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Background The oncoprotein MYC has the dual capacity to drive cell cycle progression or induce apoptosis, depending on the cellular context. BAG1 was previously identified as a transcriptional target of MYC that functions as a critical determinant of this cell fate decision. The BAG1 protein is expressed as multiple isoforms, each having an array of distinct biochemical functions; however, the specific effector function of BAG1 that directs MYC-dependent cell survival has not been defined. Methods In our studies the human osteosarcoma line U2OS expressing a conditional MYC-ER allele was used to induce oncogenic levels of MYC. We interrogated MYC-driven survival processes by modifying BAG1 protein expression. The function of the separate BAG1 isoforms was investigated by depleting cells of endogenous BAG1 and reintroducing the distinct isoforms. Flow cytometry and immunoblot assays were performed to analyze the effect of specific BAG1 isoforms on MYC-dependent apoptosis. These experiments were repeated to determine the role of the HSP70 chaperone complex in BAG1 survival processes. Finally, a proteomic approach was used to identify a set of specific pro-survival proteins controlled by the HSP70/BAG1 complex. Results Loss of BAG1 resulted in robust MYC-induced apoptosis. Expression of the larger isoforms of BAG1, BAG1L and BAG1M, were insufficient to rescue survival in cells with oncogenic levels of MYC. Alternatively, reintroduction of BAG1S significantly reduced the level of apoptosis. Manipulation of the BAG1S interaction with HSP70 revealed that BAG1S provides its pro-survival function by serving as a cofactor for the HSP70 chaperone complex. Via a proteomic approach we identified and classified a set of pro-survival proteins controlled by this HSP70/BAG1 chaperone complex that contribute to the BAG1 anti-apoptotic phenotype. Conclusions The small isoform of BAG1, BAG1S, in cooperation with the HSP70 chaperone complex, selectively mediates cell survival in MYC overexpressing tumor cells. We identified a set of specific pro-survival clients controlled by the HSP70/BAG1S chaperone complex. These clients define new nodes that could be therapeutically targeted to disrupt the survival of tumor cells driven by MYC activation. With MYC overexpression occurring in most human cancers, this introduces new strategies for cancer treatment. Electronic supplementary material The online version of this article (10.1186/s12885-019-5454-2) contains supplementary material, which is available to authorized users.

The SAGA coactivator complex is essential for eukaryotic transcription and comprises four distinct modules, one of which contains the ubiquitin hydrolase USP22. In yeast, the USP22 ortholog deubiquitylates H2B, resulting in Pol II Ser2 phosphorylation and subsequent transcriptional elongation. In contrast to this H2B‐associated role in transcription, we report here that human USP22 contributes to the early stages of stimulus‐responsive transcription, where USP22 is required for pre‐initiation complex (PIC) stability. Specifically, USP22 maintains long‐range enhancer–promoter contacts and controls loading of Mediator tail and general transcription factors (GTFs) onto promoters, with Mediator core recruitment being USP22‐independent. In addition, we identify Mediator tail subunits MED16 and MED24 and the Pol II subunit RBP1 as potential non‐histone substrates of USP22. Overall, these findings define a role for human SAGA within the earliest steps of transcription., Loss of human USP22 affects the stability of the pre‐initiation complex, rather than histone H2B ubiquitylation, during stimulus‐responsive transcription.

The unfolded protein response (UPR) is a stress-activated signalling pathway that regulates cell proliferation, metabolism and survival. The circadian clock coordinates metabolism and signal transduction with light/dark cycles. We explore how UPR signalling interfaces with the circadian clock. UPR activation induces a 10 h phase shift in circadian oscillations through induction of miR-211, a PERK-inducible microRNA that transiently suppresses both Bmal1 and Clock, core circadian regulators. Molecular investigation reveals that miR-211 directly regulates Bmal1 and Clock via distinct mechanisms. Suppression of Bmal1 and Clock has the anticipated impact on expression of select circadian genes, but we also find that repression of Bmal1 is essential for UPR-dependent inhibition of protein synthesis and cell adaptation to stresses that disrupt endoplasmic reticulum homeostasis. Our data demonstrate that c-Myc-dependent activation of the UPR inhibits Bmal1 in Burkitt's lymphoma, thereby suppressing both circadian oscillation and ongoing protein synthesis to facilitate tumour progression.

The posttranslational lysine acetylation of proteins is increasingly appreciated as a key regulatory mechanism in fundamental cellular process such as transcription, cytoskeleton dynamics, metabolic flux, and cell survival/death signaling. As empirical studies are undertaken to dissect the functional importance of specific acetylation events, methods for rapid detection of this modification on individual proteins, in different cellular contexts, is essential. Much like nucleosomal histones, the tumor suppressor protein p53 is acetylated on a number of distinct lysine residues, often with distinct functional consequences. We discuss here a number of technical considerations that facilitate the use of protein-specific antibodies to interrogate these key acetylation events.

Significance The deubiquitylase USP22 is frequently overexpressed in cancer and contributes to tumorigenesis by driving cell cycle progression. Current models define USP22 as functional mediator of gene regulation and chromatin modification, working within the SAGA transcriptional cofactor complex. Here we report a catalytic role for USP22 distinct from its well-characterized transcription regulatory capacity. USP22 directly stabilizes the essential G1-cyclin, CCND1, protecting it from proteasome-mediated degradation via deubiquitylation. Our findings reveal a pathway that regulates CCND1, while also raising the possibility that simulteously targeting USP22 may allow the use of less toxic doses of the new wave of cancer therapies that target the cyclin/CDK complex. Finally, these results provide a mechanistic explanation for the effects of USP22 in cancer cell cycle control., Overexpression of the deubiquitylase ubiquitin-specific peptidase 22 (USP22) is a marker of aggressive cancer phenotypes like metastasis, therapy resistance, and poor survival. Functionally, this overexpression of USP22 actively contributes to tumorigenesis, as USP22 depletion blocks cancer cell cycle progression in vitro, and inhibits tumor progression in animal models of lung, breast, bladder, ovarian, and liver cancer, among others. Current models suggest that USP22 mediates these biological effects via its role in epigenetic regulation as a subunit of the Spt-Ada-Gcn5-acetyltransferase (SAGA) transcriptional cofactor complex. Challenging the dogma, we report here a nontranscriptional role for USP22 via a direct effect on the core cell cycle machinery: that is, the deubiquitylation of the G1 cyclin D1 (CCND1). Deubiquitylation by USP22 protects CCND1 from proteasome-mediated degradation and occurs separately from the canonical phosphorylation/ubiquitylation mechanism previously shown to regulate CCND1 stability. We demonstrate that control of CCND1 is a key mechanism by which USP22 mediates its known role in cell cycle progression. Finally, USP22 and CCND1 levels correlate in patient lung and colorectal cancer samples and our preclinical studies indicate that targeting USP22 in combination with CDK inhibitors may offer an approach for treating cancer patients whose tumors exhibit elevated CCND1.

Recruitment of transcription factors (TFs) to repressed genes in euchromatin is essential to activate new transcriptional programs during cell differentiation. However, recruitment of all TFs, including pioneer factors, is impeded by condensed H3K27me3-containing chromatin. Single-cell and gene-specific analyses revealed that, during the first hours of induction of differentiation of mammalian embryonic stem cells (ESCs), accumulation of the repressive histone mark H3K27me3 is delayed after DNA replication, indicative of a decondensed chromatin structure in all regions of the replicating genome. This delay provides a critical "window of opportunity" for recruitment of lineage-specific TFs to DNA. Increasing the levels of post-replicative H3K27me3 or preventing S phase entry inhibited recruitment of new TFs to DNA and significantly blocked cell differentiation. These findings suggest that recruitment of lineage-specifying TFs occurs soon after replication and is facilitated by a decondensed chromatin structure. This insight may explain the developmental plasticity of stem cells and facilitate their exploitation for therapeutic purposes.

Eukaryotes control nearly every cellular process in part by modulating the transcription of genes encoded by their nuclear genome. However, these cells are faced with the added complexity of possessing a second genome, within the mitochondria, which encodes critical components of several essential processes, including energy metabolism and macromolecule biosynthesis. As these cellular processes require gene products encoded by both genomes, cells have adopted strategies for linking mitochondrial gene expression to nuclear gene expression and other dynamic cellular events. Here we discuss examples of several mechanisms that have been identified, by which eukaryotic cells link extramitochondrial signals to dynamic alterations in mitochondrial transcription. This article is part of a Special Issue entitled: Mitochondrial Gene Expression.

SAGA is a 2 MDa multi-subunit complex containing two assembly modules (TAF and SPT) and two enzymatic modules (HAT and DUB) which exhibit acetyltransferase activity via GCN5 and deubiquitylase activity via USP22. Multiple studies in yeast, flies, mice, and human systems have shown the SAGA DUB module to participate in a concerted series of biochemical steps during transcription, removing monoubiquitin from histone H2B to mediate Pol II phosphorylation and elongation downstream. High USP22 expression has been reported as a biomarker of late-stage aggressive tumors across multiple cancer types, and our previous work demonstrated that USP22 is necessary for this phenotype, as depletion of USP22 severely impairs tumor cell growth and proliferation. We utilized the ER stress response as an activator-driven transcriptional program to clarify a number of details surrounding the role of hSAGA in transcriptional regulation. ChIP-seq of endogenous human USP22 reveals a tightly regulated USP22-containing DUB module that occupies the promoters of only a handful of activated genes, despite hundreds of ER stress response genes altered within 2 hours of induction. In contrast, promoter occupancy of the HAT module subunit GCN5 is widespread and does not require activation of a transcriptional program. USP22 depletion in this system elicits significant effects on GTF recruitment and assembly of the PIC. Notably, USP22 occupancy appears entirely exclusive of chromatin-associated Ub-H2B, and shRNA-mediated depletion of USP22 fails to elicit an increase in either global or local Ub-H2B levels. These data imply that removal of monoubiquitin from histone H2B is not regulated by USP22 in higher eukaryotes. Given that several non-histone substrates of USP22 have been characterized, we performed a proteome-wide screen to measure changes in endogenous ubiquitylation levels following USP22 depletion from cells. Our data indicate that USP22-mediated deubiquitylation of Mediator tail subunits as well as the large subunit of the Pol II holoenzyme may be significant steps in efficient transcriptional activation. ChIP of core and tail Mediator subunits reveals that USP22 depletion impairs their recruitment to USP22-bound ER stress response gene promoters. Given the specialized role of USP22 in activator-driven transcriptional events and its prominence as a driver of tumor progression, we predict USP22 to be a suitable enzymatic substrate for targeted therapy in cancer. Our current efforts include high-throughput screening of compound libraries against USP22 enzymatic activity in vitro as a preliminary drug discovery campaign. Citation Format: Sabrina Butt, Timothy J. Stanek, Victoria J. Gennaro, Chris McNair, Kristen L. Pauley, Karen Knudsen, Steven B. McMahon. Divergent mechanisms of transcriptional regulation by SAGA member and epigenetic modifier USP22 [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr LB-A29.

The tumor suppressor p53 induces apo pto sis by altering the transcription of pro-apo pto tic targets in the nucleus and by a direct, nontranscriptional role at the mitochondria. Although the post-translational modifications regulating nuclear apo pto tic functions of p53 have been thoroughly characterized, little is known of how transcription-independent functions are controlled. We and others identified acetylation of the p53 DNA binding domain at lysine 120 as a critical event in apo pto sis induction. Although initial studies showed that Lys-120 acetylation plays a role in p53 function in the nucleus, we report here a role for Lys-120 acetylation in transcription-independent apo pto sis. We demonstrate that the Lys-120-acetylated isoform of p53 is enriched at mitochondria. The acetylation of Lys-120 does not appear to regulate the ability of p53 to interact with the pro-apo pto tic proteins BCL-XL and BAK. However, displacement of the inhibitory MCL-1 protein from BAK is compromised when Lys-120 acetylation is blocked. Functional studies show that mutation of Lys-120 to a nonacetylated residue, as occurs in human cancer, inhibits transcription-independent apo pto sis, and enforced acetylation of Lys-120 enhances transcription-independent apo pto sis. These data support a model whereby Lys-120 acetylation contributes to both the transcription-dependent and -independent apo pto tic pathways induced by p53.

Mammalian cells fuel their growth and proliferation through the catabolism of two main substrates: glucose and glutamine. Most of the remaining metabolites taken up by proliferating cells are not catabolized, but instead are used as building blocks during anabolic macromolecular synthesis. Investigations of phosphoinositol 3-kinase (PI3K) and its downstream effector AKT have confirmed that these oncogenes play a direct role in stimulating glucose uptake and metabolism, rendering the transformed cell addicted to glucose for the maintenance of survival. In contrast, less is known about the regulation of glutamine uptake and metabolism. Here, we report that the transcriptional regulatory properties of the oncogene Myc coordinate the expression of genes necessary for cells to engage in glutamine catabolism that exceeds the cellular requirement for protein and nucleotide biosynthesis. A consequence of this Myc-dependent glutaminolysis is the reprogramming of mitochondrial metabolism to depend on glutamine catabolism to sustain cellular viability and TCA cycle anapleurosis. The ability of Myc-expressing cells to engage in glutaminolysis does not depend on concomitant activation of PI3K or AKT. The stimulation of mitochondrial glutamine metabolism resulted in reduced glucose carbon entering the TCA cycle and a decreased contribution of glucose to the mitochondrial-dependent synthesis of phospholipids. These data suggest that oncogenic levels of Myc induce a transcriptional program that promotes glutaminolysis and triggers cellular addiction to glutamine as a bioenergetic substrate.

Polycomb genes encode critical regulators of both normal stem cells and cancer stem cells. A gene signature that includes Polycomb genes and additional genes coregulated with Polycomb genes was recently identified. The expression of this signature has been reported to identify tumors with the cancer stem cell phenotypes of aggressive growth, metastasis, and therapy resistance. Most members of this 11 gene signature encode proteins with well-defined roles in human cancer. However, the function of the signature member USP22 remains unknown. We report that USP22 is a previously uncharacterized subunit of the human SAGA transcriptional cofactor complex. Within SAGA, USP22 deubiquitylates histone H2B. Furthermore, USP22 is recruited to specific genes by activators such as the Myc oncoprotein, where it is required for transcription. In support of a functional role within the Polycomb/cancer stem cell signature, USP22 is required for appropriate progression through the cell cycle.

The ability of p53 to induce apoptosis plays an important role in tumor suppression. Here, we describe a previously unknown posttranslational modification of the DNA-binding domain of p53. This modification, acetylation of lysine 120 (K120), occurs rapidly after DNA damage and is catalyzed by the MYST family acetyltransferases hMOF and TIP60. Mutation of K120 to arginine, as occurs in human cancer, debilitates K120 acetylation and diminishes p53-mediated apoptosis without affecting cell-cycle arrest. The K120R mutation selectively blocks the transcription of proapoptotic target genes such as BAX and PUMA while the nonapoptotic targets p21 and hMDM2 remain unaffected. Consistent with this, depletion of hMOF and/or TIP60 inhibits the ability of p53 to activate BAX and PUMA transcription. Furthermore, the acetyllysine 120 (acetyl-K120) form of p53 specifically accumulates at proapoptotic target genes. These data suggest that K120 acetylation may help distinguish the cell-cycle arrest and apoptotic functions of p53.

The MYC oncoprotein is among the most potent regulators of cell cycle progression and malignant transformation in human cells. Current models suggest that much of MYC's role in these processes is related to its ability to regulate the transcription of downstream target genes that encode the ultimate effector proteins. In addition to its carboxy-terminal DNA binding and dimerization domains, an enigmatic motif in the amino terminus termed MbII is required for all of MYC's biological activities. In spite of historical observations demonstrating the absolute requirement for MbII in these biological functions, clues implicating this domain in target gene transcription have only recently appeared. Based on this emerging link between MbII and transcriptional activation, we hypothesized that the identification of individual MYC targets whose transactivation requires MbII would help define the essential downstream effectors of MYC in transformation and cell cycle progression. In hopes of directly identifying new MbII-dependent MYC target genes, an expression profiling screen was conducted. This screen resulted in our identification of ten novel downstream targets of MYC. As a proof of principle, we recently demonstrated using RNAi-mediated depletion that one of these targets, the metastasis regulator MTA1, is absolutely required for MYC mediated transformation. Here we report the identity of these previously uncharacterized MYC targets and discuss their potential roles in MYC function. In addition, we attempt to reconcile the historical and contemporary evidence linking MbII to transcriptional activation.

The c-MYC oncoprotein functions as a sequence-specific transcription factor. The ability of c-MYC to activate transcription relies in part on the recruitment of cofactor complexes containing the histone acetyltransferases mammalian GCN5 (mGCN5)/PCAF and TIP60. In addition to acetylating histones, these enzymes have been shown to acetylate other proteins involved in transcription, including sequence-specific transcription factors. This study was initiated in order to determine whether c-MYC is a direct substrate of mGCN5 and TIP60. We report here that mGCN5/PCAF and TIP60 acetylate c-MYC in vivo. By using nanoelectrospray tandem mass spectrometry to examine c-MYC purified from human cells, the major mGCN5-induced acetylation sites have been mapped. Acetylation of c-MYC by either mGCN5/PCAF or TIP60 results in a dramatic increase in protein stability. The data reported here suggest a conserved mechanism by which acetyltransferases regulate c-MYC function by altering its rate of degradation.

MYC is overexpressed by many human tumour types and has been shown to regulate cell functions that are required for tumorigenesis. It is not clear, however, which of its target genes mediate these effects. A series of recent studies have indicated that this could be a result of the fact that MYC binds and regulates up to 15% of all genes. Does MYC function as a widespread regulator of transcription or as a classical transcription factor that regulates a limited number of downstream targets?

The p53 tumor suppressor regulates the cellular response to genetic damage through its function as a sequence-specific transcription factor. Among the most well-characterized transcriptional targets of p53 is the mdm2 oncogene. Activation of mdm2 is critical in the p53 pathway because the mdm2 protein marks p53 for proteosome-mediated degradation, thereby providing a negative-feedback loop. Here we show that the ATM-related TRRAP protein functionally cooperates with p53 to activate mdm2 transcription. TRRAP is a component of several multiprotein acetyltransferase complexes implicated in both transcriptional regulation and DNA repair. In support of a role for these complexes in mdm2 expression, we show that transactivation of the mdm2 gene is augmented by pharmacological inhibition of cellular deacetylases. In vitro analysis demonstrates that p53 directly binds to a TRRAP domain previously shown to be an activator docking site. Furthermore, transfection of cells with antisense TRRAP blocks p53-dependent transcription of mdm2. Finally, using chromatin immunoprecipitation, we demonstrate direct p53-dependent recruitment of TRRAP to the mdm2 promoter, followed by increased histone acetylation. These findings suggest a model in which p53 directly recruits a TRRAP/acetyltransferase complex to the mdm2 gene to activate transcription. In addition, this study defines a novel biochemical mechanism utilized by the p53 tumor suppressor to regulate gene expression.

Nuclear receptors (NRs) regulate transcription in a ligand-dependent way through two types of coactivator complexes: the p160/CBP histone acetyl transferase (HAT) complex and the DRIP/TRAP/SMCC complex without HAT activity. Here we identified a large human (h) coactivator complex necessary for the estrogen receptor alpha (ERalpha) transactivation. This complex contains the GCN5 HAT, the c-Myc interacting protein TRRAP/PAF400, TAF(II)30, and other subunits. Similarly to known TFTC (TBP-free TAF(II)-containing)-type HAT complexes (hTFTC, hPCAF, and hSTAGA), TRRP directly interacted with liganded ER alpha, or other NRs. ER alpha transactivation was enhanced by the purified complex in vitro. Antisense TRRAP RNA inhibited estrogen-dependent cell growth of breast cancer cells. Thus, the isolated TFTC-type HAT complex acts as a third class of coactivator complex for NR function.

(Molecular Cell 9, 553–562; March 2002)Recently, we were made aware that images in Figures 1B, 2F, and 3E were inappropriately manipulated such that they did not reflect the actual experimental data they claimed to represent. The experiments and figure preparation were done in the nuclear signaling laboratory in the IMCB. Because of the data handling issues, we wish to retract this paper and to sincerely apologize to the scientific community for any potential harm we may have caused.

The E2F family of transcription factors regulates the temporal transcription of genes involved in cell cycle progression and DNA synthesis. E2F transactivation is antagonized by retinoblastoma protein (pRb), which recruits chromatin-remodeling proteins such as histone deacetylases and SWI.SNF complexes to the promoter to repress transcription. We hypothesized that E2F proteins must reverse the pRb-imposed chromatin structure to stimulate transcription. If this is true, E2F proteins should recruit proteins capable of histone acetylation. Here we map the E2F-4 transactivation domain and show that E2F-1 and E2F-4 transactivation domains bind the acetyltransferase GCN5 and cofactor TRRAP in vivo. TRRAP and GCN5 co-expression stimulated E2F-mediated transactivation, and c-Myc repressed E2F transactivation dependent on an intact TRRAP/GCN5 binding motif. The transactivation domain of E2F-4 recruited proteins with significant histone acetyltransferase activity in vivo, and this activity required catalytically active GCN5. E2F-4 proteins with subtle mutations in the transactivation domain exhibited a positive correlation among transcriptional activation and GCN5 and TRRAP binding capacity and associated acetyltransferase activity. We conclude that E2F stimulates transcription by recruiting acetyltransferase activity and the essential cofactors GCN5 and TRRAP. These results provide a mechanism for E2F transcription factors to overcome pRb-mediated dominant repression of transcription.

The ATM-related TRRAP protein is a component of several different histone acetyltransferase (HAT) complexes but lacks the kinase activity characteristic of other ATM family members. We identified a novel function for this evolutionarily conserved domain in its requirement for the assembly of a functional HAT complex. Ectopic expression of TRRAP protein with a mutation in the ATM-related domain inhibits Myc-mediated oncogenic transformation. The Myc-binding region of TRRAP maps to a separable domain, and ectopic expression of this domain inhibits cell growth. These findings demonstrate that the ATM-related domain of TRRAP forms a structural core for the assembly and recruitment of HAT complexes by transcriptional activators.

The c-Myc transactivation domain was used to affinity purify tightly associated nuclear proteins. Two of these proteins were identified as TIP49 and a novel related protein called TIP48, both of which are highly conserved in evolution and contain ATPase/helicase motifs. TIP49 and TIP48 are complexed with c-Myc in vivo, and binding is dependent on a c-Myc domain essential for oncogenic activity. A missense mutation in the TIP49 ATPase motif acts as a dominant inhibitor of c-Myc oncogenic activity but does not inhibit normal cell growth, indicating that functional TIP49 protein is an essential mediator of c-Myc oncogenic transformation. The TIP49 and TIP48 ATPase/helicase proteins represent a novel class of cofactors recruited by transcriptional activation domains that function in diverse pathways.

Initial studies of the mammalian hSAGA transcriptional coactivator complex identified the acetyltransferase hGCN5/PCAF as the only known enzymatic subunit. Recently we and others demonstrated that the ubiquitin hydrolase USP22 comprises a second enzymatic subunit of hSAGA, which is required for activator-driven transcription. USP22 is expressed with polycomb ubiquitin ligases in an 11 gene signature that defines therapy-resistant tumors. At the biochemical level, these Polycomb proteins function as global transcriptional repressors by catalyzing the ubiquitylation of histone H2A. In yeast, the USP22 homolog functions as a transcriptional coactivator by removing ubiquitin from a distinct core histones, H2B. Given that USP22 is expressed in cancer as part of an 11 gene signature that includes transcriptional repressors which ubiquitylate H2A, it seemed possible that USP22 might activate transcription in part via the deubiquitylation of this same substrate. As reported here, biochemical analysis of the substrate specificity of USP22 reveals that it deubiquitylates histone H2A in addition to H2B. This finding supports a model in which the H2A ubiquitin hydrolase USP22 is coordinately expressed with Polycomb H2A ubiquitin ligases in order that the transcription of certain critical transforming genes be maintained in the face of the global repression mediated by Polycomb.

The yeast Ada and TBP class of Spt proteins interact in multiple complexes that are required for transcriptional regulation. We have identified Tra1p as a component of these complexes through tandem mass spectrometry analysis of proteins that associate with Ngg1p/Ada3p. TRA1 is an essential gene and encodes a 3744-amino acid protein that is a member of a group of proteins including the catalytic subunit of DNA-dependent protein kinase, ATM and TRRAP, with carboxyl-terminal regions related to phosphatidylinositol 3-kinases. The interaction between Tra1p and Ada/Spt components was verified by the reciprocal coimmunoprecipitation of Ada2p and Tra1p from whole cell extracts in one or more complexes containing Spt7p. Tra1p cofractionated with Ngg1p and Spt7p through consecutive chromatography on Mono Q, DNA-cellulose, and Superose 6 columns. Binding of Tra1p to DNA-cellulose required Ada components. The association of Tra1p with two Ada.Spt complexes was suggested by its cofractionation with Ngg1p and Spt7p in two peaks on the Mono Q column. In the absence of Ada2p, the elution profile of Tra1p shifted to a distinct peak. Despite the similarity of Tra1p to a group of putative protein kinases, we have not detected protein kinase activity within immunoprecipitates of Tra1p or the Ada.Spt complexes.

Pluripotent embryonic stem cells (ESCs) undergo self-renewal until stimulated to differentiate along specific lineage pathways. Many of the transcriptional networks that drive reprogramming of a self-renewing ESC to a differentiating cell have been identified. However, fundamental questions remain unanswered about the epigenetic programs that control these changes in gene expression. Here we report that the histone ubiquitin hydrolase ubiquitin-specific protease 22 (USP22) is a critical epigenetic modifier that controls this transition from self-renewal to differentiation. USP22 is induced as ESCs differentiate and is necessary for differentiation into all three germ layers. We further report that USP22 is a transcriptional repressor of the locus encoding the core pluripotency factor sex-determining region Y-box 2 (SOX2) in ESCs, and this repression is required for efficient differentiation. USP22 occupies the Sox2 promoter and hydrolyzes monoubiquitin from ubiquitylated histone H2B and blocks transcription of the Sox2 locus. Our study reveals an epigenetic mechanism that represses the core pluripotency transcriptional network in ESCs, allowing ESCs to transition from a state of self-renewal into lineage-specific differentiation programs. Background: Ubiquitin-specific protease 22 (USP22) is a deubiquitylating enzyme with established biological functions in cancer cells. Results: USP22 drives differentiation of embryonic stem cells (ESCs) and represses sex-determining region Y-box 2 (SOX2) transcription. Conclusion: USP22 is induced during ESC differentiation to repress SOX2 transcription. Significance: Understanding the epigenetic programs that control changes in gene expression during the transition from self-renewal to differentiation.

Although many functions and targets have been attributed to the histone and protein deacetylase SIRT1, a comprehensive analysis of SIRT1 binding proteins yielding a high-confidence interaction map has not been established. Using a comparative statistical analysis of binding partners, we have assembled a high-confidence SIRT1 interactome. Employing this method, we identified the deubiquitinating enzyme ubiquitin-specific protease 22 (USP22), a component of the deubiquitinating module (DUBm) of the SAGA transcriptional coactivating complex, as a SIRT1-interacting partner. We found that this interaction is highly specific, requires the ZnF-UBP domain of USP22, and is disrupted by the inactivating H363Y mutation within SIRT1. Moreover, we show that USP22 is acetylated on multiple lysine residues and that alteration of a single lysine (K129) within the ZnF-UBP domain is sufficient to alter interaction of the DUBm with the core SAGA complex. Furthermore, USP22-mediated recruitment of SIRT1 activity promotes the deacetylation of individual SAGA complex components. Our results indicate an important role of SIRT1-mediated deacetylation in regulating the formation of DUBm subcomplexes within the larger SAGA complex.

The induction of immediate early genes in cells of the immune system is critical to determining the ultimate outcome of exposure to antigen. The importance of many of these genes relates to the role their transcription factor products play in dictating patterns of expression of downstream, function-related genes. Evidence from several systems indicates that the immediate early gene, egr-1 may be of particular importance in the immune system. Recently, the egr-1 promoter has been shown to be highly responsive to the diverse biochemical signals generated by antigen and cytokines in cells of the immune system. Furthermore, an important role for egr-1 in determining the differentiation pathway of myeloid cell precursors has been recently elaborated. Finally, potential targets of regulation by the zinc-finger transcription factor encoded by egr-1 include the interleukin-2, CD44, ICAM-1, and tumor necrosis factor genes. The role of egr-1 in regulation of the immune response will be discussed in the context of these recent studies.

Induction of the primary response gene egr-1 occurs rapidly following antigen receptor cross-linking in B lymphocytes. Antisense studies have demonstrated that this induction is necessary for their subsequent activation to this signal. The present study examines the molecular mechanism whereby the receptor-generated signals interact with the egr-1 promoter to elicit transcription. Deletion mapping and point mutations have indicated that two of the five serum response elements (SREs) in the egr-1 promoter can mediate induction. Of the two critical SREs, both are capable of mediating maximal induction even in the absence of the other SRE. Our results also indicate that adjacent Ets motifs are necessary for induction. Like the c-fos SRE, the egr-1 SRE/Ets sites are occupied by a multiprotein (ternary) complex containing a homodimer of serum response factor and an unidentified member of the Ets family of transcription factors. The identification of a ternary complex-dependent mechanism of egr-1 induction, along with selective utilization of SREs in B lymphocytes, suggests that a complicated array of signaling cascades interacts with unique combinations of regulatory elements in the egr-1 promoter in different cell types.

Aberrant MYC expression is a common oncogenic event in human cancer. Paradoxically, MYC can either drive cell cycle progression or induce apoptosis. The latent ability of MYC to induce apoptosis has been termed "intrinsic tumor suppressor activity," and reactivating this apoptotic function in tumors is widely considered a valuable therapeutic goal. As a transcription factor, MYC controls the expression of many downstream targets, and for the majority of these, it remains unclear whether or not they play direct roles in MYC function. To identify the subset of genes specifically required for biological activity, we conducted a screen for functionally important MYC targets and identified BAG1, which encodes a prosurvival chaperone protein. Expression of BAG1 is regulated by MYC in both a mouse model of breast cancer and transformed human cells. Remarkably, BAG1 induction is essential for protecting cells from MYC-induced apoptosis. Ultimately, the synthetic lethality we have identified between MYC overexpression and BAG1 inhibition establishes a new pathway that might be exploited to reactivate the latent apoptotic potential of MYC as a cancer therapy.

In unstressed cells, the p53 tumor suppressor is highly unstable. DNA damage and other forms of cellular stress rapidly stabilize and activate p53. This process is regulated by a complex array of post-translational modifications that are dynamically deposited onto p53. Recent studies show that these modifications orchestrate p53-mediated processes such as cell cycle arrest and apoptosis. Cancer cells carry inherent genetic damage, but avoid arrest and apoptosis by inactivating p53. Defining the enzymatic machinery that regulates the stress-induced modification of p53 at single-residue resolution is critical to our understanding of the biochemical mechanisms that control this critical tumor suppressor. Specifically, acetylation of p53 at lysine 120, a DNA-binding domain residue mutated in human cancer, is essential for triggering apoptosis. Given the oncogenic properties of deacetylases and the success of deacetylase inhibitors as anticancer agents, we investigated the regulation of Lys(120) deacetylation using pharmacologic and genetic approaches. This analysis revealed that histone deacetylase 1 is predominantly responsible for the deacetylation of Lys(120). Furthermore, treatment with the clinical-grade histone deacetylase inhibitor entinostat enhances Lys(120) acetylation, an event that is mechanistically linked to its apoptotic effect. These data expand our understanding of the mechanisms controlling p53 function and suggest that regulation of p53 modification status at single-residue resolution by targeted therapeutics can selectively alter p53 pathway function. This knowledge may impact the rational application of deacetylase inhibitors in the treatment of human cancer.

MicroRNA145 (miR145), a tumor suppressor miR, has been reported to inhibit growth of human cancer cells, to induce differentiation and to cause apoptosis, all conditions that result in growth arrest. In order to clarify the functional effects of miR145, we have investigated its expression in diverse conditions and different cell lines. Our results show that miR145 levels definitely increase in differentiating cells and also in growth-arrested cells, even in the absence of differentiation. Increased expression during differentiation sometimes occurs as a late event, suggesting that miR145 could be required either early or late during the differentiation process.

Activation of p53 by DNA damage results in either cell-cycle arrest, allowing DNA repair and cell survival, or induction of apoptosis. As these opposite outcomes are both mediated by p53 stabilization, additional mechanisms to determine this decision must exist. Here, we show that glycogen synthase kinase-3 (GSK-3) is required for the p53-mediated induction of the proapoptotic BH3 only-protein PUMA, an essential mediator of p53-induced apoptosis. Inhibition of GSK-3 protected from cell death induced by DNA damage and promoted increased long-term cell survival. We demonstrate that GSK-3 phosphorylates serine 86 of the p53-acetyltransferase Tip60. A Tip60(S86A) mutant was less active to induce p53 K120 acetylation, histone 4 acetylation, and expression of PUMA. Our data suggest that GSK-3 mediated Tip60S86 phosphorylation provides a link between PI3K signaling and the choice for or against apoptosis induction by p53.

Protein phosphorylation is dynamically regulated in eukaryotic cells via modulation of the enzymatic activity of kinases and phosphatases. Like phosphorylation, acetylation has emerged as a critical regulatory protein modification that is dynamically altered in response to diverse cellular cues. Moreover, acetyltransferases and deacetylases are tightly linked to cellular signaling pathways. Recent studies provide clues about the mechanisms utilized to regulate acetyltransferases and deacetylases. The therapeutic value of deacetylase inhibitors suggests that understanding acetylation pathways will directly impact our ability to rationally target these enzymes in patients. Recently discovered mechanisms which directly regulate the catalytic activity of acetyltransferases and deacetylases provide exciting new insights about these enzymes.

The p53 tumor suppressor continues to hold distinction as the most frequently mutated gene in human cancer. The ability of p53 to induce programmed cell death, or apoptosis, of cells exposed to environmental or oncogenic stress constitutes a major pathway whereby p53 exerts its tumor suppressor function. In the past decade, we have discovered that p53 is not alone in its mission to destroy damaged or aberrantly proliferating cells: it has two homologs, p63 and p73, that in various cellular contexts and stresses contribute to this process. In this review, the mechanisms whereby p53, and in some cases p63 and p73, induce apoptosis are discussed. Other reviews have focused more extensively on the contribution of individual p53-regulated genes to apoptosis induction by this protein, whereas in this review, we focus more on those factors that mediate the decision between growth arrest and apoptosis by p53, p63 and p73, and on the post-translational modifications and protein-protein interactions that influence this decision.

Stabilization of the p53 tumor suppressor is a critical event in the response to various forms of cellular stress. Two distinct signaling pathways are thought to lead to this stabilization, depending on the type of cellular stress encountered. Genotoxic stress, such as chromosomal breaks or lesions induced by chemotherapeutic agents, result in the activation of the well-characterized DNA damage response pathway. Conversely, cellular stress that results from the aberrant activation of oncogenes triggers p53 stabilization via the induction of the p19ARF pathway. While activation of the DNA damage pathway ultimately causes a complex array of post-translational modifications on p53, few if any modifications have been demonstrated to occur following activation of the p19ARF pathway. We and others have recently identified a novel modification on p53, acetylation of lysine 120 within the DNA binding domain. This acetylation event is eliminated by tumor-derived mutations in p53 and its presence is required for the tumor suppressor apoptotic function of p53. We demonstrate here that both the DNA damage response pathway and the p19ARF/oncogene stress pathway induce the acetylation of p53 at lysine 120.

The c-MYC oncoprotein is among the most potent transforming agents in human cells. Ironically, c-MYC is also capable of inducing massive apoptosis under certain conditions. A clear understanding of the distinct pathways activated by c-MYC during apoptosis induction and transformation is crucial to the design of therapeutic strategies aimed at selectively reactivating the apoptotic potential of c-MYC in cancer cells. We recently demonstrated that apoptosis induction in primary human cells strictly requires that c-MYC bind and inactivate the transcription factor MIZ-1. This presumably blocked the ability of MIZ-1 to activate the transcription of an unidentified pro-survival gene. Here we report that MIZ-1 activates the transcription of BCL2. More importantly, inhibition of the MIZ-1/BCL2 signal is an essential event during the apoptotic response. Furthermore, targeting BCL2 with short hairpin RNA or small molecule inhibitors restores the apoptotic potential of a c-MYC mutant that is defective for MIZ-1 inhibition. These observations suggest that repression of BCL2 transcription is the single essential consequence of targeting the MIZ-1 pathway during apoptosis induction. These data define a genetic pathway that helps to explain historical observations documenting cooperation between c-MYC and BCL2 overexpression in human cancer.

The c-Myc oncoprotein plays a central role in human cancer via its ability to either activate or repress the transcription of essential downstream targets. For many of the repressed target genes, down-regulation by c-Myc relies on its ability to bind and inactivate the transcription factor Miz-1. Although Miz-1 inactivation is suspected to be essential for at least some of the biological activities of c-Myc, it has been difficult to demonstrate this requirement experimentally. Using a combination of short hairpin RNA-mediated knockdown and a previously characterized mutant of c-Myc that is defective for Miz-1 inactivation, we examined whether this inactivation is critical for three of the most central biological functions of c-Myc, cell cycle progression, transformation, and apoptosis. The results of this analysis demonstrated that in the in vitro assays utilized here, Miz-1 inactivation is dispensable for c-Myc-induced cell cycle progression and transformation. In marked contrast, the ability of c-Myc to induce apoptosis in primary diploid human fibroblasts in response to growth factor withdrawal is entirely dependent on its ability to inactivate Miz-1. These data have a significant impact on our understanding of the biochemical mechanisms dictating how c-Myc mediates opposing biological functions, such as transformation and apoptosis, and demonstrate the first requirement for Miz-1 inactivation in any of the biological functions of c-Myc.

In this issue, Trinidad et al. (2013) show that CCT/TRiC is a chaperone required for p53 folding, thus providing another layer of regulation of p53 function, with implications for cancer therapeutics targeting the p53 pathway.

We demonstrate that transformation-transactivation domain-associated protein (TRRAP) binding and the recruitment of histone H3 and H4 acetyltransferase activities are required for the transactivation of a silent telomerase reverse transcriptase (TERT) gene in exponentially growing human fibroblasts by c-Myc or N-Myc protein. However, recruitment of TRRAP by c- or N-Myc is dispensable for the partial induction of several basally expressed genes in exponentially growing primary and immortalized fibroblasts. Furthermore, recruitment of TRRAP is required for c-Myc- or N-Myc-mediated oncogenic transformation but not for the partial restoration of the growth defect in myc-null fibroblasts. A segment of the adenovirus E1A protein fused to a transformation-defective N-Myc protein carrying a small deletion in the transactivation domain specifically restores interaction with TRRAP, activates the silent TERT gene, induces acetylation of histones H3 and H4 at the TERT promoter, and transforms primary cells. Accordingly, wild-type L-Myc is much less efficient in TRRAP binding, activation of the silent TERT gene, and transformation of primary fibroblasts. Nevertheless, L-Myc is a potent activator of several basally expressed genes and can fully restore the growth defect of myc-null cells. These results suggest a differential requirement for TRRAP for several Myc-mediated activities.

ING1 proteins are nuclear, growth inhibitory, and regulate apoptosis in different experimental systems. Here we show that similar to their yeast homologs, human ING1 proteins interact with proteins associated with histone acetyltransferase (HAT) activity, such as TRRAP, PCAF, CBP, and p300. Human ING1 immunocomplexes contain HAT activity, and overexpression of p33ING1b, but not of p47ING1a, induces hyperacetylation of histones H3 and H4, in vitro and in vivo at the single cell level. p47ING1a inhibits histone acetylation in vitro and in vivo and binds the histone deacetylase HDAC1. Finally, we present evidence indicating that p33ING1b affects the degree of physical association between proliferating cell nuclear antigen (PCNA) and p300, an association that has been proposed to link DNA repair to chromatin remodeling. Together with the finding that human ING1 proteins bind PCNA in a DNA damage-dependent manner, these data suggest that ING1 proteins provide a direct linkage between DNA repair, apoptosis, and chromatin remodeling via multiple HAT·ING1·PCNA protein complexes.

MYC expression is tightly correlated with cell-cycle progression in normal tissues, whereas unchecked MYC expression is among the most prominent hallmarks of the hyperproliferation associated with most forms of cancer. At first glance it might seem counterintuitive that MYC is also among the most robust agents of programmed cell death (apoptosis) in mammalian cells. However it is clearly beneficial for a multicellular organism to have a mechanism for triggering death in cells that express potentially oncogenic levels of MYC. Decades of intense study have begun to provide an understanding of the mechanisms that regulate MYC's seemingly split personality. Key features of MYC-induced apoptosis will be discussed here along with examples of how our understanding of this pathway might be exploited for the therapeutic benefit of cancer patients.

Dynamic lysine acetylation regulates proteins involved in diverse cellular processes, with individual enzymes often acetylating multiple substrates. Here, Li et al. (2009) show that the substrate specificity of hMOF/MYST1/KAT8 is controlled by differential interaction with two mutually exclusive partners.

The c-Myc protein functions as a transcription factor to facilitate oncogenic transformation; however, the biochemical and genetic pathways leading to transformation remain undefined. We demonstrate here that the recently described c-Myc cofactor TRRAP recruits histone acetylase activity, which is catalyzed by the human GCN5 protein. Since c-Myc function is inhibited by recruitment of histone deacetylase activity through Mad family proteins, these opposing biochemical activities are likely to be responsible for the antagonistic biological effects of c-Myc and Mad on target genes and ultimately on cellular transformation.

The MYC oncoprotein drives cell cycle progression in both normal cells and human tumors. In fact, MYC overexpression provides such a proliferative advantage that a remarkable percentage of human tumors exhibit elevated MYC levels.1 Hyper-proliferation requires that cells ramp up biosynthetic pathways for protein and lipid production, energy metabolism and DNA replication. Evidence for MYC controlling multiple points in the pathways leading to protein synthesis and energy metabolism has come from a variety of sources in recent years.2 The study by Mannava published in this issue, along with similar findings from the Dang group, now firmly links MYC to the production of nucleotides required as building blocks for DNA synthesis. After finding that human melanoma cell lines required MYC for optimal proliferation, Mannava et al. identified MYC target genes that might explain this requirement by using expression profiling. Among the targets they identified were genes encoding enzymes that regulate several rate-limiting steps in nucleotide biosynthesis. At least three of these enzymes, thymidylate synthase (TS), inosine monophosphate dehydrogenase 2 (IMPDH2) and phosphoribosyl pyrophosphate synthetase 2 (PRPS2), are encoded by direct MYC target genes. As predicted by this set of observations, MYC depletion resulted in decreased cellular dNTP levels. Using the gold standard for epistatis experiments, Mannava ultimately show that ectopic expression of TS, IMPDH2 and PRPS2 partially rescues the proliferative defect caused by MYC depletion in melanoma cells. These observations are strengthened by the independent findings of the Dang group. Dang et al. also identified IMPDH2 (and IMPDH1) as a MYC target using an unbiased screen for MYC binding sites in the genome.3 These studies were conducted in different cell types than the melanomas used by Manneva et al. suggesting that the link between MYC and nucleotide biosynthesis holds true across very distinct lineages. More broadly, the Dang group, unlike Manneva et al. found that genes involved in nucleotide metabolism were statistically overrepresented among their list of MYC targets. These two exciting studies appear against the backdrop of previous work linking MYC to this pathway. A number of years ago, similar excitement was generated by evidence that MYC controlled transcription of CAD,4 the gene encoding the first three enzymes required for pyrimidine biosynthesis. While CAD is clearly regulated by MYC in many settings, subsequent studies showed that its levels were not linked to MYC in Burkitt’s lymphoma.5 Thus, these new studies linking MYC to nucleotide biosynthesis renew optimism that we might now have the mechanistic explanation for how MYC provides one of the essential sets of building blocks necessary as it pushes cell proliferation. Relevant to these findings, a study from the Dalla-Favera group last year showed a role for MYC in the function of origins of DNA replication.6 Coupled with a role for MYC in generating the nucleotides necessary for DNA synthesis, this finding implicates MYC in controlling multiple nodes in the pathway leading to genome duplication. This seems to be MYC’s favorite modus operandi, as it often regulates critical pathways by regulating a number of important steps. The true litmus test for the importance of these findings will be when studies are conducted examining levels of these proteins in more human tumor types driven by MYC overexpression (eg. Burkitt’s lymphoma, breast cancer, colon cancer). Certainly drugs like mycophenolic acid, which inhibit nucleotide biosynthesis, have been effective chemotherapeutics for cancer and other hyper-proliferative diseases,7 and the Dang group show that mycophenolic acid treatment blocks MYC induced proliferation.3 In the clinic, drugs blocking this pathway in transplant recipients in order to squelch the immune response, led to an unanticipated decrease in cancer incidence.8 While it is not surprising that depleting nucleotide pools blocks proliferation, knowing specific enzymatic steps that are regulated by MYC may allow for more selective therapy. Since MYC appears to transform cells by exacting modest increases in a wide number of pathways, studies like those described here provide a critical knowledge base that will ultimately inform the design of combination therapy to simultaneously inhibit multiple MYC-regulated pathways.

The c-Myc and E2F transcription factors are among the most potent regulators of cell cycle progression in higher eukaryotes. This report describes the isolation of a novel, highly conserved 434 kDa protein, designated TRRAP, which interacts specifically with the c-Myc N terminus and has homology to the ATM/PI3-kinase family. TRRAP also interacts specifically with the E2F-1 transactivation domain. Expression of transdominant mutants of the TRRAP protein or antisense RNA blocks c-Myc- and E1A-mediated oncogenic transformation. These data suggest that TRRAP is an essential cofactor for both the c-Myc and E1A/E2F oncogenic transcription factor pathways.

Cellular responses to growth and differentiation factors involve a complex cascade of signals that begins at the cell surface, traverses the cytoplasm, and eventually enters the nucleus. Although a great deal is known about the surface and cytoplasmic stages of this cascade, the nuclear events involved in transducing and translating growth and differentiation signals are less well understood. Recent work has implicated a set of genes known as primary response genes as critical for this process. To propagate the activation signal, these genes possess the ability not only to directly respond to upstream biochemical events, but also to transmit the signal downstream by modulating the unique changes in gene expression necessary for a particular cellular response. In this review we discuss how transcription factors encoded by primary response genes may be responsible for regulating tissue- or stimulus-specific responses associated with cellular activation.

Cyclin D1 elicits transcriptional effects through inactivation of the retinoblastoma protein and direct association with transcriptional regulators. The current work reveals a molecular relationship between cyclin D1/CDK4 kinase and protein arginine methyltransferase 5 (PRMT5), an enzyme associated with histone methylation and transcriptional repression. Primary tumors of a mouse lymphoma model exhibit increased PRMT5 methyltransferase activity and histone arginine methylation. Analyses demonstrate that MEP50, a PRMT5 co-regulatory factor, is a CDK4 substrate, and phosphorylation increases PRMT5/MEP50 activity. Increased PRMT5 activity mediates key events associated with cyclin D1-dependent neoplastic growth including CUL4 repression, CDT1 overexpression, and DNA re-replication. Importantly, human cancers harboring mutations in Fbx4, the cyclin D1 E3 ligase, exhibit nuclear cyclin D1 accumulation and increased PRMT5 activity.