RESUMEN
AIRE expression in thymus is downregulated by estrogen after puberty, what probably renders women more susceptible to autoimmune disorders. Here we investigated the effects of minipuberty on male and female infant human thymic tissue in order to verify if this initial transient increase in sex hormones - along the first six months of life - could affect thymic transcriptional network regulation and AIRE expression. Gene co-expression network analysis for differentially expressed genes and miRNA-target analysis revealed sex differences in thymic tissue during minipuberty, but such differences were not detected in the thymic tissue of infants aged 7-18 months, i.e. the non-puberty group. AIRE expression was essentially the same in both sexes in minipuberty and in non-puberty groups, as assessed by genomic and immunohistochemical assays. However, AIRE-interactors networks showed several differences in all groups regarding gene-gene expression correlation. Therefore, minipuberty and genomic mechanisms interact in shaping thymic sexual dimorphism along the first six months of life.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica , Redes Reguladoras de Genes , MicroARNs/genética , Caracteres Sexuales , Timo/metabolismo , Factores de Transcripción/genética , Estrógenos/metabolismo , Femenino , Perfilación de la Expresión Génica , Ontología de Genes , Humanos , Lactante , Masculino , MicroARNs/clasificación , MicroARNs/metabolismo , Anotación de Secuencia Molecular , Factores Sexuales , Timo/crecimiento & desarrollo , Factores de Transcripción/metabolismo , Proteína AIRERESUMEN
Somatic cell reprogramming by transcription factors and other modifiers such as microRNAs has opened broad avenues for the study of developmental processes, cell fate determination, and interplay of molecular mechanisms in signaling pathways. However, many of the mechanisms that drive nuclear reprogramming itself remain yet to be elucidated. Here, we analyzed the role of miR-29 during reprogramming in more detail. Therefore, we evaluated miR-29 expression during reprogramming of fibroblasts transduced with lentiviral OKS and OKSM vectors and we show that addition of c-MYC to the reprogramming factor cocktail decreases miR-29 expression levels. Moreover, we found that transfection of pre-miR-29a strongly decreased OKS-induced formation of GFP+-colonies in MEF-cells from Oct4-eGFP reporter mouse, whereas anti-miR-29a showed the opposite effect. Furthermore, we studied components of two pathways which are important for reprogramming and which involve miR-29 targets: active DNA-demethylation and Wnt-signaling. We show that inhibition of Tet1, Tet2 and Tet3 as well as activation of Wnt-signaling leads to decreased reprogramming efficiency. Moreover, transfection of pre-miR-29 resulted in elevated expression of ß-Catenin transcriptional target sFRP2 and increased TCF/LEF-promoter activity. Finally, we report that Gsk3-ß is a direct target of miR-29 in MEF-cells. Together, our findings contribute to the understanding of the molecular mechanisms by which miR-29 influences reprogramming.
Asunto(s)
Reprogramación Celular , MicroARNs/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Vía de Señalización Wnt/fisiología , Regiones no Traducidas 3' , Animales , Antagomirs/metabolismo , Secuencia de Bases , Línea Celular , Proteínas de Unión al ADN/antagonistas & inhibidores , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Regulación hacia Abajo , Fibroblastos/citología , Fibroblastos/metabolismo , Vectores Genéticos/genética , Vectores Genéticos/metabolismo , Glucógeno Sintasa Quinasa 3 beta/antagonistas & inhibidores , Glucógeno Sintasa Quinasa 3 beta/genética , Glucógeno Sintasa Quinasa 3 beta/metabolismo , Células HEK293 , Humanos , Lentivirus/genética , Ratones , MicroARNs/antagonistas & inhibidores , MicroARNs/genética , Regiones Promotoras Genéticas , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas/genética , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Alineación de Secuencia , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
During the early thymus colonization, Notch signaling activation on hematopoietic progenitor cells (HPCs) drives proliferation and T cell commitment. Although these processes are driven by transcription factors such as HOXB4 and GATA3, there is no evidence that Notch directly regulates their transcription. To evaluate the role of NOTCH and TNF signaling in this process, human CD34+ HPCs were cocultured with OP9-DL1 cells, in the presence or absence of TNF. The use of a Notch signaling inhibitor and a protein synthesis inhibitor allowed us to distinguish primary effects, mediated by direct signaling downstream Notch and TNF, from secondary effects, mediated by de novo synthesized proteins. A low and physiologically relevant concentration of TNF promoted T lymphopoiesis in OP9-DL1 cocultures. TNF positively modulated the expression of both transcripts in a Notch-dependent manner; however, GATA3 induction was mediated by a direct mechanism, while HOXB4 induction was indirect. Induction of both transcripts was repressed by a GSK3ß inhibitor, indicating that activation of canonical Wnt signaling inhibits rather than induces their expression. Our study provides novel evidences of the mechanisms integrating Notch and TNF-alpha signaling in the transcriptional induction of GATA3 and HOXB4. This mechanism has direct implications in the control of self-renewal, proliferation, commitment, and T cell differentiation.