Biomedical subjects

William P Tansey

Publications and source records attributed to William P Tansey.

At least 19 recordsLinked to original sources

PubMed2025

MYC Regulates a DNA Repair Gene Expression Program in Small Cell Carcinoma of the Ovary, Hypercalcemic Type.

BACKGROUND/OBJECTIVES: SCCOHT is an aggressive and often fatal cancer that belongs to the ~20% of cancers defined by mutations to subunits of the SWI/SNF chromatin remodeling complex. In SCCOHT, mutations to the SMARCA4 gene, which encodes the SWI/SNF ATPase BRG1, are sufficient to impair SWI/SNF function. This single genetic lesion leads to a cascade of events that promote tumorigenesis, some of which may involve the intersection of SWI/SNF with oncogenic pathways such as those regulated by the MYC oncogene. In SCCOHT tumors and other cancers marked by SWI/SNF subunit mutation, MYC target genes are recurrently activated, pointing to a relationship between SWI/SNF and MYC that has yet to be fully explored. METHODS: In this study, we investigate the contribution of MYC to SCCOHT biology by performing a combination of chromatin binding and transcriptome assays in genetically engineered SCCOHT cell lines, with subsequent validation using patient tumor expression data. RESULTS: We find that MYC binds to thousands of active promoters in the BIN-67 SCCOHT cell line and that the depletion of MYC results in a broad range of gene expression changes with a notable effect on the expression of genes related to DNA repair. We uncover an MYC-regulated DNA repair gene expression program in BIN-67 cells that is antagonized by BRG1 reintroduction. Finally, we identify a DNA repair gene signature that is upregulated in SCCOHT tumors and in tumors defined by loss of the SWI/SNF subunit SNF5. CONCLUSIONS: Collectively, these data implicate MYC as a robust regulator of DNA repair gene expression in SCCOHT and lay a foundation for future studies focused on interrogating the relationship between BRG1 and MYC.

ATR↗

PubMed2006

SCFCdc4-mediated degradation of the Hac1p transcription factor regulates the unfolded protein response in Saccharomyces cerevisiae.

The Saccharomyces cerevisiae basic leucine zipper transcription factor Hac1p is synthesized in response to the accumulation of unfolded polypeptides in the lumen of the endoplasmic reticulum (ER), and it is responsible for up-regulation of approximately 5% of all yeast genes, including ER-resident chaperones and protein-folding catalysts. Hac1p is one of the most short-lived yeast proteins, having a half-life of approximately 1.5 min. Here, we have shown that Hac1p harbors a functional PEST degron and that degradation of Hac1p by the proteasome involves the E2 ubiquitin-conjugating enzyme Ubc3/Cdc34p and the SCF(Cdc4) E3 complex. Consistent with the known nuclear localization of Cdc4p, rapid degradation of Hac1p requires the presence of a functional nuclear localization sequence, which we demonstrated to involve basic residues in the sequence (29)RKRAKTK(35). Two-hybrid analysis demonstrated that the PEST-dependent interaction of Hac1p with Cdc4p requires Ser146 and Ser149. Turnover of Hac1p may be dependent on transcription because it is inhibited in cell mutants lacking Srb10 kinase, a component of the SRB/mediator module of the RNA polymerase II holoenzyme. Stabilization of Hac1p by point mutation or deletion, or as the consequence of defects in components of the degradation pathway, results in increased unfolded protein response element-dependent transcription and improved cell viability under ER stress conditions.

Amino Acid Motifs↗

PubMed2006

The proteasome: a utility tool for transcription?

A growing body of evidence demonstrates that the components of the proteasome are involved directly and mechanistically in the regulation of gene transcription. Proteolytic activities of the proteasome are important for establishing limits for transcription, for promoting the exchange of transcription factors on chromatin, and possibly for facilitating multiple rounds of transcription initiation. Non-proteolytic activities of the proteasome are important for co-activator recruitment, transcriptional elongation, and histone modification. Here, we discuss different ways in which the proteasome can influence transcription, and argue that its unique combination of biological activities makes it ideally suited to act at multiple stages in the transcription process.

Animals↗

PubMed2006

Chromatin immunoprecipitation to study protein-DNA interactions in budding yeast.

The accurate replication and expression of genetic information is ultimately governed by the interaction of regulatory proteins with specific sites on chromosomes. In recent years, our understanding of how these interactions occur in vivo has advanced considerably, in large part owing to the widespread application of chromatin immunoprecipitation (ChIP), a technique that allows quantification of protein-DNA interactions within the context of native chromatin. The ChIP assay involves three main steps: (1) chemical crosslinking of protein-DNA complexes in intact cells; (2) recovery of specific proteins by immunoprecipitation; and (3) detection of co-precipitating DNA sequences, usually by the polymerase chain reaction (PCR). Here, we provide a detailed description of a ChIP procedure that is commonly used to detect protein-DNA interactions in the yeast Saccharomyces cerevisiae, and discuss various methods for quantifying co-precipitating DNAs.

Chromatin↗

PubMed2005

The proteasome regulatory particle alters the SAGA coactivator to enhance its interactions with transcriptional activators.

Promoter recruitment of the Saccharomyces cerevisiae SAGA histone acetyltransferase complex is required for RNA polymerase II-dependent transcription of several genes. SAGA is targeted to promoters through interactions with sequence-specific DNA binding transcriptional activators and facilitates preinitiation-complex assembly and transcription. Here, we show that the 19S proteasome regulatory particle (19S RP) alters SAGA to stimulate its interaction with transcriptional activators. The ATPase components of the 19S RP are required for stimulation of SAGA/activator interactions and enhance SAGA recruitment to promoters. Proteasomal ATPases genetically interact with SAGA, and their inhibition reduces global histone H3 acetylation levels and SAGA recruitment to target promoters in vivo. These results indicate that the 19S RP modulates SAGA complex using its ATPase components, thereby facilitating subsequent transcription events at promoters.

Acetylation↗

PubMed2005

Evasion of the p53 tumour surveillance network by tumour-derived MYC mutants.

The c-Myc oncoprotein promotes proliferation and apoptosis, such that mutations that disable apoptotic programmes often cooperate with MYC during tumorigenesis. Here we report that two common mutant MYC alleles derived from human Burkitt's lymphoma uncouple proliferation from apoptosis and, as a result, are more effective than wild-type MYC at promoting B cell lymphomagenesis in mice. Mutant MYC proteins retain their ability to stimulate proliferation and activate p53, but are defective at promoting apoptosis due to a failure to induce the BH3-only protein Bim (a member of the B cell lymphoma 2 (Bcl2) family) and effectively inhibit Bcl2. Disruption of apoptosis through enforced expression of Bcl2, or loss of either Bim or p53 function, enables wild-type MYC to produce lymphomas as efficiently as mutant MYC. These data show how parallel apoptotic pathways act together to suppress MYC-induced transformation, and how mutant MYC proteins, by selectively disabling a p53-independent pathway, enable tumour cells to evade p53 action during lymphomagenesis.

Adoptive Transfer↗

PubMed2005

The F box protein Dsg1/Mdm30 is a transcriptional coactivator that stimulates Gal4 turnover and cotranscriptional mRNA processing.

We report here that the prototypical yeast transcription factor Gal4 undergoes two distinct modes of ubiquitin-mediated proteolysis: one that occurs independent of transcription and restricts Gal4 function, and another that is transcription coupled and essential for productive activation of Gal4 target genes. Destruction of transcriptionally active Gal4 depends on an F box protein called Dsg1/Mdm30. In the absence of Dsg1, Gal4 is stable, nonubiquitylated, and unable to productively stimulate transcription. Analysis of the phenotype of dsg1-null yeast reveals a striking disconnect between GAL gene RNA and protein levels; in the absence of Dsg1, Gal4 target genes are transcribed, but the resulting RNAs are not translated. The translational defects of these RNAs are related to defects in phosphorylation of the RNA polymerase II carboxy-terminal domain, which in turn affects recruitment of RNA processing machinery. We propose that Gal4 ubiquitylation and destruction are required for initiation-competent transcription complexes to transition to fully mature elongating complexes capable of appropriate mRNA processing.

DNA-Binding Proteins↗

PubMed2005

A conserved element in Myc that negatively regulates its proapoptotic activity.

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.

Animals↗

PubMed2004

Multiple cell-type-specific elements regulate Myc protein stability.

Myc is a highly unstable transcription factor that is destroyed by ubiquitin (Ub)-mediated proteolysis. We have previously identified an amino-terminal 'degron' within Myc that signals its destruction; this degron spans the transcriptional activation domain of Myc, and includes two highly conserved regions called Myc boxes I and II. We now report the identification of a second element--the D-element--which is also required for Myc proteolysis. The centrally located D-element is distinct from the PEST domain in Myc, but includes Myc box III, a third highly conserved region with no previously known function. We show that deletion of the D-element stabilizes the Myc protein without affecting its ubiquitylation, and report that the D-element and the degron act in a cell-type-specific manner to direct Myc proteolysis. These data thus demonstrate that Myc stability is regulated at both the ubiquitylation and postubiquitylation levels, and reveal that substrates of the Ub-proteasome system can be targeted for destruction differently in different cell types.

Amino Acid Sequence↗

PubMed2004

From silencing to gene expression: real-time analysis in single cells.

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.

Animals↗

PubMed2004

Proteasomal ATPases link ubiquitylation of histone H2B to methylation of histone H3.

In Saccharomyces cerevisiae, methylation of histone H3 at active genes is an epigenetic mark that distinguishes active from silent chromatin and functions as a short-term "memory" of recent transcription. Methylation of H3 at lysine residues K4 and K79 depends on ubiquitylation of histone H2B, but the mechanisms linking H2B ubiquitylation to H3 methylation are unknown. Here, we demonstrate that proteasomal ATPases Rpt4 and Rpt6 function to connect these two histone modifications. We show that recruitment of proteasome subunits to chromatin depends on H2B ubiquitylation and that mutations in Rpt4 and Rpt6 disrupt H3 methylation at K4 and K79 but leave H2B ubiquitylation intact. Consistent with their role in H3 methylation, we also find that mutations in Rpt4 and 6-but not components of the 20S proteasome-disrupt telomeric gene silencing. These data reveal that proteasome subunits function in epigenetic gene regulation by linking chromatin modifications that establish the histone code.

Adenosine Triphosphatases↗

PubMed2004

Crashing waves of destruction: the cell cycle and APC(Cdh1) regulation of SCF(Skp2).

Coordination of events required for cell cycle progression is orchestrated in large part by the ubiquitin (Ub)-mediated destruction of key regulatory proteins such as cyclins and their inhibitors. Until now, the G1/S and mitotic phases of the cell cycle were thought to be controlled by discrete families of multisubunit Ub-ligases: SCF ligases controlled the G1 to S transition, whereas APC ligases controlled the onset and exit from mitosis. New work, published in the March 11 issue of Nature, challenges this concept by revealing that an essential function of APC is to limit SCF activity during the G1 phase of the cell cycle.

Anaphase-Promoting Complex-Cyclosome↗

PubMed2004

A nonconserved surface of the TFIIB zinc ribbon domain plays a direct role in RNA polymerase II recruitment.

The general transcription factor TFIIB is a highly conserved and essential component of the eukaryotic RNA polymerase II (pol II) transcription initiation machinery. It consists of a single polypeptide with two conserved structural domains: an amino-terminal zinc ribbon structure (TFIIB(ZR)) and a carboxy-terminal core (TFIIB(CORE)). We have analyzed the role of the amino-terminal region of human TFIIB in transcription in vivo and in vitro. We identified a small nonconserved surface of the TFIIB(ZR) that is required for pol II transcription in vivo and for different types of basal pol II transcription in vitro. Consistent with a general role in transcription, this TFIIB(ZR) surface is directly involved in the recruitment of pol II to a TATA box-containing promoter. Curiously, although the amino-terminal human TFIIB(ZR) domain can recruit both human pol II and yeast (Saccharomyces cerevisiae) pol II, the yeast TFIIB amino-terminal region recruits yeast pol II but not human pol II. Thus, a critical process in transcription from many different promoters-pol II recruitment-has changed in sequence specificity during eukaryotic evolution.

Amino Acid Sequence↗

PubMed2003

Skp2 regulates Myc protein stability and activity.

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.

Animals↗

PubMed2003

How the ubiquitin-proteasome system controls transcription.

Gene transcription and ubiquitin-mediated proteolysis are two processes that have seemingly nothing in common: transcription is the first step in the life of any protein and proteolysis the last. Despite the disparate nature of these processes, a growing body of evidence indicates that ubiquitin and the proteasome are intimately involved in gene control. Here, we discuss the deep mechanistic connections between transcription and the ubiquitin-proteasome system, and highlight how the intersection of these processes tightly controls expression of the genetic information.

Animals↗

PubMed2002

Stable and unstable pools of Myc protein exist in human cells.

The oncoprotein transcription factor Myc plays a crucial role in the control of cell growth and proliferation. Consistent with its potent growth-promoting properties, cells have evolved a number of mechanisms to limit the activity and accumulation of the Myc protein. One of the most striking of these mechanisms is ubiquitin (Ub)-mediated proteolysis, which typically destroys Myc within minutes of its synthesis. Here we show that, despite the extreme instability of the Myc protein, cells contain a pool of Myc that is metabolically stable. Entry of Myc into the stable pool is signaled by an element within the carboxy-terminus of the protein, and is a cell-specific process that is regulated during mitosis and by interaction with Max. These data demonstrate that - even for a rapidly turned-over protein such as Myc - metabolically stable and unstable forms of a protein can co-exist in cells, and suggest that the rate of destruction of Myc molecules is linked to their specific functions.

Cell Cycle↗

PubMed2002

Regulation of c-myc stability by selective stress conditions and by MEKK1 requires aa 127-189 of c-myc.

C-myc availability is central for its ability to serve as a regulator of cell growth and death. Here we study the regulation of c-myc protein stability and identify domains of c-myc that are important for its stabilization in response to stress kinases activated following selective stress conditions. UV-irradiation elicited an increase in c-myc protein levels, which could be attenuated by inhibitors of stress kinases but also by actinomycin D-inhibitor of transcription. Inhibition of protein synthesis results in a noticeable decrease in c-myc levels, further pointing to the short half-life of the protein. However, in combination with tumor necrosis factor-alpha (TNF-alpha), cycloheximide efficiently increases steady-state levels of c-myc, suggesting that selective stress conditions are required to increase c-myc protein stability. Expression of MEKK1, an upstream regulator of protein kinases that has been implicated in mediating the response to diverse stress conditions, also results in an efficient increase in the half-life of c-myc protein. To map c-myc domains that are responsive to stress kinases, we monitored changes in the level of c-myc deletion mutants following MEKK1 expression. Of the seven c-myc deletion mutants analysed, the domain spanning amino acids 127-189 was found to be required for MEKK1-dependent increase in c-myc stability. In all, the present study identifies a novel domain that is important for the regulation of c-myc stability by stress kinases in response to selective stress conditions.

Antineoplastic Agents↗

PubMed2002

Human origin recognition complex large subunit is degraded by ubiquitin-mediated proteolysis after initiation of DNA replication.

Eukaryotic cells possess overlapping mechanisms to ensure that DNA replication is restricted to the S phase of the cell cycle. The levels of hOrc1p, the largest subunit of the human origin recognition complex, vary during the cell division cycle. In rapidly proliferating cells, hOrc1p is expressed and targeted to chromatin as cells exit mitosis and prereplicative complexes are formed. Later, as cyclin A accumulates and cells enter S phase, hOrc1p is ubiquitinated on chromatin and then degraded. hOrc1p destruction occurs through the proteasome and is signaled in part by the SCF(Skp2) ubiquitin-ligase complex. Other hORC subunits are stable throughout the cell cycle. The regulation of hOrc1p may be an important mechanism in maintaining the ploidy in human cells.

Animals↗