RESUMEN
Runx1 transcription factor is highly expressed at a CD4/CD8-double-negative (DN) stage of thymocyte development but is down-regulated when cells proceed to the double-positive (DP) stage. In the present study, we examined whether the down-regulation of Runx1 is necessary for thymocyte differentiation from the DN to DP stage. When Runx1 was artificially over-expressed in thymocytes by Lck-driven Cre, the DN3 population was unaffected, as exemplified by proper pre-T-cell receptor expression, whereas the DN4 population was perturbed as shown by the decrease in the CD27(hi) sub-fraction. In parallel, the growth rate of DN4 cells was reduced by half, as measured by bromodeoxyuridine incorporation. These events impaired the transition of DN4 cells to the DP stage, resulting in the drastic reduction of the number of DP thymocytes. The Runx1 gene has two promoters, a proximal and a distal promoter; and, in thymocytes, endogenous Runx1 was mainly transcribed from the distal promoter. Interestingly, only distal, but not proximal, Runx1 over-expression exhibited an inhibitory effect on thymocyte differentiation, suggesting that the distal Runx1 protein may fulfil a unique function. Our collective results indicate that production of the distal Runx1 protein must be adequately down-regulated for thymocytes to transit from the DN to the DP stage, a critical step in the massive expansion of the T-cell lineage.
Asunto(s)
Diferenciación Celular/inmunología , Subunidad alfa 2 del Factor de Unión al Sitio Principal/inmunología , Regulación hacia Abajo/inmunología , Regiones Promotoras Genéticas/inmunología , Linfocitos T/inmunología , Timo/inmunología , Animales , Diferenciación Celular/genética , Subunidad alfa 2 del Factor de Unión al Sitio Principal/biosíntesis , Subunidad alfa 2 del Factor de Unión al Sitio Principal/genética , Regulación hacia Abajo/genética , Ratones , Ratones Transgénicos , Regiones Promotoras Genéticas/genética , Linfocitos T/citología , Linfocitos T/metabolismo , Timo/citología , Timo/metabolismo , Transcripción Genética/genética , Transcripción Genética/inmunología , Miembro 7 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/inmunología , Miembro 7 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/metabolismoRESUMEN
The heterodimeric transcription factor PEBP2/CBF is composed of a DNA-binding subunit, called Runx1, and a non-DNA-binding subunit, called PEBP2beta/CBFbeta. The Runx1 protein is detected exclusively in the nuclei of most cells and tissues, whereas PEBP2beta is located in the cytoplasm. We addressed the mechanism by which PEBP2beta localizes to the cytoplasm and found that it is associated with filamin A, an actin-binding protein. Filamin A retains PEBP2beta in the cytoplasm, thereby hindering its engagement as a Runx1 partner. The interaction with filamin A is mediated by a region within PEBP2beta that includes amino acid residues 68 to 93. The deletion of this region or the repression of filamin A enables PEBP2beta to translocate to the nucleus. Based on these observations, we propose that PEBP2beta has two distinct domains, a newly defined regulatory domain that interacts with filamin A and the previously identified Runx1-binding domain.
Asunto(s)
Núcleo Celular/metabolismo , Proteínas Contráctiles/metabolismo , Citoplasma/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas de Microfilamentos/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Factores de Transcripción/metabolismo , Animales , Subunidad alfa 2 del Factor de Unión al Sitio Principal , Filaminas , Células HeLa , Humanos , Ratones , Unión Proteica , Estructura Terciaria de Proteína , ARN Interferente Pequeño/metabolismo , Factor de Transcripción AP-2 , Técnicas del Sistema de Dos HíbridosRESUMEN
The Runx family of transcription factors is thought to regulate the differentiation of thymocytes. Runx3 protein is detected mainly in the CD4(-)8(+) subset of T lymphocytes. In the thymus of Runx3-deficient mice, CD4 expression is de-repressed and CD4(-)8(+) thymocytes do not develop. This clearly implicates Runx3 in CD4 silencing, but does not necessarily prove its role in the differentiation of CD4(-)8(+) thymocytes per se. In the present study, we created transgenic mice that overexpress Runx3 and analyzed the development of thymocytes in these animals. In the Runx3-transgenic thymus, the number of CD4(-)8(+) cells was greatly increased, whereas the numbers of CD4(+)8(+) and CD4(+)8(-) cells were reduced. The CD4(-)8(+) transgenic thymocytes contained mature cells with a TCR(high)HSA(low) phenotype. These cells were released from the thymus and contributed to the elevated level of CD4(-)8(+) cells relative to CD4(+)8(-) cells in the spleen. Runx3 overexpression also increased the number of mature CD4(-)8(+) thymocytes in mice with class II-restricted, transgenic TCR and in mice with a class I-deficient background, both of which are favorable for CD4(+)8(-) lineage selection. Thus, Runx3 can drive thymocytes to select the CD4(-)8(+) lineage. This activity is likely to be due to more than a simple silencing of CD4 gene expression.