Apoptotic cells contain nuclear autoantigens that may initiate a systemic autoimmune response. To explore the mechanism of antibody binding to apoptotic cells, 3H9, a murine autoantibody with dual specificity for phospholipids and DNA, was used. H chain mutants of 3H9 were constructed, expressed as single-chain Fv (scFv) in Escherichia coli, and assessed for binding to phosphatidylserine, an antigen expressed on apoptotic cells. Both 3H9 and its germline revertant bound to dioleoyl phosphatidylserine in ELISA, and binding was enhanced by ␤2 glycoprotein I (␤2GPI), a plasma protein that selectively binds to apoptotic cells. Higher relative affinity for DOPS-␤2GPI was achieved by the introduction of Arg residues into the 3H9 H chain variable region at positions previously shown to mediate DNA binding. Specificity of the two structurally most diverse scFv for apoptotic cells was shown by flow cytometry, and two populations of scFv-bound cells were identified by differences in propidium iodide staining. The results suggest that, in autoimmunity, B cells with Ig receptors for apoptotic cells and DNA are positively selected, and that the antibodies they produce have the potential to affect the clearance and processing of apoptotic cells. C ells undergoing apoptosis package autoantigens, including nuclear proteins and DNA, in vesicles located beneath the surface of the plasma membrane (1). The contents of such surface vesicles may serve to enforce tolerance or, under a different set of circumstances, they may induce an autoimmune response (2-4). However, it is not obvious how autoantigens from apoptotic cells might activate the adaptive immune system, given that recognition and clearance of dying cells by mononuclear scavenger cells, such as monocytes and macrophages, is rapid and noninflammatory (5).One of the earliest signals for the uptake of apoptotic cells by phagocytes is the exposure of phosphatidylserine on the outer membrane leaflet, a consequence of the loss of membrane asymmetry in apoptotic cells (6). Phagocyte recognition of apoptotic cells occurs by several alternative mechanisms, including binding via the phosphatidylserine receptor (7), class A scavenger receptors (8), vitronectin receptors (9), macrosialin (10), CD14 (11), and CD36 (12). Moreover, serum proteins such as ␤2 glycoprotein I (␤2GPI; refs. 13 and 14), and C1q, the first component of complement (15), bind apoptotic cells and enhance their uptake.An important clue linking cell death to the onset of autoimmunity is provided by autoantibodies that bind apoptotic cells (16) and recognize surface epitopes that include complexes of phospholipid and ␤2GPI (17-19). The role of such autoantibodies is not known, although it has been postulated that they may preserve tissue homeostasis by enhancing the removal of dead or dying cells (20). In autoimmunity, it is possible that autoantibodies to apoptotic cells arise secondary to an increased load of apoptotic cells (18, 21), a decreased capacity for apoptotic cell removal (22, 23), or other as yet unknow...

We screened a chicken liver cDNA expression library with a probe spanning the distal region of the chicken vitellogenin II (VTGH) gene promoter and isolated clones for a transcription factor that we have named VBP (for vitellogenin gene-binding protein). VBP binds to one of the most important positive elements in the VTGH promoter and appears to play a pivotal role in the estrogen-dependent regulation of this gene. The protein sequence of VBP was deduced from a nearly full length cDNA copy and was found to contain a basic/zipper (bZIP) motif. As expected for a bZIP factor, VBP binds to its target DNA site as a dimer. Moreover, VBP is a stable dimer free in solution. A data base search revealed that VBP is related to rat DBP. However, despite the fact that the basic/hinge regions of VBP and DBP differ at only three amino acid positions, the DBP binding site in the rat albumin promoter is a relatively poor binding site for VBP. Thus, the optimal binding sites for VBP and DBP may be distinct. Similarities between the VBP and DBP leucine zippers are largely confined to only four of the seven helical spokes. Nevertheless, these leucine zippers are functionally compatible and appear to define a novel subfamily. In contrast to the bZIP regions, other portions of VBP and DBP are markedly different, as are the expression profiles for these two genes. In particular, expression of the VBP gene commences early in liver ontogeny and is not subject to circadian control.Genetic and biochemical studies have established that transcription by RNA polymerase II requires the assembly of a stable preinitiation complex over the proximal promoter region of each target gene (6,32 which a nonfunctional partner suppresses the activity of a functional partner. In addition to DNA binding domains (and, in some cases, dimerization domains), transcription factors also contain one or more transactivation domains that are required to mediate positive effects on the general transcriptional machinery. Subclasses of transactivation domains have been identified, and it is likely that other novel domains will be found as more transcription factors are cloned and analyzed. Much current work is directed at understanding how these activation domains function, and the notion that additional bridging factors may be involved has been advanced from several recent studies (reviewed in reference 25).Studies in our laboratory are focused on a molecular understanding of the estrogen-dependent and liver-specific transcriptional regulation of the chicken vitellogenin II (VTGII) gene. The estrogen-dependent aspect of this regulation was shown to be due to the presence of two upstream estrogen response elements, and the ability of the VTGII promoter to be activated by these elements was found to be cell type specific (5, 7). A linker scanner mutational analysis of the VTGII promoter using transient expression assays in chicken hepatoma (LMH) cells (18) and chicken embryo fibroblast cells revealed that this promoter has multiple positive elements as well as a negat...