The maintenance of transcriptional silencing at HM mating-type loci and telomeres in yeast requires the SIR2, SIR3, and SIR4 proteins, none of which appear to be DNA-binding proteins. Here we show that SIR3 and SIR4 interact with a carboxy-terminal domain of the silencer, telomere, and UAS-binding protein RAP1. We identified SIR3 and SIR4 in a two-hybrid screen for RAPl-interacting factors and showed that SIR3 interacts both with itself and with SIR4. The interaction between RAP1 and SIR3 can be observed in vitro in the absence of other yeast proteins. Consistent with the notion that native SIR proteins interact with the RAP1 carboxyl terminus, we show that mutation of the endogenous SIR3 and SIR4 genes increases transcriptional activation by LexA/RAP1 hybrids. To test the importance of the RAP1-SIR3 interaction for silencing, we identified mutations in the RAP1 carboxyl terminus that either diminish or abolish this interaction. When introduced into the native RAP1 protein, these mutations cause corresponding defects in silencing at both HMR and telomeres. We propose that RAP1 acts in the initiation of transcriptional silencing by recruiting a complex of SIR proteins to the chromosome via protein-protein interactions. These data are consistent with a model in which SIR3 and SIR4 play a structural role in the maintenance of silent chromatin and indicate that their action is initiated at the silencer itself.

The extracellular domains of a diverse group of membrane proteins are shed in response to protein kinase C activators such as phorbol 12-myristate 13-acetate (PMA). The lack of sequence similarity in the cleavage sites suggests the involvement of many proteases of diverse specificity in this process. However, a mutant Chinese hamster ovary cell line recently isolated for being defective in PMA-activated shedding of the membraneanchored growth factor transforming growth factor ␣ precursor (proTGF-␣) is concomitantly defective in the shedding of many other unrelated membrane proteins. Here we show that independent mutagenesis and selection experiments yield shedding mutants having the same recessive phenotype and belonging to the same genetic complementation group. Furthermore, two structurally distinct agents, TAPI-2 and 1,10-phenanthroline, which are known to inhibit metalloproteases, block PMA-activated shedding of proTGF-␣, cell adhesion receptor L-selectin, interleukin 6 receptor ␣ subunit, ␤-amyloid precursor protein, and an entire set of anonymous Chinese hamster ovary cell surface proteins. Certain serine protease inhibitors prevent release of these proteins by interfering with their maturation and transport to the cell surface but do not inhibit ectodomain shedding from the cell surface. The results suggest the existence of a common system for membrane protein ectodomain shedding involving one or several proteolytic activities sensitive to metalloprotease inhibitors, whose ability to act can be disrupted by recessive mutations in a single gene.

A DNA-binding factor with properties of NF-KB and another similar activity are rapidly induced when growth-arrested BALB/c 3T3 ceUs are stimulated with serum growth factors. Induction of these DNA-binding activities is not inhibited by pretreatment of quiescent ceUs with the protein synthesis inhibitor cycloheximide. Interestingly, the major NF-KB-like activity is not detected in nuclear extracts of proliferating ceUs, and thus its expression appears to be limited to the Go-to-G1 transition in 3T3 ceUs. These DNA-binding activities bind many of the expected NF-cB target sequences, including elements in the class I major histocompatibility complex and human immunodeficiency virus enhancers, as weUl as a recently identified NF-cB binding site upstream of the c-myc gene. Furthermore, both the class I major histocompatibility complex and c-myc NF-KcB binding sites confer inducibility on a minimal promoter in 3T3 cells stimulated with serum growth factors. The results demonstrate that NF-KB-like activities are immediate-early response proteins in 3T3 cells and suggest a role for these factors in the Go-to-G1 transition.Much of our knowledge concerning the signals that are activated following the interaction of serum growth factors with their receptors and that are involved in the stimulation of cell growth has been generated by using serum-deprived BALB/c 3T3 fibroblasts. In the absence of serum growth factors, 3T3 cells enter a state called Go in which growth and cell division stop. The addition of growth factors initiates a complex series of events which causes cells to enter G1 and which culminates in the synthesis of DNA and cell division (1,13,15,16,18,29,41). During the GO-to-G1 transition, the expression of several transcription factors, including those encoded by certain proto-oncogenes, is activated, and these gene products play a critical role in initiating and controlling cell growth and DNA synthesis (13, 15-18, 28, 32, 38, 41, 48, 50, 58, 64). Aberrant expression of these proto-oncogenes, such as c-fos or c-myc, is implicated in the pathogenesis of a variety of neoplasms (21,39).NF-KB is a DNA-binding protein originally identified as a B-cell-specific factor involved in the control of expression of immunoglobulin kappa genes (59). Subsequently, regulatory elements in or upstream of various genes involved in immune function or in inflammation and elements in certain viruses such as the human immunodeficiency virus (HIV) have been shown to interact with NF-KB and to be critical for appropriate regulated or cell-type-specific gene expression (7,11,20,23,30,34,42,43,45,49,53,55,67). NF-KB DNA-binding activity can be induced in certain cell types with the cytokines tumor necrosis factor alpha and interleukin-1 (34, 42, 46, 52, 61), with the phorbol ester phorbol myristate acetate (PMA) (60), by virus infection, and by double-stranded RNA treatment (23,42,43,67). Furthermore, NF-KB DNA-binding activity is activated by treatment of pre-B cells and Jurkat T cells with mitogens (11,20,49,60 inhibitor IKB (2,3,...

A DNA-binding factor with properties of NF-kappa B and another similar activity are rapidly induced when growth-arrested BALB/c 3T3 cells are stimulated with serum growth factors. Induction of these DNA-binding activities is not inhibited by pretreatment of quiescent cells with the protein synthesis inhibitor cycloheximide. Interestingly, the major NF-kappa B-like activity is not detected in nuclear extracts of proliferating cells, and thus its expression appears to be limited to the G0-to-G1 transition in 3T3 cells. These DNA-binding activities bind many of the expected NF-kappa B target sequences, including elements in the class I major histocompatibility complex and human immunodeficiency virus enhancers, as well as a recently identified NF-kappa B binding site upstream of the c-myc gene. Furthermore, both the class I major histocompatibility complex and c-myc NF-kappa B binding sites confer inducibility on a minimal promoter in 3T3 cells stimulated with serum growth factors. The results demonstrate that NF-kappa B-like activities are immediate-early response proteins in 3T3 cells and suggest a role for these factors in the G0-to-G1 transition.