-expressing groups. Cytokine production was compared following stimulation through natural cytotoxicity receptors or through CD16. Cytotoxicity was measured by anti-CD16-redirected lysis and by classical antibody-dependent cell-mediated cytotoxicity (ADCC) against anti-class I human leukocyte antigen (HLA) antibody-coated cells. Our data indicate highly similar HCMV-driven NK cell differentiation in HIV infection with or without NKG2C. While the fraction of mature (CD57pos) NK cells expressing CD2 (p = 0.009) or co-expressing CD2 and CD16 (p = 0.03) was significantly higher in NKG2Cnull HIV-infected individuals, there were no significant differences in NKG2A, FcεRIγ, or PLZF expression. The general phenotypic and functional equivalency observed suggests NKG2C-independent routes of HCMV-driven NK cell differentiation, which may involve increased CD2 expression.


Assuntos
Infecções por Citomegalovirus/imunologia , Citomegalovirus/patogenicidade , Infecções por HIV/imunologia , Células Matadoras Naturais/virologia , Subfamília C de Receptores Semelhantes a Lectina de Células NK/genética , Adulto , Citotoxicidade Celular Dependente de Anticorpos , Diferenciação Celular , Coinfecção/imunologia , Coinfecção/virologia , Citocinas/imunologia , Feminino , Proteínas Ligadas por GPI/genética , Proteínas Ligadas por GPI/imunologia , Humanos , Células Matadoras Naturais/citologia , Ativação Linfocitária , Masculino , Pessoa de Meia-Idade , Subfamília C de Receptores Semelhantes a Lectina de Células NK/imunologia , Proteína com Dedos de Zinco da Leucemia Promielocítica/genética , Proteína com Dedos de Zinco da Leucemia Promielocítica/imunologia , Receptores de IgE/genética , Receptores de IgE/imunologia , Receptores de IgG/genética , Receptores de IgG/imunologia
11.
Genesis ; 57(3): e23281, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30628160

RESUMO

The promyelocytic leukemia zinc finger (PLZF) transcription factor mediates a wide-range of biological processes. Accordingly, perturbation of PLZF function results in a myriad of physiologic defects, the most conspicuous of which is abnormal skeletal patterning. Although whole body knockout of Plzf in the mouse (Plzf KO ) has significantly expanded our understanding of Plzf function in vivo, a conditional knockout mouse model that enables tissue or cell-type specific ablation of Plzf has not been developed. Therefore, we used CRISPR/Cas 9 gene editing to generate a mouse model in which exon 2 of the murine Plzf gene is specifically flanked (or floxed) by LoxP sites (Plzf f/f ). Crossing our Plzf f/f mouse with a global cre-driver mouse to generate the Plzf d/d bigenic mouse, we demonstrate that exon 2 of the Plzf gene is ablated in the Plzf d/d bigenic. Similar to the previously reported Plzf KO mouse, the Plzf d/d mouse exhibits a severe defect in skeletal patterning of the hindlimb, indicating that the Plzf f/f mouse functions as designed. Therefore, studies in this short technical report demonstrate that the Plzf f/f mouse will be useful to investigators who wish to explore the role of the Plzf transcription factor in a specific tissue or cell-type.


Assuntos
Sistemas CRISPR-Cas , Proteína com Dedos de Zinco da Leucemia Promielocítica/genética , Animais , Padronização Corporal , Osso e Ossos/anormalidades , Osso e Ossos/embriologia , Técnicas de Inativação de Genes/métodos , Membro Posterior/anormalidades , Membro Posterior/embriologia , Camundongos , Camundongos Endogâmicos C57BL , Proteína com Dedos de Zinco da Leucemia Promielocítica/deficiência
12.
Oncol Rep ; 41(2): 1007-1018, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30431129

RESUMO

Promyelocytic leukemia zinc finger (PLZF) plays important roles in tumorigenic and developmental processes of various types of cancers. However, the expression of PLZF in gastric cancer (GC) has not been reported. The aim of the present study was to investigate the expression level and potential status of PLZF in GC as well as its possible mechanism. In the present study, we found that PLZF was downregulated in the majority of GC cell lines and tumor tissues and that alteration of PLZF expression was closely correlated with a malignant phenotype, epithelial­mesenchymal transformation and overall survival. Evaluation of in vitro proliferation, colony information, migration and invasion indicated that PLZF gene transduction induced a less malignant phenotype, which was also confirmed through in vivo studies performed in athymic nude mice. Furthermore, we assessed the expression levels of the lncRNA ANRIL in GC and found that it was negatively associated with the level of PLZF and that ANRIL indirectly methylated PLZF to suppress its expression via binding with polycomb repressive complex 2. When GC cells were treated with the methylation inhibitor 5­Aza­2'­deoxycytidine, the expression of PLZF increased, which further confirmed that PLZF was methylated. These results indicated that constitutive ANRIL activation was a possible cause of the lack of PLZF expression in GC cells. Coupled deregulation of PLZF and ANRIL may account for most of the alterations described in GC, and PLZF may become a potential target of GC therapy.


Assuntos
Proliferação de Células/genética , Transição Epitelial-Mesenquimal/genética , Proteína com Dedos de Zinco da Leucemia Promielocítica/genética , RNA Longo não Codificante/metabolismo , Neoplasias Gástricas/genética , Animais , Carcinogênese/genética , Linhagem Celular Tumoral , Metilação de DNA/genética , Regulação para Baixo , Feminino , Regulação Neoplásica da Expressão Gênica , Histonas/genética , Histonas/metabolismo , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Pessoa de Meia-Idade , Complexo Repressor Polycomb 2/metabolismo , Regiões Promotoras Genéticas/genética , Proteína com Dedos de Zinco da Leucemia Promielocítica/metabolismo , RNA Longo não Codificante/genética , Estômago/patologia , Estômago/cirurgia , Neoplasias Gástricas/mortalidade , Neoplasias Gástricas/patologia , Neoplasias Gástricas/cirurgia , Análise de Sobrevida , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
13.
Epigenomics ; 10(9): 1243-1257, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-30212243

RESUMO

AIM: Decidualization is essential for embryo implantation and placental development. We aimed to obtain transcriptome and epigenome profiles for primary endometrial stromal cells (ESCs) and in vitro decidualized cells. MATERIALS & METHODS: ESCs isolated from human endometrial tissues remained untreated (D0), or decidualized for 4 days (D4) and 8 days (D8) in the presence of 8-bromo-cAMP and progesterone. RESULTS: Among the epigenetic modifications examined (DNA methylation, H3K27ac, H3K9me3 and H3K27me3), the H3K27ac patterns changed most dramatically, with a moderate correlation with gene expression changes, upon decidualization. Subsets of up- and down-regulated genes upon decidualization were associated with reciprocal changes of H3K27ac and H3K27me3 modifications at their promoter region, and were enriched with genes essential for decidualization such as WNT4, ZBTB16, PROK1 and GREB1. CONCLUSION: Our dataset is useful to further elucidate the molecular mechanisms underlying decidualization.


Assuntos
Decídua/metabolismo , Implantação do Embrião/genética , Epigênese Genética , Histonas/metabolismo , Placentação/genética , Células Cultivadas , Metilação de DNA , Decídua/citologia , Feminino , Hormônios Gastrointestinais/genética , Humanos , Proteínas de Neoplasias/genética , Gravidez , Regiões Promotoras Genéticas , Proteína com Dedos de Zinco da Leucemia Promielocítica/genética , Células Estromais/metabolismo , Fator de Crescimento do Endotélio Vascular Derivado de Glândula Endócrina/genética , Proteína Wnt4/genética
14.
Exp Cell Res ; 373(1-2): 71-79, 2018 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-30266657

RESUMO

During spermatogenesis, a group of undifferentiated spermatogonia undergoes an essential transition to a differentiating stage, which involves gain of Kit receptor. In the current study, we showed that a small non-coding RNA, miRNA-26b could induce transition from Kit- to Kit+ and inhibit proliferation of spermatogonia. A key transcriptional factor for undifferentiated spermatogonia, Plzf, was proven as a direct target of miR-26b. When undifferentiated spermatogonia were treated with Retinoic acid (RA), miR-26b was increased, further promoting RA-induced differentiation of spermatogonia. In addition, miR-26b could repress 5-hydroxymethylcytosine (5hmC) via repression of Tet3 in spermatogonia. These findings demonstrate that miR-26b might play a role in promoting the transition from Kit- to Kit+ SSCs.


Assuntos
MicroRNAs/fisiologia , Espermatogênese , Espermatogônias/metabolismo , 5-Metilcitosina/análogos & derivados , 5-Metilcitosina/metabolismo , Animais , Apoptose , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células , Células Cultivadas , Proteínas de Ligação a DNA/metabolismo , Masculino , Camundongos , MicroRNAs/metabolismo , Proteína com Dedos de Zinco da Leucemia Promielocítica/genética , Proteína com Dedos de Zinco da Leucemia Promielocítica/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-kit/análise , Espermatogônias/citologia , Espermatogônias/efeitos dos fármacos , Tretinoína/farmacologia
15.
Cell Physiol Biochem ; 48(6): 2528-2538, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30121655

RESUMO

BACKGROUND/AIMS: Our study aims to characterize functions of ZBTB16 gene in the process of intramuscular fat (IMF) deposition and metabolism of bovine, thereby providing insights into mechanisms for the use of ZBTB16 in fat management. METHODS: Primary preadipocytes derived from bovine IMF tissue were isolated and used as the in vitro cell model. An adenovirus Ad-ZBTB16 was transfected into bovine preadipocytes to overexpress the ZBTB16 gene. By using real-time quantitative PCR (RT-qPCR), western blotting, Oil Red-O staining, glycerol-3-phosphate dehydrogenase (GPDH) activity assay, and cell counting kit-8 (CCK-8) test, adipogenic and proliferative signals in adipocytes were monitored to investigate effects of ZBTB16 on adipogenesis of bovine preadipocytes. RESULTS: After transfection, mRNA and protein levels of ZBTB16 gene were significantly increased. Enhanced ZBTB16 significantly promoted preadipocyte differentiation, as evidenced by accelerated lipid accumulation, enhanced GPDH activity, consistently increased mRNA expressions of adipogenic key transcription factors PPARγ, C/EBPα, FABP4, and ADIPOQ, and markedly increased protein expressions of PPARγ and FABP4. No difference was observed concerning proliferation of preadipocytes after treatment with Ad-ZBTB16. Furthermore, relative mRNA levels of brown adipocyte selective genes (PRDM16, UCP1, Cidea, Cox8b, and PGC-1α) and beige adipocyte selective genes (CD137, TMEM26, and Tbx1) as well as UCP1 protein expression were significantly increased by Ad-ZBTB16. Meanwhile, Ad-ZBTB16 treatment remarkably induced mitochondrial biogenesis and increased relative mitochondrial DNA (mtDNA) copy number in bovine adipocytes. CONCLUSION: These results suggest that ZBTB16 overexpression can promote white adipogenesis and induce brown-like adipocyte formation for bovine white intramuscular preadipocytes.


Assuntos
Adipogenia , Tecido Adiposo Marrom/metabolismo , Tecido Adiposo Branco/metabolismo , Proteína com Dedos de Zinco da Leucemia Promielocítica/metabolismo , Adenoviridae/genética , Adipócitos/citologia , Adipócitos/metabolismo , Adiponectina/metabolismo , Animais , Proteína alfa Estimuladora de Ligação a CCAAT/metabolismo , Bovinos , Proliferação de Células , Células Cultivadas , DNA Mitocondrial/metabolismo , Proteínas de Ligação a Ácido Graxo/metabolismo , Vetores Genéticos/genética , Vetores Genéticos/metabolismo , Metabolismo dos Lipídeos , PPAR gama/metabolismo , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/genética , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo , Proteína com Dedos de Zinco da Leucemia Promielocítica/genética , Proteína Desacopladora 1/genética , Proteína Desacopladora 1/metabolismo , Regulação para Cima
16.
Nat Commun ; 9(1): 2819, 2018 07 19.
Artigo em Inglês | MEDLINE | ID: mdl-30026551

RESUMO

The role of stem cells in tissue maintenance is appreciated and hierarchical models of stem cell self-renewal and differentiation often proposed. Stem cell activity in the male germline is restricted to undifferentiated A-type spermatogonia (Aundiff); however, only a fraction of this population act as stem cells in undisturbed testis and Aundiff hierarchy remains contentious. Through newly developed compound reporter mice, here we define molecular signatures of self-renewing and differentiation-primed adult Aundiff fractions and dissect Aundiff heterogeneity by single-cell analysis. We uncover an unappreciated population within the self-renewing Aundiff fraction marked by expression of embryonic patterning genes and homeodomain transcription factor PDX1. Importantly, we find that PDX1 marks a population with potent stem cell capacity unique to mature, homeostatic testis and demonstrate dynamic interconversion between PDX1+ and PDX1- Aundiff states upon transplant and culture. We conclude that Aundiff exist in a series of dynamic cell states with distinct function and provide evidence that stability of such states is dictated by niche-derived cues.


Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/genética , Espermatogônias/metabolismo , Células-Tronco/metabolismo , Testículo/metabolismo , Transativadores/genética , Animais , Diferenciação Celular , Linhagem da Célula/genética , Efeito Fundador , Perfilação da Expressão Gênica , Genes Reporter , Proteínas de Homeodomínio/metabolismo , Integrases/genética , Integrases/metabolismo , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Masculino , Camundongos , Camundongos Transgênicos , Fator 3 de Transcrição de Octâmero/genética , Fator 3 de Transcrição de Octâmero/metabolismo , Proteína com Dedos de Zinco da Leucemia Promielocítica/genética , Proteína com Dedos de Zinco da Leucemia Promielocítica/metabolismo , Proteínas Proto-Oncogênicas c-kit/genética , Proteínas Proto-Oncogênicas c-kit/metabolismo , Análise de Célula Única , Espermatogônias/citologia , Células-Tronco/citologia , Testículo/citologia , Testículo/crescimento & desenvolvimento , Transativadores/metabolismo
17.
J Immunol ; 201(5): 1452-1459, 2018 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-30012846

RESUMO

γδ NKT cells are neonatal-derived γδ T lymphocytes that are grouped together with invariant NKT cells based on their shared innate-like developmental program characterized by the transcription factor PLZF (promyelocytic leukemia zinc finger). Previous studies have demonstrated that the population size of γδ NKT cells is tightly controlled by Id3-mediated inhibition of E-protein activity in mice. However, how E proteins promote γδ NKT cell development and expansion remains to be determined. In this study, we report that the transcription factor Egr2, which also activates PLZF expression in invariant NKT cells, is essential for regulating γδ NKT cell expansion. We observed a higher expression of Egr family genes in γδ NKT cells compared with the conventional γδ T cell population. Loss of function of Id3 caused an expansion of γδ NKT cells, which is accompanied by further upregulation of Egr family genes as well as PLZF. Deletion of Egr2 in Id3-deficient γδ NKT cells prevented cell expansion and blocked PLZF upregulation. We further show that this Egr2-mediated γδ NKT cell expansion is dependent on c-Myc. c-Myc knockdown attenuated the proliferation of Id3-deficient γδ NKT cells, whereas c-Myc overexpression enhanced the proliferation of Id3/Egr2-double-deficient γδ NKT cells. Therefore, our data reveal a regulatory circuit involving Egr2-Id3-E2A, which normally restricts the population size of γδ NKT cells by adjusting Egr2 dosage and c-Myc expression.


Assuntos
Proliferação de Células/fisiologia , Proteína 2 de Resposta de Crescimento Precoce/imunologia , Regulação da Expressão Gênica/imunologia , Proteínas Inibidoras de Diferenciação/imunologia , Células T Matadoras Naturais/imunologia , Proteínas Proto-Oncogênicas c-myc/imunologia , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/imunologia , Proteína 2 de Resposta de Crescimento Precoce/genética , Proteínas Inibidoras de Diferenciação/genética , Camundongos , Camundongos Knockout , Células T Matadoras Naturais/citologia , Proteína com Dedos de Zinco da Leucemia Promielocítica/genética , Proteína com Dedos de Zinco da Leucemia Promielocítica/imunologia , Proteínas Proto-Oncogênicas c-myc/genética , Receptores de Antígenos de Linfócitos T gama-delta
18.
Dev Cell ; 46(1): 112-125.e4, 2018 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-29974860

RESUMO

Zebrafish is a powerful model for forward genetics. Reverse genetic approaches are limited by the time required to generate stable mutant lines. We describe a system for gene knockout that consistently produces null phenotypes in G0 zebrafish. Yolk injection of sets of four CRISPR/Cas9 ribonucleoprotein complexes redundantly targeting a single gene recapitulated germline-transmitted knockout phenotypes in >90% of G0 embryos for each of 8 test genes. Early embryonic (6 hpf) and stable adult phenotypes were produced. Simultaneous multi-gene knockout was feasible but associated with toxicity in some cases. To facilitate use, we generated a lookup table of four-guide sets for 21,386 zebrafish genes and validated several. Using this resource, we targeted 50 cardiomyocyte transcriptional regulators and uncovered a role of zbtb16a in cardiac development. This system provides a platform for rapid screening of genes of interest in development, physiology, and disease models in zebrafish.


Assuntos
Técnicas de Inativação de Genes/métodos , Coração/embriologia , Proteína com Dedos de Zinco da Leucemia Promielocítica/genética , Proteínas de Peixe-Zebra/genética , Peixe-Zebra/genética , Animais , Sequência de Bases , Sistemas CRISPR-Cas/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Subunidades alfa G12-G13 de Proteínas de Ligação ao GTP/genética , Engenharia Genética/métodos , Morfolinos/genética , Miócitos Cardíacos/citologia , Transcrição Genética/genética , Peixe-Zebra/embriologia
19.
Int J Mol Sci ; 19(3)2018 Mar 07.
Artigo em Inglês | MEDLINE | ID: mdl-29518903

RESUMO

Natural Killer T cells (NKT cells) are emerging as critical regulators of pro- and anti-tumor immunity, both at baseline and in therapeutic settings. While type I NKT cells can promote anti-tumor immunity, their activity in the tumor microenvironment may be limited by negative regulators such as inhibitory immune checkpoints. We observed dominant expression of B- and T-lymphocyte attenuator (BTLA) on type I NKT cells in polyoma middle T oncogene-driven (PyMT) murine autochthonous mammary tumors. Other immune checkpoint receptors, such as programmed cell death 1 (PD-1) were equally distributed among T cell populations. Interference with BTLA using neutralizing antibodies limited tumor growth and pulmonary metastasis in the PyMT model in a therapeutic setting, correlating with an increase in type I NKT cells and expression of cytotoxic marker genes. While therapeutic application of an anti-PD-1 antibody increased the number of CD8+ cytotoxic T cells and elevated IL-12 expression, tumor control was not established. Expression of ZBTB16, the lineage-determining transcription factor of type I NKT cells, was correlated with a favorable patient prognosis in the METABRIC dataset, and BTLA levels were instrumental to further distinguish prognosis in patents with high ZBTB16 expression. Taken together, these data support a role of BTLA on type I NKT cells in limiting anti-tumor immunity.


Assuntos
Neoplasias da Mama/imunologia , Neoplasias da Mama/metabolismo , Regulação Neoplásica da Expressão Gênica , Células T Matadoras Naturais/imunologia , Células T Matadoras Naturais/metabolismo , Receptores Imunológicos/genética , Animais , Biomarcadores , Biomarcadores Tumorais , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Modelos Animais de Doenças , Regulação para Baixo , Feminino , Imunofenotipagem , Contagem de Linfócitos , Camundongos , Prognóstico , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Proteína com Dedos de Zinco da Leucemia Promielocítica/genética , Proteína com Dedos de Zinco da Leucemia Promielocítica/metabolismo , Receptores Imunológicos/metabolismo , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo
20.
Cell Death Dis ; 9(2): 200, 2018 02 08.
Artigo em Inglês | MEDLINE | ID: mdl-29422670

RESUMO

Chromatin conformation plays a key role in regulating gene expression and controlling cell differentiation. However, the whole-genome chromatin conformation changes that occur during leukemia cell differentiation are poorly understood. Here, we characterized the changes in chromatin conformation, histone states, chromatin accessibility, and gene expression using an all-trans retinoic acid (ATRA)-induced HL-60 cell differentiation model. The results showed that the boundaries of topological associated domains (TADs) were stable during differentiation; however, the chromatin conformations within several specific TADs were obviously changed. By combining H3K4me3, H3K27ac, and Hi-C signals, we annotated the differential gene-regulatory chromatin interactions upon ATRA induction. The gains and losses of the gene-regulatory chromatin interactions are significantly correlated with gene expression and chromatin accessibility. Finally, we found that the loss of GATA2 expression and DNA binding are crucial for the differentiation process, and changes in the chromatin structure around the GATA2 regulate its expression upon ATRA induction. This study provided both statistical insights and experimental details regarding the relationship between chromatin conformation changes and transcription regulation during leukemia cell differentiation, and the results suggested that the chromatin conformation is a new type of potential drug target for cancer therapy.


Assuntos
Cromatina/metabolismo , Leucemia/patologia , Tretinoína/farmacologia , Antineoplásicos/farmacologia , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Cromatina/genética , Fator de Transcrição GATA2/biossíntese , Fator de Transcrição GATA2/genética , Fator de Transcrição GATA2/metabolismo , Regulação Leucêmica da Expressão Gênica/efeitos dos fármacos , Células HL-60 , Humanos , Leucemia/genética , Leucemia/metabolismo , Conformação Molecular/efeitos dos fármacos , Regiões Promotoras Genéticas , Proteína com Dedos de Zinco da Leucemia Promielocítica/genética , RNA Mensageiro/biossíntese , RNA Mensageiro/genética
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