Cytokines in the tumor necrosis factor (TNF) family regulate development and function of the immune system. We have isolated a new member of this family, designated Apo-2 ligand (Apo-2L), via an expressed sequence tag. Apo-2L is a 281-amino acid protein, related most closely to Fas/Apo-1 ligand. Transfected Apo-2L is expressed at the cell surface with its C terminus exposed, indicating a type II transmembrane protein topology. Like Fas/Apo-1 ligand and TNF, the C-terminal extracellular region of Apo-2L (amino acids 114 -281) exhibits a homotrimeric subunit structure. Soluble Apo-2L induces extensive apoptosis in lymphoid as well as non-lymphoid tumor cell lines. The effect of Apo-2L is not inhibited by soluble Fas/Apo-1 and TNF receptors; moreover, expression of human Fas/Apo-1 in mouse fibroblasts, which confers sensitivity to induction of apoptosis by agonistic anti-Fas/Apo-1 antibody, does not confer sensitivity to Apo-2L. Hence, Apo-2L acts via a receptor which is distinct from Fas/Apo-1 and TNF receptors. These results suggest that, along with other family members such as Fas/Apo-1 ligand and TNF, Apo-2L may serve as an extracellular signal that triggers programmed cell death. TNF1 family cytokines modulate host defense mechanisms via a corresponding family of receptors (1-3). Most TNF family cytokines are expressed as type II transmembrane proteins, whose C-terminal extracellular domain is processed proteolytically to form a soluble homotrimeric molecule. In contrast, the members of the TNF receptor (TNFR) family are type I transmembrane proteins. In both families, sequence homology is found mainly in the extracellular regions, which mediate ligand-receptor binding.Members of the TNF family have diverse biological actions, including T cell co-stimulation, induction of B cell proliferation and differentiation, and macrophage activation (1-3). In addition, certain TNF family members regulate the elimination of unwanted immune cells by inducing programmed cell death (apoptosis). For example, Fas/Apo-1 ligand (Fas/Apo-1L) plays a key role in peripheral deletion of self-reactive lymphocytes, as suggested by the autoimmune phenotype of the mouse mutations lpr and gld, which occur, respectively, in the Fas/Apo-1 receptor or ligand genes (4, 5). In addition, Fas/Apo-1L is involved in apoptosis of B cells and CD4 ϩ T cells subsequent to stimulation by antigen, while TNF mediates a similar function in CD8ϩ T cells (4 -7). In this article, we describe a new member of the TNF cytokine family. We have designated this protein Apo-2L, because it resembles the Fas/Apo-1L in its amino acid sequence, as well as in its ability to induce apoptosis. Apo-2L appears to act via a receptor which is distinct from Fas/Apo-1 and TNF receptors, suggesting that its biological role is unique. EXPERIMENTAL PROCEDURESCloning of Apo-2L cDNA-To isolate a full-length cDNA that contains the sequence of expressed sequenced tag (EST) HHEA47M, a gt11 bacteriophage library of human placental cDNA (ϳ1 ϫ 10 6 clones) (HL1075b, Clontech) was screene...

Members of a family of murine octamer-binding proteins interact specifically with the octamer motif, a transcription regulatory element found in the promoter and enhancer regions of many genes. Oct-4 is a maternally expressed protein that is also present in the pre-implantation mouse embryo. Although many regulatory proteins are expressed in post-implantation embryos, transcription factors regulating pre-implantation processes have remained elusive. The Oct-4 gene is therefore a prime candidate for an early developmental control gene. Here we report the complementary DNA cloning of the mouse Oct-4 gene, and the characterization of the encoded protein(s) by sequential in vitro transcription, translation, DNA-binding and protease-clipping analysis. Deletion analysis shows that the DNA-binding activity is mediated by a POU domain encoded in an open reading frame corresponding to a 324-amino-acid protein. Sequence comparison with known POU domains reveals that Oct-4 is a novel member of the POU-family.

The complementary DNA coding for a lymphocyte-specific transcription factor binding to the DNA 'octamer' sequence TNATTTGCAT has been cloned. The nucleotide sequence shows homology to the homoeobox domain. Expression of this cDNA in HeLa cells is sufficient for a strong transcriptional activation of B-cell-specific promoters.

Basal transcription by human RNA polymerase II requires the coordinate action of several ancillary factors (TFIIA-J) and can be regulated by various promoter-specific DNA binding proteins. An additional class of factors, called coactivators, are dispensable for basal transcription but are indispensable for regulation by transcriptional activators. Biochemical studies established that some coactivators are associated with the TATA-binding protein (TBP) to form the TFIID complex. We therefore set out to define the relationship between TBP and these TBP-associated factors (TAFs). Here we describe the cloning, expression and properties of the first human TAF, hTAFII250. The hTAFII250 gene is identical to a gene, CCG1, (ref 7,8), implicated in cell-cycle progression. Recombinant hTAFII250 binds directly to TBP both in vitro and in yeast, and participates in the formation of the TFIID complex. This largest TAF may therefore play a central role in TFIID assembly by interacting with both TBP and other TAFs, as well as serving to link the control of transcription to the cell cycle.

Some TAF subunits of transcription factor TFIID play a pivotal role in transcriptional activation by mediating protein-protein interactions, whereas other TAFs direct promoter selectivity via protein-DNA recognition. Here, we report that purified recombinant TAFII250 is a protein serine kinase that selectively phosphotylates RAP74 but not other basal transcription factors or common phosphoacceptor proteins. The phosphorylation of RAP74 also occurs in the context of the complete TFIID complex. Deletion analysis revealed that TAFII250 contains two distinct kinase domains each capable of autophosphorylation. However, both the N- and C-terminal kinase domains of TAFII250 are required for efficient transphosphorylation of RAP74 on serine residues. These findings suggest that the targeted phosphorylation of RAP74 by TAFII250 may provide a mechanism for signaling between components within the initiation complex to regulate transcription.