Myc is an oncoprotein transcription factor that plays a prominent role in cancer. Like many transcription factors, Myc is an unstable protein that is destroyed by ubiquitin (Ub)-mediated proteolysis. Here, we report that the oncoprotein and Ub ligase Skp2 regulates Myc ubiquitylation and stability. Because of the growing number of Ub ligases that function as transcriptional coactivators, we speculated that Skp2 might also regulate Myc's transcriptional activity. Consistent with this model, we also show that Skp2 is a transcriptional coactivator for Myc, recognizing an essential element within the Myc activation domain and activating Myc target genes. These data suggest that Skp2 functions to connect Myc activity and destruction, and reveal an unexpected oncoprotein connection that may play an important role in controlling cell growth in normal and cancer cells.

The human proto-oncogene c-myc encodes a highly unstable transcription factor that promotes cell proliferation. Although the extreme instability of Myc plays an important role in preventing its accumulation in normal cells, little is known about how Myc is targeted for rapid destruction. Here, we have investigated mechanisms regulating the stability of Myc. We show that Myc is destroyed by ubiquitin-mediated proteolysis, and define two elements in Myc that oppositely regulate its stability: a transcriptional activation domain that promotes Myc destruction, and a region required for association with the POZ domain protein Miz-1 that stabilizes Myc. We also show that Myc is stabilized by cancer-associated and transforming mutations within its transcriptional activation domain. Our data reveal a complex network of interactions regulating Myc destruction, and imply that enhanced protein stability contributes to oncogenic transformation by mutant Myc proteins.

We have developed an inducible system to visualize gene expression at the levels of DNA, RNA and protein in living cells. The system is composed of a 200 copy transgene array integrated into a euchromatic region of chromosome 1 in human U2OS cells. The condensed array is heterochromatic as it is associated with HP1, histone H3 methylated at lysine 9, and several histone methyltransferases. Upon transcriptional induction, HP1alpha is depleted from the locus and the histone variant H3.3 is deposited suggesting that histone exchange is a mechanism through which heterochromatin is transformed into a transcriptionally active state. RNA levels at the transcription site increase immediately after the induction of transcription and the rate of synthesis slows over time. Using this system, we are able to correlate changes in chromatin structure with the progression of transcriptional activation allowing us to obtain a real-time integrative view of gene expression.

The ability of transcriptional activation domains (TADs) to signal ubiquitin-mediated proteolysis suggests an involvement of the ubiquitin-proteasome pathway in transcription. To probe this involvement, we asked how ubiquitylation regulates the activity of a transcription factor containing the VP16 TAD. We show that the VP16 TAD signals ubiquitylation through the Met30 ubiquitin-ligase and that Met30 is also required for the VP16 TAD to activate transcription. The requirement for Met30 in transcription is circumvented by fusion of ubiquitin to the VP16 activator, demonstrating that activator ubiquitylation is essential for transcriptional activation. We propose that ubiquitylation regulates TAD function by serving as a dual signal for activation and activator destruction.

Many transcription factors, particularly those involved in the control of cell growth, are unstable proteins destroyed by ubiquitin-mediated proteolysis. In a previous study of sequences targeting the transcription factor Myc for destruction, we observed that the region in Myc signaling ubiquitin-mediated proteolysis overlaps closely with the region in Myc that activates transcription. Here, we present evidence that the overlap of these two activities is not unique to Myc, but reflects a more general phenomenon. We show that a similar overlap of activation domains and destruction elements occurs in other unstable transcription factors and report a close correlation between the ability of an acidic activation domain to activate transcription and to signal proteolysis. We also show that destruction elements from yeast cyclins, when tethered to a DNA-binding domain, activate transcription. The intimate overlap of activation domains and destruction elements reveals an unexpected convergence of two very different processes and suggests that transcription factors may be destroyed because of their ability to activate transcription.

The human immunodeficiency virus type 1 nef gene induces endocytosis of CD4 antigen and disrupts the association between CD4 and p56Ick protein-tyrosine kinase (EC 2.7.1.112). We demonstrate that in T cells these effects of the viral protein require a cluster of hydrophobic amino acids in a membrane-proximal region of the CD4 cytoplasmic tail; other amino acids in the C-terminal segment of CD4 cytoplasmic tail also contribute to the interaction. Mutations in CD4 that prevent down-modulation by Nef also decrease CD4 association with p56lck and prevent Nef-induced disruption of CD4-p56lck complexes. Together, the overlap in CD4 sequences required for interaction with Nef and p56lck and the tight correlation between Nef-induced CD4 down-modulation and disruption of CD4-p56Ick association suggest that Nef, or cellular factors recruited by Nef, interact with this segment of CD4 to displace p56lck from the complex and induce CD4 endocytosis.nef genes of human and simian immunodeficiency viruses (HIV, SIV) encode related N-terminally myristoylated cytoplasmic proteins (1, 2). nef is essential for high viral load and pathogenesis in vivo (3) but is not required for viral replication under commonly used in vitro conditions (3-5). The mechanism by which Nef stimulates viral replication in vivo is unknown but may involve down-modulation of CD4 antigen expression on the cell surface, as has been observed with a large fraction of nef alleles derived from laboratory HIV and SIV isolates and from peripheral blood leukocytes of HIVinfected patients (6-11). Nef-induced down-modulation of CD4 on the cell surface reflects accelerated CD4 endocytosis and its degradation in the lysosomal compartment (12, 13).CD4 is a transmembrane glycoprotein expressed at high levels on helper T cells and is essential for their ontogeny and antigen-specific responses (14-17). In T cells, the short cytoplasmic domain of CD4 is involved in at least two interactions.One is with the p56lck protein-tyrosine kinase (EC 2.7.1.112) (18,19) and this association is required for antigen-specific signaling (15). The CD4-p56lck association requires cysteine motifs in the CD4 cytoplasmic tail and the N terminus of p56lck and may involve direct binding of the two proteins (20,21). The cysteine motif in CD4 is not sufficient for binding p56lck, as a deletion of the membrane-proximal segment of the CD4 cytoplasmic tail prevents association of the two proteins (13,21).The other interactions involve CD4 endocytosis induced by Nef and/or phorbol ester [phorbol 12-myristate 13-acetate (PMA)]. and Nef-induced CD4 endocytosis (12) and CD4 targeting to the lysosomal compartment require a di-leucine motif in the membrane-proximal region of CD4 cytoplasmic tail. CD4 endocytosis induced by PMA is initiated by phosphorylation of serines located in a close proximity to the di-leucine motif, an event mediated by protein kinase C (22, 23). In contrast, the effect of Nef on CD4 does not involve serine phosphorylation, or formation of stable complexes between the two prot...

Myc is an oncoprotein transcription factor that promotes cell proliferation and apoptosis. Analysis of highly conserved elements within vertebrate Myc proteins has been instrumental in defining the functions of the Myc protein. Here, we probe the role of a highly conserved, but little studied, element within the central region of c-Myc, termed 'Myc box III' (MbIII). We show that MbIII is important for transcriptional repression by Myc, and for transformation both in vitro and in a mouse model of lymphomagenesis. Curiously, disruption of MbIII decreases transformation activity by increasing the efficiency with which Myc can induce apoptosis, suggesting that MbIII is a negative regulator of programmed cell death. These findings reveal a role for MbIII in Myc biology, and establish that the oncogenic capacity of Myc is linked directly to its ability to temper the apoptotic response.

Emerging evidence suggests that components of the ubiquitin-proteasome system are involved in the regulation of gene expression. A variety of factors, including transcriptional activators, coactivators, and histones, are controlled by ubiquitylation, but the mechanisms through which this modification can function in transcription are generally unknown. Here, we report that the Saccharomyces cerevisiae protein Asr1 is a RING finger ubiquitin-ligase that binds directly to RNA polymerase II via the carboxyl-terminal domain (CTD) of the largest subunit of the enzyme. We show that interaction of Asr1 with the CTD depends on serine-5 phosphorylation within the CTD and results in ubiquitylation of at least 2 subunits of the enzyme, Rpb1 and Rpb2. Ubiquitylation by Asr1 leads to the ejection of the Rpb4/Rpb7 heterodimer from the polymerase complex and is associated with inactivation of polymerase function. Our data demonstrate that ubiquitylation can directly alter the subunit composition of a core component of the transcriptional machinery and provide a paradigm for how ubiquitin can influence gene activity.transcription ͉ ubiquitin T he correct regulation of gene transcription depends on mechanisms that regulate the formation and dynamics of large multiprotein complexes during various stages of the transcription process. One of the most prominent of these mechanisms is posttranslational protein modification. Phosphorylation is frequently used to promote and stabilize the interaction of various proteins; recruitment of capping and splicing factors to elongating RNA polymerase II (pol II), for example, is signaled by phosphorylation within the carboxyl-terminal domain (CTD) of its largest subunit (1), although modifications such as methylation (2) and acetylation (3) can also influence critical protein-protein interactions. One modification that has received attention in recent years is ubiquitylation, as it has become evident that modification of transcription proteins by ubiquitin (Ub) (4, 5) plays a role in diverse aspects of gene regulation.Ub is a 76-amino acid protein that is covalently linked to other proteins by the action of an enzymatic cascade, the last step of which is mediated via a Ub-protein ligase (or E3) that recognizes a specific element within the substrate and promotes transfer of Ub to a lysine residue(s) within that protein (6). The utility of ubiquitylation stems from its specificity and its ability to function as either a ''classic'' modification or as a signal for substrate destruction by the 26S proteasome. By varying the extent of protein ubiquitylation, the type of poly-Ub chains, or the sites of ubiquitylation in the substrate, Ub can act as either a reversible modifier of protein function or an irreversible mechanism for limiting protein levels.Several recent sets of studies have revealed that ubiquitylation influences multiple steps in the transcription process. Our particular interest has centered on the connection between ubiquitylation of transcriptional activators and the regulation of...