LEF-1 negatively controls interleukin-4 expression through a proximal promoter regulatory element

Lymphoid enhancer-binding factor 1 (LEF-1) and T cell factor (TCF-1) are downstream effectors of the Wnt signaling pathway and are involved in the regulation of T cell development in the thymus. LEF-1 and TCF-1 are also expressed in mature peripheral primary T cells, but their expression is down-regulated following T cell activation. Although the decisive roles of LEF-1 and TCF-1 in the early stages of T cell development are well documented, the functions of these factors in mature peripheral T cells are largely unknown. Recently, LEF-1 was shown to suppress Th2 cytokines interleukin-4 (IL-4), -5, and -13 expression from the developing Th2 cells that overexpress LEF-1 through retrovirus gene transduction. In this study, we further investigated the expression and functions of LEF-1 and TCF-1 in peripheral CD4+ T cells and revealed that LEF-1 is dominantly expressed in Th1 but not in Th2 cells. We identified a high affinity LEF-1-binding site in the negative regulatory element of the IL-4 promoter. Knockdown LEF-1 expression by LEF-1-specific small interfering RNA resulted in an increase in the IL-4 mRNA expression. This study further confirms a negative regulatory role of LEF-1 in mature peripheral T cells. Furthermore, we found that IL-4 stimulation possesses a negative effect on the expressions of LEF-1 and TCF-1 in primary T cells, suggesting a positive feedback effect of IL-4 on IL4 gene expression.     

Alx1, a member of the Cart1/Alx3/Alx4 subfamily of Paired-class homeodomain proteins,is an essential component of the gene network controlling skeletogenic fate specification in the sea urchin embryo

In the sea urchin embryo, the large micromeres and their progeny function as a critical signaling center and execute a complex morphogenetic program. We have identified a new and essential component of the gene network that controls large micromere specification, the homeodomain protein Alx1. Alx1 is expressed exclusively by cells of the large micromere lineage beginning in the first interphase after the large micromeres are born. Morpholino studies demonstrate that Alx1 is essential at an early stage of specification and controls downstream genes required for epithelial-mesenchymal transition and biomineralization. Expression of Alx1 is cell autonomous and regulated maternally through beta-catenin and its downstream effector, Pmar1. Alx1 expression can be activated in other cell lineages at much later stages of development, however, through a regulative pathway of skeletogenesis that is responsive to cell signaling. The Alx1 protein is highly conserved among euechinoid sea urchins and is closely related to the Cart1/Alx3/Alx4 family of vertebrate homeodomain proteins. In vertebrates, these proteins regulate the formation of skeletal elements of the limbs, face and neck. Our findings suggest that the ancestral deuterostome had a population of biomineral-forming mesenchyme cells that expressed an Alx1-like protein.     

Characterization of the interaction between the baculovirus replication factors LEF-1 and LEF-2.

The Autographa californica multinucleocapsid nuclear polyhedrosis virus has six genes required and three genes stimulatory for transient DNA replication. We demonstrate that the products of two of these genes, LEF-1 and LEF-2, interact in both two-hybrid assays using Saccharomyces cerevisiae and glutathione S-transferase fusion affinity assays. Using yeast-two-hybrid assays, we mapped the interaction domain of LEF-2 to amino acids between positions 20 and 60. Extensive deletion analyses of LEF-1 failed to reveal a delimited interaction domain, suggesting that there may be essential secondary structural elements that are inactivated by these deletions. All clones expressing LEF-1 and LEF-2 that were unable to interact also failed to support significant levels of transient DNA replication, suggesting that this interaction is required for DNA replication. Sequence analysis of LEF-1 revealed a primase-like motif, WVVDAD. When this motif was mutated to WVVQAD, LEF-1 no longer supported transient DNA replication.     

Identification of domains in Autographa californica multiple nucleopolyhedrovirus late expression factor 3 required for nuclear transport of P143

Autographa californica multiple nucleopolyhedrovirus (AcMNPV) late expression factor 3 (LEF-3) is an essential protein for DNA replication in transient assays. P143, a large DNA-binding protein with DNA-unwinding activity, is also essential for viral DNA replication in vivo. Both LEF-3 and P143 are found in the nucleus of AcMNPV-infected cells, but only LEF-3 localizes to the nucleus when expressed in transfected cells on its own from a plasmid expression vector. P143 requires LEF-3 as a transporter to enter the nucleus. To investigate the possibility that LEF-3 carries a nuclear localization signal domain, we constructed a series of LEF-3 deletion mutants and examined the intracellular localization of the products in plasmid-transfected cells. We discovered that the N-terminal 56 amino acid residues of LEF-3 were sufficient for nuclear localization and that this domain, when fused with either the green fluorescent protein reporter gene or P143, was able to direct these proteins to the nucleus. Transient DNA replication assays demonstrated that fusing the LEF-3 nuclear localization signal domain to P143 did not alter the function of P143 in supporting DNA replication but was not sufficient to substitute for whole LEF-3. These data show that although one role for LEF-3 during virus infection is to transport P143 to the nucleus, LEF-3 performs other essential replication functions once inside the nucleus.