Down-regulation of Runx1 Expression by TCR Signal Involves an Autoregulatory Mechanism and Contributes to IL-2 Production*

Runx1 transcription factor plays multiple roles in T cell development, differentiation, and function. However, the regulatory mechanisms and functional significance of high Runx1 protein expression in resting peripheral CD4+ T cells is not well understood. Here, we demonstrate that T-cell receptor (TCR) activation down-regulates distal Runx1 transcription, resulting in a significant reduction of Runx1 protein. Interestingly, this down-regulation of distal Runx1 transcription appears to be mediated through a negative auto-regulatory mechanism, whereby Runx1 protein binds to a Runx consensus site in the distal promoter. Through the use of Runx1-overexpressing cells from transgenic mice, we demonstrate that interference with TCR-mediated Runx1 down-regulation inhibits IL-2 production and proliferation in activated CD4+ T cells. In contrast, using Runx1-deficient cells prepared from targeted mice, we show that the absence of Runx1 in unstimulated CD4+ T cells results in IL-2 derepression. In summary, we propose that high levels of Runx1 in resting CD4+ T cells functions negatively in the regulation of IL-2 transcription, and that TCR activation-mediated down-regulation of Runx1 involves negative auto-regulation of the distal Runx1 promoter and contributes to IL-2 production

Transcription Factors Runx1 and Runx3 Suppress Keratin Expression in Undifferentiated Keratinocytes

The Runt-related transcription factor (Runx) family has been suggested to play roles in stem cell regulation, tissue development, and oncogenesis in various tissues/organs. In this study, we investigated the possible functions of Runx1 and Runx3 in keratinocyte differentiation. Both Runx1 and Runx3 proteins were detected in primary cultures of mouse keratinocytes. Proteins were localized in the nuclei of undifferentiated keratinocytes but translocated to the cytoplasm of differentiated cells. The siRNA-mediated inhibition of Runx1 and Runx3 expression increased expression of keratin 1 and keratin 10, which are early differentiation markers of keratinocytes. In contrast, overexpression of Runx1 and Runx3 suppressed keratin 1 and keratin 10 expression. Endogenous Runx1 and Runx3 proteins were associated with the promoter sequences of keratin 1 and keratin 10 genes in undifferentiated but not differentiated keratinocytes. In mouse skin, the inhibition of Runx1 and Runx3 expression by keratinocyte-specific gene targeting increased the ratios of keratin 1- and keratin 10-positive cells in the basal layer of the epidermis. On the other hand, inhibition of Runx1 and Runx3 expression did not alter the proliferation capacity of cultured or epidermal keratinocytes. These results suggest that Runx1 and Runx3 likely function to directly inhibit differentiation-induced expression of keratin 1 and keratin 10 genes but are not involved in the regulation of keratinocyte proliferation

Filamin A-Bound PEBP2β/CBFβ Is Retained in the Cytoplasm and Prevented from Functioning as a Partner of the Runx1 Transcription Factor

The heterodimeric transcription factor PEBP2/CBF is composed of a DNA-binding subunit, called Runx1, and a non-DNA-binding subunit, called PEBP2β/CBFβ. The Runx1 protein is detected exclusively in the nuclei of most cells and tissues, whereas PEBP2β is located in the cytoplasm. We addressed the mechanism by which PEBP2β localizes to the cytoplasm and found that it is associated with filamin A, an actin-binding protein. Filamin A retains PEBP2β in the cytoplasm, thereby hindering its engagement as a Runx1 partner. The interaction with filamin A is mediated by a region within PEBP2β that includes amino acid residues 68 to 93. The deletion of this region or the repression of filamin A enables PEBP2β to translocate to the nucleus. Based on these observations, we propose that PEBP2β has two distinct domains, a newly defined regulatory domain that interacts with filamin A and the previously identified Runx1-binding domain