Interaction of MYC with host cell factor-1 is mediated by the evolutionarily conserved Myc box IV motif.

The MYC family of oncogenes encodes a set of three related transcription factors that are overexpressed in many human tumors and contribute to the cancer-related deaths of more than 70,000 Americans every year. MYC proteins drive tumorigenesis by interacting with co-factors that enable them to regulate the expression of thousands of genes linked to cell growth, proliferation, metabolism and genome stability. One effective way to identify critical co-factors required for MYC function has been to focus on sequence motifs within MYC that are conserved throughout evolution, on the assumption that their conservation is driven by protein-protein interactions that are vital for MYC activity. In addition to their DNA-binding domains, MYC proteins carry five regions of high sequence conservation known as Myc boxes (Mb). To date, four of the Mb motifs (MbI, MbII, MbIIIa and MbIIIb) have had a molecular function assigned to them, but the precise role of the remaining Mb, MbIV, and the reason for its preservation in vertebrate Myc proteins, is unknown. Here, we show that MbIV is required for the association of MYC with the abundant transcriptional coregulator host cell factor-1 (HCF-1). We show that the invariant core of MbIV resembles the tetrapeptide HCF-binding motif (HBM) found in many HCF-interaction partners, and demonstrate that MYC interacts with HCF-1 in a manner indistinguishable from the prototypical HBM-containing protein VP16. Finally, we show that rationalized point mutations in MYC that disrupt interaction with HCF-1 attenuate the ability of MYC to drive tumorigenesis in mice. Together, these data expose a molecular function for MbIV and indicate that HCF-1 is an important co-factor for MYC.

A common functional consequence of tumor-derived mutations within c-MYC.

The relevance of changes to the coding sequence of the c-MYC oncogene to malignancy is controversial. Overexpression of a pristine form of MYC is observed in many cancers and is sufficient to drive tumorigenesis in most contexts. Yet missense changes to MYC are found in ~50% of Burkitt's lymphomas, aggregate within an amino-terminal degron important for proteasomal destruction of MYC, and where examined profoundly enhance the tumorigenic properties of MYC in vitro and in vivo. Much of the controversy surrounding these mutants stems from the limited number of mutations that have been evaluated and their clustering within a single region of the MYC protein; the highly-conserved Myc box I (MbI) element. Here, by analysis of extant genomic data sets, we identify a previously unrecognized hotspot for tumor-associated MYC mutations, located in a conserved central portion of the protein. We show that, despite their distal location in MYC, mutations in this region precisely phenocopy those in MbI in terms of stability, in vitro transformation, growth-promoting properties, in vivo tumorigenesis and ability to escape p53-dependent tumor surveillance mechanisms. The striking parallels between the behavior of tumor-derived mutations in disparate regions of the MYC protein reveals that a common molecular process is disrupted by these mutations, implying an active role for these mutations in tumorigenesis and suggesting that different therapeutic strategies may be needed for treatment of lymphomas expressing wild type versus mutant forms of MYC protein.

Transcriptional control and the ubiquitin-proteasome system.

Regulation of transcription is a critically important process that controls development, differentiation, and the maintenance of cellular homeostasis. Cells have evolved numerous mechanisms to keep gene transcription tightly in check, some of which involve the ubiquitin-proteasome system. In this chapter, we review evidence supporting the concept that ubiquitin and the proteasome not only control transcription, but provide the biochemical means to drive key steps in the transcription process forward.

Regulation of transcriptional activation domain function by ubiquitin.

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.

Transcriptional activation: risky business.

Transcriptional regulation is all about getting RNA polymerase to the right place on the gene at the right time and making sure that it is competent to conduct transcription. Traditional views of this process place most of their emphasis on the events that precede initiation of transcription. We imagine a promoter-bound transcriptional activator (or collection of activators) recruiting components of the basal transcriptional machinery to the DNA, eventually leading to the recruitment of RNA polymerase II and the onset of gene transcription. Although these events play a crucial role in regulating gene expression, they are only half the story. Correct regulation of transcription requires that polymerase not only initiates when and where it should, but that it stops initiating when no longer appropriate. But how are the signals from transcriptional activators, telling RNA polymerase to fire, terminated? Is this process governed by chance, with activators simply falling off the promoter at a certain frequency? Or is there some more direct mechanism, whereby activators are aggressively limited from uncontrolled promoter activation? A new article by suggests the latter may be true, and provides a mechanism for how a component of the basal transcription machinery can mark the activators it has encountered, sentencing them to an early death or banishing them from the nucleus. The ability of the basal transcriptional apparatus to mark activators provides an efficient way to limit activator function and ensures that continuing transcription initiation at a promoter is coupled to the continuing synthesis and activation of transcriptional activators.

Functional overlap of sequences that activate transcription and signal ubiquitin-mediated proteolysis.

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.

HAM: a new epitope-tag for in vivo protein labeling.

The ability to metabolically label proteins with 35S-methionine is critical for the analysis of protein synthesis and turnover. Despite the importance of this approach, however, efficient labeling of proteins in vivo is often limited by a low number of available methionine residues, or by deleterious side-effects associated with protein overexpression. To overcome these limitations, we have created a methionine-rich variant of the widely used HA tag, called HAM, for use with ectopically expressed proteins. Here we describe the development of a series of vectors, and corresponding antisera, for the expression and detection of HAM-tagged proteins in mammalian cells. We show that the HAM tag dramatically improves the sensitivity of 35S-methionine labeling, and permits the analysis of Myc oncoprotein turnover even when HAM-tagged Myc is expressed at levels comparable to that of the endogenous protein. Because of the improved sensitivity provided by the HAM tag, the vectors and antisera described here should be useful for the analysis of protein synthesis and destruction at physiological levels of protein expression.

Destruction of Myc by ubiquitin-mediated proteolysis: cancer-associated and transforming mutations stabilize Myc.

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.

Selective use of TBP and TFIIB revealed by a TATA-TBP-TFIIB array with altered specificity.

Interaction between the TATA box-binding protein TBP and TFIIB is critical for transcription in vitro. An altered-specificity TBP-TFIIB interaction was rationally designed and linked in sequence to an altered-specificity TATA box-TBP interaction to study how TBP and TFIIB function together to support transcription in human cells. The activity of this altered-specificity TATA-TBP-TFIIB array demonstrated that many activators use the known TBP-TFIIB interaction to stimulate transcription. One activator, however, derived from a glutamine-rich activation domain of Sp1, activated transcription independently of this interaction. These results reveal that selectivity in activator function in vivo can be achieved through differential use of TBP and TFIIB.

Mutations in the carboxy-terminal domain of TBP affect the synthesis of human immunodeficiency virus type 1 full-length and short transcripts similarly.

The human immunodeficiency virus type 1 promoter generates two types of RNA molecules, full-length transcripts and short transcripts. Synthesis of the short transcripts depends on the inducer of short transcripts (IST), an element located downstream of the start site. In the presence of the viral activator Tat, the synthesis of full-length transcripts is up-regulated while that of short transcripts is down-regulated. Full-length and short transcripts are probably generated by different types of transcription complexes. The first is IST independent, capable of efficient elongation, and up-regulated by Tat. The second is IST dependent, incapable of efficient elongation, and down-regulated by Tat. We have used an in vivo assay to assess the role of TBP in human immunodeficiency virus type I transcription and to test the effect of mutations in TBP on synthesis of full-length and short transcripts. We find that TBP bound to the TATA box is required for the synthesis of short and full-length transcripts as well as for Tat activation and that both yeast TBP and the carboxy-terminal domain of human TBP can replace full-length human TBP for these processes. Mutations in TBP affect the synthesis of short and full-length transcripts as well as Tat activation similarly, and these effects correlate with the previously described effects of these mutations on binding of TBP to the TBP-associated factor TAFII250 in vitro. Together, these results suggest that if short and full-length transcripts are generated by variant transcription complexes, these complexes use TBP similarly, probably as part of the TFIID complex.

The ability to associate with activation domains in vitro is not required for the TATA box-binding protein to support activated transcription in vivo.

The TATA box-binding protein (TBP) interacts in vitro with the activation domains of many viral and cellular transcription factors and has been proposed to be a direct target for transcriptional activators. We have examined the functional relevance of activator-TBP association in vitro to transcriptional activation in vivo. We show that alanine substitution mutations in a single loop of TBP can disrupt its association in vitro with the activation domains of the herpes simplex virus activator VP16 and of the human tumor suppressor protein p53; these mutations do not, however, disrupt the transcriptional response of TBP to either activation domain in vivo. Moreover, we show that a region of VP16 distinct from its activation domain can also tightly associate with TBP in vitro, but fails to activate transcription in vivo. These data suggest that the ability of TBP to interact with activation domains in vitro is not directly relevant to its ability to support activated transcription in vivo.

Renin gene promoter activity in GC cells is regulated by cAMP and thyroid hormone through Pit-1-dependent mechanisms.

Transcriptional activity of human renin gene (hREN) 5'-flanking DNA sequences in pituitary cells is highly dependent on binding of the pituitary-specific transcription factor Pit-1. Pit-1 has been implicated in cAMP regulation of a number of pituitary genes and has also been shown to interact with thyroid hormone (T3) receptors in mediating T3 responsiveness of the rat growth hormone gene. In the present study we examine the effects of forskolin and T3 on the expression of luciferase hybrid genes containing hREN 5'-flanking DNAs (hREN.luc) transiently transfected into the pituitary cell line GC. Basal activities of all hREN.luc constructs transfected into cells grown in media containing serum stripped of hormones were low. Addition of forskolin stimulated expression up to 48-fold, depending on the hREN sequences present. The hREN sequence -148 to +18 was sufficient for both maximal expression and maximal stimulation by forskolin. Mutagenesis of the Pit-1 site between -82 and -58 reduced forskolin induction 4-5-fold. In addition to the Pit-1 site, the sequence between -148 and -98 was also required for maximal activity and forskolin induction. T3 on its own had no effect on hREN promoter activity in GC cells, but suppressed the effects of forskolin. Gel mobility shift and Western blot analyses indicated that forskolin treatment had no effect on Pit-1 DNA binding or Pit-1 levels. However, T3 reduced Pit-1 levels which was reflected in lower DNA binding under the conditions employed. Taken together, these findings emphasize the importance of cAMP-dependent mechanisms in directing renin gene expression.

Multiple regions of TBP participate in the response to transcriptional activators in vivo.

We used mutant yeast and human TBP molecules with an altered DNA-binding specificity to examine the role of TBP in transcriptional activation in vivo. We show that yeast TBP is functionally equivalent to human TBP for response to numerous transcriptional activators in human cells, including those that do not function in yeast. Despite the extensive conservation of TBP, its ability to respond to transcriptional activators in vivo is curiously resistant to clustered sets of alanine substitution mutations in different regions of the protein, including those that disrupt DNA binding and basal transcription in vitro. Combined sets of these mutations, however, can attenuate the in vivo activity of TBP and can differentially affect response to different activation domains. Although the activity of TBP mutants in vivo did not correlate with DNA binding or basal transcription in vitro, it did correlate with binding in vitro to the largest subunit of TFIID, hTAFII250. Together, these data suggest that TBP utilizes multiple interactions across its surface to respond to RNA polymerase II transcriptional activators in vivo; some of these interactions appear to involve recruitment of TBP into TFIID, whereas others are involved in response to specific types of transcriptional activators.

Distance-dependent interactions between basal, cyclic AMP, and thyroid hormone response elements in the rat growth hormone promoter.

Developmental stage- and tissue-specific expression of the rat growth hormone (rGH) gene is conferred by DNA sequences within 237 base pairs of the transcription start site. Although binding of a number of transcription factors including Pit-1, Sp1, GHF3, and thyroid hormone receptor (T3R) stimulates rGH expression, several studies have suggested that interactions between these factors are important in determining cell specificity and responsiveness to extracellular signals. We have directly tested this hypothesis by creating a set of nested insertional mutations at two positions in the rGH promoter. Sequences were inserted at either position -148, separating GHF-3 and T3R binding sites from the downstream Pit-1 and Sp 1 binding sites, or at -51, separating the above elements from the TATA box. All insertions were made in the context of the rGH gene -237/+8 5'-flanking DNA, linked to a chloramphenicol acetyltransferase reporter gene and tested for activity by transient transfection in GC pituitary tumor cells. Insertions at both -148 and -51 caused sharp distance-dependent reductions in serum-stimulated expression such that insertions of 23 base pairs at -51 or 44 base pairs at -148 were sufficient to isolate the effects of sequences upstream of the insertion point. Insertions at -148 reduced T3 responsiveness severalfold but had little or no effect on stimulation by forskolin, whereas insertions at -51 reduced both T3 and forskolin responsiveness. Our results are consistent with the hypothesis that expression and regulation of the rGH gene is dependent on short-range protein-protein interactions, which are more critically dependent on spacing than the relative orientation of the transcription factor binding sites.

Transcriptional and posttranscriptional activation of urokinase plasminogen activator gene expression in metastatic tumor cells.

Urokinase plasminogen activator (uPA) is a serine protease which has frequently been implicated in the process of tumor cell invasion and metastasis. The degree of expression and mode(s) of regulation of the uPA gene in metastatic compared with nonmetastatic tumor cells have not yet been addressed. We have cloned and sequenced a full-length rat uPA complementary DNA and utilized Northern blot analysis to report that the uPA gene is expressed at levels 3.5- to 70-fold higher in metastatic cell lines than in nonmetastatic cell lines derived from two independent rat mammary adenocarcinomas. Nuclear run-on assays and RNA half-life estimations indicated that metastatic MAT 13762 rat mammary adenocarcinoma cells expressed 3.5-fold higher levels of uPA RNA than a nonmetastatic derivative (J-clone), due to a combined increase in uPA gene transcription and cytoplasmic RNA stability. By contrast, uPA RNA (and enzyme) levels were elevated by up to 70-fold in metastatic clones of dimethylbenz(a)anthracene-induced rat mammary adenocarcinoma (DMBA-8) due to predominantly posttranscriptional mechanisms. Moreover, treatment of nonmetastatic DMBA-8 cell lines with protein synthesis inhibitors led to an increase in nuclear and cytoplasmic uPA RNA levels, without altering the rate of uPA gene transcription. These results suggest that in addition to gene transcription, posttranscriptional events localized in the nucleus and cytoplasm are key determinants of uPA gene activation in rat mammary adenocarcinomas.

Sp1 and thyroid hormone receptor differentially activate expression of human growth hormone and chorionic somatomammotropin genes.

Activation of hGH-1 expression is mediated by the pituitary-specific transcription factor GHF-I/Pit-I which binds the 5'-flanking DNA at two sites: I (-96/-70) and II (-134/-106). Although the factor(s) which direct the placental-specific expression of hCS-1 are not known, hCS-1 sequences are transcriptionally active in pituitary cells. In the present study we examined the effects of sequence differences between hGH-1 and hCS-1 5'-flanking DNAs in determining their basal and thyroid hormone-regulated promoter activities. We showed that Sp1 is a major determinant of both hGH-1 and hCS-1 promoter activities and that in hGH-1, binding and activation by Sp1 are modulated by interference from GHF-I/Pit-1 binding at the adjacent site II sequence. A single base which differed in site II of hCS-1 greatly reduced GHF-1/Pit-1 binding and thus facilitated binding and activation by Sp1. Further differences in promoter activity of hGH-1 and hCS-1 sequences were accounted for by a thyroid hormone-responsive element between -62/-48 in the hCS-1 gene. However, induction by T3 was independent of either Sp1 or GH-1/Pit-1 binding in the site II region. These data demonstrate that a small number of base changes between hGH and hCS promoter sequences subserve a number of mechanisms which may differentially modulate the expression of hGH and hCS genes.