In budding yeast genes that encode G1 cyclins and proteins involved in DNA synthesis are transcriptionally activated in late G1. A transcription factor, called SBF, is composed of Swi4 and Swi6 proteins and activates transcription of G1 cyclin genes. A different, but related, complex called MBF binds to MCB elements (Mlu I cell cycle box) found in the promoter of most DNA synthesis genes. MBF contains Swi6 and a 120-kilodalton protein (p120). MBF was purified and the gene encoding p120 (termed MBP1) was cloned. A deletion of MBP1 was not lethal but led to deregulated expression of DNA synthesis genes, indicating a direct regulatory role for MBF in MCB-driven transcription. Mbp1 is related to Swi4. Strains deleted for both MBP1 and SWI4 were inviable, demonstrating that transcriptional activation by MBF and SBF has an important role in the transition from G1 to S phase.

No abstract

Most genes involved in DNA replication in the yeast Saccharomyces cerevisiae are transcribed transiently during late G1 as cells become committed to a new cell cycle at Start. Their promoters all contain one or more versions of an 8-base-pair motif (ACGCGTNA) containing an MluI restriction enzyme site and called the MluI cell-cycle box (MCB). MCBs are both necessary and sufficient for the late G1-specific transcription of the TMP1 thymidylate synthase and POL1 DNA polymerase genes. A different late G1-specific 8-base-pair transcription element called the SCB (CACGAAAA; ref. 5) is bound by a factor containing the Swi4 and Swi6 proteins. We describe here the formation in vitro of complexes on TMP1 MCBs that contain the Swi6 protein and, we suggest, a protein of relative molecular mass 120,000 (p120) that is distinct from Swi4. Transcription due to SCBs and MCBs occurs in the absence of Swi6 but it is no longer correctly regulated in the cell cycle. We suggest that Swi6 is an essential regulatory subunit of two different Start-dependent transcription factors. One factor (SBF) contains Swi4 and binds to SCBs, whereas the other (MBF) contains the protein p120 and binds MCBs.

We have shown recently that mouse Th1 cells but not Th2 cells are selectively recruited into inflamed sites of a delayed-type hypersensitivity (DTH) reaction of the skin. This migration was blocked by monoclonal antibodies (mAb) against P- and E-selectin. Here we show that Th1 cells bind to P-selectin via the P-selectin glycoprotein ligand-1 (PSGL-1). This is the only glycoprotein ligand that was detectable by affinity isolation with a P-selectin–Ig fusion protein. Binding of Th1 cells to P-selectin, as analyzed by flow cytometry and in cell adhesion assays, was completely blocked by antibodies against PSGL-1. The same antibodies blocked partially the migration of Th1 cells into cutaneous DTH reactions. This blocking activity, in combination with that of a mAb against E-selectin, was additive. PSGL-1 on Th2 cells, although expressed at similar levels as on Th1 cells, did not support binding to P-selectin. Thus, the P-selectin–binding form of PSGL-1 distinguishes Th1 cells from Th2 cells. Furthermore, PSGL-1 is relevant for the entry of Th1 cells into inflamed areas of the skin. This is the first demonstration for the importance of PSGL-1 for mouse leukocyte recruitment in vivo.

It has been suggested that T cell immunoglobulin mucin (Tim)-1 expressed on T cells serves to positively costimulate T cell responses. However, crosslinking of Tim-1 by its ligand Tim-4 resulted in either activation or inhibition of T cell responses, thus raising the issue of whether Tim-1 can have a dual function as a costimulator. To resolve this issue, we tested a series of monoclonal antibodies specific for Tim-1 and identified two antibodies that showed opposite functional effects. One anti–Tim-1 antibody increased the frequency of antigen-specific T cells, the production of the proinflammatory cytokines IFN-γ and IL-17, and the severity of experimental autoimmune encephalomyelitis. In contrast, another anti–Tim-1 antibody inhibited the generation of antigen-specific T cells, production of IFN-γ and IL-17, and development of autoimmunity, and it caused a strong Th2 response. Both antibodies bound to closely related epitopes in the IgV domain of the Tim-1 molecule, but the activating antibody had an avidity for Tim-1 that was 17 times higher than the inhibitory antibody. Although both anti–Tim-1 antibodies induced CD3 capping, only the activating antibody caused strong cytoskeletal reorganization and motility. These data indicate that Tim-1 regulates T cell responses and that Tim-1 engagement can alter T cell function depending on the affinity/avidity with which it is engaged.