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

Combined chemical and genetic approach to inhibit proteolysis by the proteasome

Regulated protein destruction by the proteasome is crucial for the maintenance of normal cellular homeostasis. Much of our understanding of proteasome function stems from the use of drugs that inhibit its activity. Curiously, despite the importance of proteasomal proteolysis, previous studies have found that proliferation of the yeast Saccharomyces cerevisiae is relatively resistant to the effects of proteasome inhibitors such as MG132, even in the presence of mutations that increase inhibitor levels in cells. We reasoned that part of the resistance of S. cerevisiae to proteasome inhibitors stems from the fact that most proteasome inhibitors preferentially target the chymotryptic activity of the proteasome, and that the caspase-like and tryptic sites within the 20S core could compensate for proteasome function under these conditions. To test this hypothesis, we generated a strain of yeast in which the gene encoding the drug efflux pump Pdr5 is deleted, and the tryptic and caspase-like proteasome activities are inactivated by mutation. We find that this strain has dramatically increased sensitivity to the proteasome inhibitor MG132. Under these conditions, treatment of yeast with MG132 blocks progression through the cell cycle, increases the accumulation of polyubiquitylated proteins and decreases the ability to induce transcription of certain genes. These results highlight the contribution of the caspase-like and tryptic activities of the proteasome to its function, and provide a strategy to potently block proteasomal proteolysis in yeast that has practical applications

Interaction of the oncoprotein transcription factor MYC with its chromatin cofactor WDR5 is essential for tumor maintenance.

The oncoprotein transcription factor MYC is overexpressed in the majority of cancers. Key to its oncogenic activity is the ability of MYC to regulate gene expression patterns that drive and maintain the malignant state. MYC is also considered a validated anticancer target, but efforts to pharmacologically inhibit MYC have failed. The dependence of MYC on cofactors creates opportunities for therapeutic intervention, but for any cofactor this requires structural understanding of how the cofactor interacts with MYC, knowledge of the role it plays in MYC function, and demonstration that disrupting the cofactor interaction will cause existing cancers to regress. One cofactor for which structural information is available is WDR5, which interacts with MYC to facilitate its recruitment to chromatin. To explore whether disruption of the MYC-WDR5 interaction could potentially become a viable anticancer strategy, we developed a Burkitt\u27s lymphoma system that allows replacement of wild-type MYC for mutants that are defective for WDR5 binding or all known nuclear MYC functions. Using this system, we show that WDR5 recruits MYC to chromatin to control the expression of genes linked to biomass accumulation. We further show that disrupting the MYC-WDR5 interaction within the context of an existing cancer promotes rapid and comprehensive tumor regression in vivo. These observations connect WDR5 to a core tumorigenic function of MYC and establish that, if a therapeutic window can be established, MYC-WDR5 inhibitors could be developed as anticancer agents

Gal4 turnover and transcription activation

Growing evidence supports the notion that proteasome-mediated destruction of transcriptional activators can be intimately coupled to their function. Recently, Nalley et al. challenged this view by reporting that the prototypical yeast activator Gal4 does not dynamically associate with chromatin, but rather 'locks in' to stable promoter complexes that are resistant to competition. Here we present evidence that the assay used to reach this conclusion is unsuitable, and that promoter-bound, active Gal4 is indeed susceptible to competition in vivo. Our data challenge the key evidence that Nalley et al. used to reach their conclusion, and indicate that Gal4 functions in vivo within the context of dynamic promoter complexes

A Conditional Yeast E1 Mutant Blocks the Ubiquitin–Proteasome Pathway and Reveals a Role for Ubiquitin Conjugates in Targeting Rad23 to the Proteasome

E1 ubiquitin activating enzyme catalyzes the initial step in all ubiquitin-dependent processes. We report the isolation of uba1-204, a temperature-sensitive allele of the essential Saccharomyces cerevisiae E1 gene, UBA1. Uba1-204 cells exhibit dramatic inhibition of the ubiquitin–proteasome system, resulting in rapid depletion of cellular ubiquitin conjugates and stabilization of multiple substrates. We have employed the tight phenotype of this mutant to investigate the role ubiquitin conjugates play in the dynamic interaction of the UbL/UBA adaptor proteins Rad23 and Dsk2 with the proteasome. Although proteasomes purified from mutant cells are intact and proteolytically active, they are depleted of ubiquitin conjugates, Rad23, and Dsk2. Binding of Rad23 to these proteasomes in vitro is enhanced by addition of either free or substrate-linked ubiquitin chains. Moreover, association of Rad23 with proteasomes in mutant and wild-type cells is improved upon stabilizing ubiquitin conjugates with proteasome inhibitor. We propose that recognition of polyubiquitin chains by Rad23 promotes its shuttling to the proteasome in vivo

Integrative network analysis identified key genes and pathways in the progression of hepatitis C virus induced hepatocellular carcinoma

Background: Incidence of hepatitis C virus (HCV) induced hepatocellular carcinoma (HCC) has been increasing in the United States and Europe during recent years. Although HCV-associated HCC shares many pathological characteristics with other types of HCC, its molecular mechanisms of progression remain elusive. Methods: To investigate the underlying pathology, we developed a systematic approach to identify deregulated biological networks in HCC by integrating gene expression profiles with high-throughput protein-protein interaction data. We examined five stages including normal (control) liver, cirrhotic liver, dysplasia, early HCC and advanced HCC. Results: Among the five consecutive pathological stages, we identified four networks including precancerous networks (Normal-Cirrhosis and Cirrhosis-Dysplasia) and cancerous networks (Dysplasia-Early HCC, Early-Advanced HCC). We found little overlap between precancerous and cancerous networks, opposite to a substantial overlap within precancerous or cancerous networks. We further found that the hub proteins interacted with HCV proteins, suggesting direct interventions of these networks by the virus. The functional annotation of each network demonstrates a high degree of consistency with current knowledge in HCC. By assembling these functions into a module map, we could depict the stepwise biological functions that are deregulated in HCV-induced hepatocarcinogenesis. Additionally, these networks enable us to identify important genes and pathways by developmental stage, such as LCK signalling pathways in cirrhosis, MMP genes and TIMP genes in dysplastic liver, and CDC2-mediated cell cycle signalling in early and advanced HCC. CDC2 (alternative symbol CDK1), a cell cycle regulatory gene, is particularly interesting due to its topological position in temporally deregulated networks. Conclusions: Our study uncovers a temporal spectrum of functional deregulation and prioritizes key genes and pathways in the progression of HCV induced HCC. These findings present a wealth of information for further investigation

MYC regulates ribosome biogenesis and mitochondrial gene expression programs through its interaction with host cell factor-1.

The oncoprotein transcription factor MYC is a major driver of malignancy and a highly validated but challenging target for the development of anticancer therapies. Novel strategies to inhibit MYC may come from understanding the co-factors it uses to drive pro-tumorigenic gene expression programs, providing their role in MYC activity is understood. Here we interrogate how one MYC co-factor, host cell factor (HCF)-1, contributes to MYC activity in a human Burkitt lymphoma setting. We identify genes connected to mitochondrial function and ribosome biogenesis as direct MYC/HCF-1 targets and demonstrate how modulation of the MYC-HCF-1 interaction influences cell growth, metabolite profiles, global gene expression patterns, and tumor growth in vivo. This work defines HCF-1 as a critical MYC co-factor, places the MYC-HCF-1 interaction in biological context, and highlights HCF-1 as a focal point for development of novel anti-MYC therapies

Tg2576 Cortical Neurons That Express Human Ab Are Susceptible to Extracellular Aβ-Induced, K+ Efflux Dependent Neurodegeneration

Background: One of the key pathological features of AD is the formation of insoluble amyloid plaques. The major constituent of these extracellular plaques is the beta-amyloid peptide (Aβ), although Aβ is also found to accumulate intraneuronally in AD. Due to the slowly progressive nature of the disease, it is likely that neurons are exposed to sublethal concentrations of both intracellular and extracellular Aβ for extended periods of time. Results: In this study, we report that daily exposure to a sublethal concentration of Aβ1-40 (1 μM) for six days induces substantial apoptosis of cortical neurons cultured from Tg2576 mice (which express substantial but sublethal levels of intracellular Aβ). Notably, untreated Tg2576 neurons of similar age did not display any signs of apoptosis, indicating that the level of intracellular Aβ present in these neurons was not the cause of toxicity. Furthermore, wildtype neurons did not become apoptotic under the same chronic Aβ1-40 treatment. We found that this apoptosis was linked to Tg2576 neurons being unable to maintain K⁺ homeostasis following Aβ treatment. Furthermore, blocking K⁺ efflux protected Tg2576 neurons from Aβ-induced neurotoxicity. Interestingly, chronic exposure to 1 μM Aβ1-40 caused the generation of axonal swellings in Tg2576 neurons that contained dense concentrations of hyperphosphorylated tau. These were not observed in wildtype neurons under the same treatment conditions. Conclusions: Our data suggest that when neurons are chronically exposed to sublethal levels of both intra- and extra-cellular Aβ, this causes a K⁺-dependent neurodegeneration that has pathological characteristics similar to AD.9 page(s

Dephosphorylation of Nucleophosmin by PP1β Facilitates pRB Binding and Consequent E2F1-dependent DNA Repair

We report a new pathway through which PP1β signals to nucleophosmin (NPM) in response to DNA damage. UV induces dephosphorylation of NPM at multiple sites, leading to enhancement of complex formation between NPM and retinoblastoma tumor suppressor protein and the subsequent upregulation of E2F1. Consequently, such signaling pathway potentiates the cellular DNA repair capacity

Proteasome Nuclear Import Mediated by Arc3 Can Influence Efficient DNA Damage Repair and Mitosis in Schizosaccharomyces Pombe

Proteasomes must efficiently remove their substrates throughout the cells in a timely manner as many of these proteins can be toxic. This study shows that proteasomes can do so efficiently because they are highly mobile. Furthermore this study uncovers that proteasome mobility requires functional Arc3, a subunit of the Arp2/3 complex