Search

SF1: Analysis of USP22 expression with known drivers of disease progression; SF2: USP22 modulates DNA repair factors expression and survival after DNA damage; SF3: Genetically engineered mouse model of tumor-associated USP22 expression; SF4: The USP22-sensitive ubiquitylome reveals altered modification of DNA repair proteins; SF5: USP22 deubiquitylates the NER protein XPC, modulating foci formation.

Emerging evidence indicates the deubiquitinase USP22 regulates transcriptional activation and modification of target substrates to promote pro-oncogenic phenotypes. Here, in vivo characterization of tumor-associated USP22 upregulation and unbiased interrogation of USP22-regulated functions in vitro demonstrated critical roles for USP22 in prostate cancer. Specifically, clinical datasets validated that USP22 expression is elevated in prostate cancer, and a novel murine model demonstrated a hyperproliferative phenotype with prostate-specific USP22 overexpression. Accordingly, upon overexpression or depletion of USP22, enrichment of cell-cycle and DNA repair pathways was observed in the USP22-sensitive transcriptome and ubiquitylome using prostate cancer models of clinical relevance. Depletion of USP22 sensitized cells to genotoxic insult, and the role of USP22 in response to genotoxic insult was further confirmed using mouse adult fibroblasts from the novel murine model of USP22 expression. As it was hypothesized that USP22 deubiquitylates target substrates to promote protumorigenic phenotypes, analysis of the USP22-sensitive ubiquitylome identified the nucleotide excision repair protein, XPC, as a critical mediator of the USP22-mediated response to genotoxic insult. Thus, XPC undergoes deubiquitylation as a result of USP22 function and promotes USP22-mediated survival to DNA damage. Combined, these findings reveal unexpected functions of USP22 as a driver of protumorigenic phenotypes and have significant implications for the role of USP22 in therapeutic outcomes.Significance:The studies herein present a novel mouse model of tumor-associated USP22 overexpression and implicate USP22 in modulation of cellular survival and DNA repair, in part through regulation of XPC.

PDF file - 38K, Supplemental Table 1. Genes binding both P53 and DACH1 in ChIP Seq.

PDF file - 176K, Supplementary Figure S1: USP22 Specifically Promotes AR Recruitment to Target Loci. Supplementary Figure 2: Depletion of USP22 can be mediated by multiple sequences.Supplementary Figure S3: AR Ubiquitylation Levels are not Altered in Response to USP22 Depletion.

PDF file - 293K, Supplemental Figure 1. Reduced DACH1 mRNA in NSCLC. Supplemental Figure 2. DACH1 and p53 co-localized in a nuclear extranucleolar location.Supplemental Figure 3. Western blot analysis of H1299 cells treated with 5-Aza-2'- deoxycytidine (5MM) for 5 days and Trichostatin A (1MM, 8hrs).Supplemental Figure 4. EGFR and DACH1 expression in NSCLC vs. SCLC.

PDF file - 72K

PDF file - 50K, Primer sequences used in manuscript.

Increasing evidence links deregulation of the ubiquitin-specific proteases 22 (USP22) deubitiquitylase to cancer development and progression in a select group of tumor types, but its specificity and underlying mechanisms of action are not well defined. Here we show that USP22 is a critical promoter of lethal tumor phenotypes that acts by modulating nuclear receptor and oncogenic signaling. In multiple xenograft models of human cancer, modeling of tumor-associated USP22 deregulation demonstrated that USP22 controls androgen receptor accumulation and signaling, and that it enhances expression of critical target genes coregulated by androgen receptor and MYC. USP22 not only reprogrammed androgen receptor function, but was sufficient to induce the transition to therapeutic resistance. Notably, in vivo depletion experiments revealed that USP22 is critical to maintain phenotypes associated with end-stage disease. This was a significant finding given clinical evidence that USP22 is highly deregulated in tumors, which have achieved therapeutic resistance. Taken together, our findings define USP22 as a critical effector of tumor progression, which drives lethal phenotypes, rationalizing this enzyme as an appealing therapeutic target to treat advanced disease. Cancer Res; 74(1); 272–86. ©2013 AACR.

Sporadic colorectal cancers arise from initiating mutations in APC, producing oncogenic β-catenin/TCF-dependent transcriptional reprogramming. Similarly, the tumor suppressor axis regulated by the intestinal epithelial receptor GUCY2C is among the earliest pathways silenced in tumorigenesis. Retention of the receptor, but loss of its paracrine ligands, guanylin and uroguanylin, is an evolutionarily conserved feature of colorectal tumors, arising in the earliest dysplastic lesions. Here, we examined a mechanism of GUCY2C ligand transcriptional silencing by β-catenin/TCF signaling.We performed RNA sequencing analysis of 4 unique conditional human colon cancer cell models of β-catenin/TCF signaling to map the core Wnt-transcriptional program. We then performed a comparative analysis of orthogonal approaches, including luciferase reporters, chromatin immunoprecipitation sequencing, CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats) knockout, and CRISPR epigenome editing, which were cross-validated with human tissue chromatin immunoprecipitation sequencing datasets, to identify functional gene enhancers mediating GUCY2C ligand loss.RNA sequencing analyses reveal the GUCY2C hormones as 2 of the most sensitive targets of β-catenin/TCF signaling, reflecting transcriptional repression. The GUCY2C hormones share an insulated genomic locus containing a novel locus control region upstream of the guanylin promoter that mediates the coordinated silencing of both genes. Targeting this region with CRISPR epigenome editing reconstituted GUCY2C ligand expression, overcoming gene inactivation by mutant β-catenin/TCF signaling.These studies reveal DNA elements regulating corepression of GUCY2C ligand transcription by β-catenin/TCF signaling, reflecting a novel pathophysiological step in tumorigenesis. They offer unique genomic strategies that could reestablish hormone expression in the context of canonical oncogenic mutations to reconstitute the GUCY2C axis and oppose transformation.

Lung cancer is the leading cause of cancer-related deaths in the USA, and alterations in the tumor suppressor gene TP53 are the most frequent somatic mutation among all histologic subtypes of lung cancer. Mutations in TP53 frequently result in a protein that exhibits not only loss of tumor suppressor capability but also oncogenic gain-of-function (GOF). The canonical p53 hotspot mutants R175H and R273H, for example, confer upon tumors a metastatic phenotype in murine models of mutant p53. To the best of our knowledge, GOF phenotypes of the less often studied V157, R158 and A159 mutants—which occur with higher frequency in lung cancer compared with other solid tumors—have not been defined. In this study, we aimed to define whether the lung mutants are simply equivalent to full loss of the p53 locus, or whether they additionally acquire the ability to drive new downstream effector pathways. Using a publicly available human lung cancer dataset, we characterized patients with V157, R158 and A159 p53 mutations. In addition, we show here that cell lines with mutant p53-V157F, p53-R158L and p53-R158P exhibit a loss of expression of canonical wild-type p53 target genes. Furthermore, these lung-enriched p53 mutants regulate genes not previously linked to p53 function including PLAU. Paradoxically, mutant p53 represses genes associated with increased cell viability, migration and invasion. These findings collectively represent the first demonstration that lung-enriched p53 mutations at V157 and R158 regulate a novel transcriptome in human lung cancer cells and may confer de novo function.

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.

For recognition of specific regulatory sequences in the genome (i.e., response elements, REs), the tumor suppressor protein 53 kDa (p53) exhibits dose-dependent selectivity. In general, binding to REs linked to target genes involved in the positive regulation of cell death requires higher levels of p53 than those connected to cell survival. Our recent findings provide a mechanistic explanation for this phenomenon. Specifically, we demonstrate that subtle differences in DNA shape, encoded in RE DNA sequence, determine the utilization of two biochemically distinct DNA-binding modes, ultimately connected to different biological outcomes.

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.

Emerging evidence indicates the deubiquitinase USP22 regulates transcriptional activation and modification of target substrates to promote pro-oncogenic phenotypes. Here, in vivo characterization of tumor-associated USP22 upregulation and unbiased interrogation of USP22-regulated functions in vitro demonstrated critical roles for USP22 in prostate cancer. Specifically, clinical datasets validated that USP22 expression is elevated in prostate cancer, and a novel murine model demonstrated a hyperproliferative phenotype with prostate-specific USP22 overexpression. Accordingly, upon overexpression or depletion of USP22, enrichment of cell-cycle and DNA repair pathways was observed in the USP22-sensitive transcriptome and ubiquitylome using prostate cancer models of clinical relevance. Depletion of USP22 sensitized cells to genotoxic insult, and the role of USP22 in response to genotoxic insult was further confirmed using mouse adult fibroblasts from the novel murine model of USP22 expression. As it was hypothesized that USP22 deubiquitylates target substrates to promote protumorigenic phenotypes, analysis of the USP22-sensitive ubiquitylome identified the nucleotide excision repair protein, XPC, as a critical mediator of the USP22-mediated response to genotoxic insult. Thus, XPC undergoes deubiquitylation as a result of USP22 function and promotes USP22-mediated survival to DNA damage. Combined, these findings reveal unexpected functions of USP22 as a driver of protumorigenic phenotypes and have significant implications for the role of USP22 in therapeutic outcomes. Significance: The studies herein present a novel mouse model of tumor-associated USP22 overexpression and implicate USP22 in modulation of cellular survival and DNA repair, in part through regulation of XPC.

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.

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.

// Amanda R. Oran 1 , Clare M. Adams 1 , Xiao-yong Zhang 1 , Victoria J. Gennaro 1 , Harla K. Pfeiffer 1 , Hestia S. Mellert 2 , Hans E. Seidel 3 , Kirsten Mascioli 1 , Jordan Kaplan 1 , Mahmoud R. Gaballa 1 , Chen Shen 4,5 , Isidore Rigoutsos 1 , Michael P. King 6 , Justin L. Cotney 7 , Jamie J. Arnold 8 , Suresh D. Sharma 8 , Ubaldo E. Martinez-Outschoorn 1 , Christopher R. Vakoc 4 , Lewis A. Chodosh 3 , James E. Thompson 9 , James E. Bradner 10 , Craig E. Cameron 8 , Gerald S. Shadel 11,12 , Christine M. Eischen 1 and Steven B. McMahon 1 1 Department of Cancer Biology, Thomas Jefferson University, Philadelphia, PA, USA 2 Biomedical Graduate Studies, University of Pennsylvania, Philadelphia, PA, USA 3 Department of Cancer Biology and Abramson Family Cancer Research Institute, University of Pennsylvania School of Medicine, Philadelphia, PA, USA 4 Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, USA 5 Molecular and Cellular Biology Program, Stony Brook University, Stony Brook, NY, USA 6 Department of Biochemistry, Thomas Jefferson University, Philadelphia, PA, USA 7 Genetics and Genome Sciences, University of Connecticut Health, Farmington, CT, USA 8 Department of Biochemistry & Molecular Biology, The Pennsylvania State University, University Park, PA, USA 9 Leukemia Service, Department of Medicine, Roswell Park Cancer Institute, Buffalo, NY, USA 10 Department of Medical Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA,USA 11 Department of Pathology, Yale School of Medicine, New Haven, CT, USA 12 Department of Genetics, Yale School of Medicine, New Haven, CT, USA Correspondence to: Steven B. McMahon, email: // Keywords : MYC, mitochondria, mitochondrial gene expression, tigecycline, synthetic lethality Received : June 08, 2016 Accepted : August 25, 2016 Published : August 31, 2016 Abstract Despite ubiquitous activation in human cancer, essential downstream effector pathways of the MYC transcription factor have been difficult to define and target. Using a structure/function-based approach, we identified the mitochondrial RNA polymerase (POLRMT) locus as a critical downstream target of MYC. The multifunctional POLRMT enzyme controls mitochondrial gene expression, a process required both for mitochondrial function and mitochondrial biogenesis. We further demonstrate that inhibition of this newly defined MYC effector pathway causes robust and selective tumor cell apoptosis, via an acute, checkpoint-like mechanism linked to aberrant electron transport chain complex assembly and mitochondrial reactive oxygen species (ROS) production. Fortuitously, MYC-dependent tumor cell death can be induced by inhibiting the mitochondrial gene expression pathway using a variety of strategies, including treatment with FDA-approved antibiotics. In vivo studies using a mouse model of Burkitt’s Lymphoma provide pre-clinical evidence that these antibiotics can successfully block progression of MYC-dependent tumors.

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.

Lung cancer, the leading cause of cancer-related mortality in the United States, occurs primarily due to prolonged exposure to an array of carcinogenic compounds in cigarette smoke. These carcinogens create bulky DNA adducts, inducing alterations including missense mutations in the tumor suppressor gene TP53. TP53 is the most commonly mutated gene in many human cancers, and a specific set of these variants are enriched in lung cancer (at amino acid residues V157, R158, and A159). This perspective postulates that lung-enriched mutations can be explained, in part, by biological selection for oncogenic gain-of-function (GOF) mutant p53 alleles at V157, R158, and A159. This hypothesis explaining tissue-specific TP53 mutations is further supported by mouse model studies of the canonical TP53 hotspots showing that tumor spectra and GOF activities are altered with mutation type. Therefore, although smoking-related lung cancer unequivocally arises due to the mutagenic environment induced by tobacco carcinogens, this perspective provides a rationale for the preferential selection of lung-enriched V157, R158, and A159 mutant p53.

Telomeres and tumor suppressor protein TP53 (p53) function in genome protection, but a direct role of p53 at telomeres has not yet been described. Here, we have identified non-canonical p53-binding sites within the human subtelomeres that suppress the accumulation of DNA damage at telomeric repeat DNA. These non-canonical subtelomeric p53-binding sites conferred transcription enhancer-like functions that include an increase in local histone H3K9 and H3K27 acetylation and stimulation of subtelomeric transcripts, including telomere repeat-containing RNA (TERRA). p53 suppressed formation of telomere-associated γH2AX and prevented telomere DNA degradation in response to DNA damage stress. Our findings indicate that p53 provides a direct chromatin-associated protection to human telomeres, as well as other fragile genomic sites. We propose that p53-associated chromatin modifications enhance local DNA repair or protection to provide a previously unrecognized tumor suppressor function of p53.

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.

Increasing evidence links deregulation of the ubiquitin-specific proteases 22 (USP22) deubitiquitylase to cancer development and progression in a select group of tumor types, but its specificity and underlying mechanisms of action are not well defined. Here we show that USP22 is a critical promoter of lethal tumor phenotypes that acts by modulating nuclear receptor and oncogenic signaling. In multiple xenograft models of human cancer, modeling of tumor-associated USP22 deregulation demonstrated that USP22 controls androgen receptor accumulation and signaling, and that it enhances expression of critical target genes coregulated by androgen receptor and MYC. USP22 not only reprogrammed androgen receptor function, but was sufficient to induce the transition to therapeutic resistance. Notably, in vivo depletion experiments revealed that USP22 is critical to maintain phenotypes associated with end-stage disease. This was a significant finding given clinical evidence that USP22 is highly deregulated in tumors, which have achieved therapeutic resistance. Taken together, our findings define USP22 as a critical effector of tumor progression, which drives lethal phenotypes, rationalizing this enzyme as an appealing therapeutic target to treat advanced disease. Cancer Res; 74(1); 272–86. ©2013 AACR.

Breast cancer is a leading form of cancer in the world. Initially cloned as a dominant inhibitor of the hyperactive EGFR, Ellipse, in Drosophila, the mammalian DACH1 regulates expression of target genes in part through interacting with DNA-binding transcription factors (c-Jun, Smads, Six, ERα), and in part through intrinsic DNA-sequence specific binding to Forkhead binding sites. The Drosophila dac gene is a key member of the retinal determination gene network (RDGN), which also includes eyes absent (eya), ey, twin of eyeless (toy), teashirt (tsh) and sin oculis (so), that specifies eye tissue identity. Several lines of evidence suggest DACH1 may function as a tumor suppressor. Clinical studies have demonstrated a correlation between poor prognosis and reduced expression of the cell-fate determination factor DACH1 in breast cancer, and loss of DACH1 expression has been observed in prostate and endometrial cancer. DACH1 inhibits breast cancer tumor metastasis and reduces breast cancer stem cell expansion via Sox2/Nanog. Although these studies suggest DACH1 may function as a tumor suppressor, the molecular mechanisms remain poorly defined. Herein, endogenous DACH1 co-localized with p53 in a nuclear, extranucleolar compartment and bound to p53 in human breast cancer cell lines, p53 and DACH1 bound common genes in ChIP-Seq. Full inhibition of breast cancer contact-independent growth by DACH1 required p53. The p53 breast cancer mutants R248Q and R273H, evaded DACH1 binding. DACH1 phosphorylation at serine residue (S439) inhibited p53 binding and phosphorylation at p53 amino-terminal sites (S15, S20) enhanced DACH1 binding. DACH1 binding to p53 was inhibited by NAD-dependent deacetylation via DACH1 K628. DACH1 repressed p21CIP1 and induced RAD51, an association found in basal breast cancer. DACH1 inhibits breast cancer cellular growth in an NAD and p53 dependent manner through direct protein-protein association. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P5-11-04.

// Ke Chen 1 , Kongming Wu 1 , Michael Gormley 1 , Adam Ertel 1 , Jing Wang 1 ,Wei Zhang 1 , Jie Zhou 1 , Gabriele DiSante 1 , Zhiping Li 1 , Hallgeir Rui 1 , Andrew A. Quong 1 , Steven B. McMahon 1,2 , Haiteng Deng 3 , Michael P. Lisanti 2 , Chenguang Wang 1,2 , Richard G. Pestell 1,2 1 Department of Cancer Biology, Thomas Jefferson University, Philadelphia, PA, USA 2 Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, PA, USA 3 Proteomics Resource Center, Rockefeller University, New York, NY, USA Correspondence: Richard G. Pestell, email: // Kongming Wu, email: // Keywords : p53, breast cancer, cell fate, stem cells, dach Received : June 13, 2013 Accepted : June 21, 2013 Published : June 21, 2013 Abstract Breast cancer is a leading form of cancer in the world. The Drosophila Dac gene was cloned as an inhibitor of the hyperactive epidermal growth factor (EGFR), ellipse . Herein, endogenous DACH1 co-localized with p53 in a nuclear, extranucleolar compartment and bound to p53 in human breast cancer cell lines, p53 and DACH1 bound common genes in Chip-Seq. Full inhibition of breast cancer contact-independent growth by DACH1 required p53. The p53 breast cancer mutants R248Q and R273H, evaded DACH1 binding. DACH1 phosphorylation at serine residue (S439) inhibited p53 binding and phosphorylation at p53 amino-terminal sites (S15, S20) enhanced DACH1 binding. DACH1 binding to p53 was inhibited by NAD-dependent deacetylation via DACH1 K628. DACH1 repressed p21 CIP1 and induced RAD51, an association found in basal breast cancer. DACH1 inhibits breast cancer cellular growth in an NAD and p53-dependent manner through direct protein-protein association.

The human telomerase reverse transcriptase (hTERT) gene is repressed in most somatic cells, whereas the expression of the mouse mTert gene is widely detected. To understand the mechanisms of this human-specific repression, we constructed bacterial artificial chromosome (BAC) reporters using human and mouse genomic DNAs encompassing the TERT genes and neighboring loci. Upon chromosomal integration, the hTERT, but not the mTert, reporter was stringently repressed in telomerase-negative human cells in a histone deacetylase (HDAC)-dependent manner, replicating the expression of their respective endogenous genes. In chimeric BACs, the mTert promoter became strongly repressed in the human genomic context, but the hTERT promoter was highly active in the mouse genomic context. Furthermore, an unrelated herpes simplex virus-thymidine kinase (HSV-TK) promoter was strongly repressed in the human, but not in the mouse, genomic context. These results demonstrated that the repression of hTERT gene was dictated by distal elements and its chromatin environment. This repression depended on class I HDACs and involved multiple corepressor complexes, including HDAC1/2-containing Sin3B, nucleosome remodeling and histone deacetylase (NuRD), and corepressor of RE1 silencing transcription factor (CoREST) complexes. Together, our data indicate that the lack of telomerase expression in most human somatic cells results from its repressive genomic environment, providing new insight into the mechanism of long-recognized differential telomerase regulation in mammalian species.-Cheng, D., Zhao, Y., Wang, S., Zhang, F., Russo, M., McMahon, S. B., Zhu, J. Repression of telomerase gene promoter requires human-specific genomic context and is mediated by multiple HDAC1-containing corepressor complexes.

The p53 tumor suppressor gene encodes a sequence-specific transcription factor. Mutations in the coding sequence of p53 occur frequently in human cancer and often result in single amino acid substitutions (missense mutations) in the DNA binding domain (DBD), blocking normal tumor suppressive functions. In addition to the loss of canonical functions, some missense mutations in p53 confer gain-of-function (GOF) activities to tumor cells. While many missense mutations in p53 cluster at six “hotspot” amino acids, the majority of mutations in human cancer occur elsewhere in the DBD and at a much lower frequency. We report here that mutations at K120, a non-hotspot DNA contact residue, confer p53 with the previously unrecognized ability to bind and activate the transcription of the pro-survival TNFAIP8 gene. Mutant K120 p53 binds the TNFAIP8 locus at a cryptic p53 response element that is not occupied by wild-type p53. Furthermore, induction of TNFAIP8 is critical for the evasion of apoptosis by tumor cells expressing the K120R variant of p53. These findings identify induction of pro-survival targets as a mechanism of gain-of-function activity for mutant p53 and will likely broaden our understanding of this phenomenon beyond the limited number of GOF activities currently reported for hotspot mutants.

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.

The MYST protein lysine acetyltransferases are evolutionarily conserved throughout eukaryotes and acetylate proteins to regulate diverse biological processes including gene regulation, DNA repair, cell-cycle regulation, stem cell homeostasis and development. Here, we demonstrate that MYST protein acetyltransferase activity requires active site lysine autoacetylation. The X-ray crystal structures of yeast Esa1 (yEsa1/KAT5) bound to a bisubstrate H4K16CoA inhibitor and human MOF (hMOF/KAT8/MYST1) reveal that they are autoacetylated at a strictly conserved lysine residue in MYST proteins (yEsa1-K262 and hMOF-K274) in the enzyme active site. The structure of hMOF also shows partial occupancy of K274 in the unacetylated form, revealing that the side chain reorients to a position that engages the catalytic glutamate residue and would block cognate protein substrate binding. Consistent with the structural findings, we present mass spectrometry data and biochemical experiments to demonstrate that this lysine autoacetylation on yEsa1, hMOF and its yeast orthologue, ySas2 (KAT8) occurs in solution and is required for acetylation and protein substrate binding in vitro. We also show that this autoacetylation occurs in vivo and is required for the cellular functions of these MYST proteins. These findings provide an avenue for the autoposttranslational regulation of MYST proteins that is distinct from other acetyltransferases but draws similarities to the phosphoregulation of protein kinases.

While the post-translational modification of histones by the addition of ubiquitin was discovered decades ago, it has only recently been appreciated that the dynamic regulation of histone ubiquitylation patterns is an important mechanism for controlling a variety of biological processes. The processes include transcription, the recognition and repair of genomic damage and DNA replication, among others. Enzymes that catalyze the addition of ubiquitin to histones, such as the polycomb family, have been well-studied. In contrast, the enzymes that remove ubiquitin from histones are less well understood. The assay strategies described here provide a platform for the thorough in vitro and in vivo analysis of histone deubiquitylation. In some cases, these poorly characterized enzymes are likely to provide new opportunities for therapeutic targeting and a detailed understanding of their biochemical and biological activities is a prerequisite to these clinical advances.

Rationale: Cancers of the lung and bronchus are responsible for nearly 30% of all cancer-related deaths in the US, and mutations in the tumor-suppressor gene TP53 occur in 54% of lung adenocarcinomas and 86% of squamous cell carcinomas. Compared with other cancers, TP53 mutational hotspots in lung cancer more often include missense mutations at codons V157, R158, and A159, with frequencies approaching that of the canonical mutational hotspot R273. The resulting mutant p53 (mutp53) protein often exhibits not only loss of tumor suppressor capability but also gain of oncogenic function (GOF). Our previous work identified a novel, p53-dependent transcriptome including alterations in expression of urokinase plasminogen activator (uPA), a key regulator of the plasminogen activation system. When activated, uPA converts plasminogen to plasmin, which cleaves extracellular matrix components and promotes cell migration. We evaluated uPA expression and cell migration in human lung cancer cell lines expressing wild-type or mutant p53. Methods: Human lung cancer cell lines with wild-type p53 (A549) or with somatic mutations at the endogenous p53 locus (NCI-H1781 and NCI-H2087, V157F; NCI-H441, R158L; NCI-H2110, R158P; NCI-H2009, R273L) were obtained from ATCC. Using RNAi (shRNA and siRNA) cells were depleted of p53 as confirmed by puromycin selection of cells infected with lentivirus, in the case of shRNA. Whole-cell lysates were harvested for protein and RNA purification, and conditioned medium was harvested for measurement of secreted proteins. Immunoblotting and qRT-PCR were used to assess expression of protein and mRNA, respectively. A wound healing assay was performed following siRNA transfection to determine cell migration in cells expressing vs. depleted of wild-type or mutant p53. Results: Silencing of p53 by shRNA and siRNA targeted to TP53 was confirmed by Western blot and qRT-PCR. With p53 depletion, protein and mRNA expression of the uPA pro-enzyme pro-uPA and its activated B- and A-chains increased in the H2087 (V157F mutp53) cell line. No p53-dependent change was seen in uPA expression in H2009 cells (R273L mutp53) despite a similar relative quantity of uPA protein. Secreted uPA also increased as measured in conditioned media from H2087 cells (V157F mutp53). A wound healing assay showed increased migration by mutp53-depleted H2087 cells compared with mutp53-expressing H2087 cells, while A549 cells (wt p53) showed no p53-dependent difference in migration rate. Conclusions: To our knowledge, GOF phenotypes in lung cancer cells carrying V157F mutp53 have not been characterized. We have shown here that mutp53 represses uPA expression in human lung cancer cells with endogenous V157F mutp53, and depletion of p53 in these cells leads to an increase in migration. These findings prompt a reexamination of the signaling pathways associated with uPA. Further study is needed to elucidate the mechanism of the p53-dependent alterations in uPA expression and to establish additional biologic effects of V157F mutp53. Citation Format: Julie A. Barta, Steven B. McMahon. Urokinase plasminogen activator expression is regulated by p53 harboring the lung cancer-specific mutation V157F [abstract]. In: Proceedings of the Fifth AACR-IASLC International Joint Conference: Lung Cancer Translational Science from the Bench to the Clinic; Jan 8-11, 2018; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(17_Suppl):Abstract nr A25.

Activation of the MYC oncoprotein is among the most ubiquitous events in human cancer. MYC functions in part as a sequence-specific regulator of transcription. Although early searches for direct downstream target genes that explain MYC's potent biological activity were met with enthusiasm, the postgenomic decade has brought the realization that MYC regulates the transcription of not just a manageably small handful of target genes but instead up to 15% of all active loci. As the dust has begun to settle, two important concepts have emerged that reignite hope that understanding MYC's downstream targets might still prove valuable for defining critical nodes for therapeutic intervention in cancer patients. First, it is now clear that MYC target genes are not a random sampling of the cellular transcriptome but instead fall into specific, critical biochemical pathways such as metabolism, chromatin structure, and protein translation. In retrospect, we should not have been surprised to discover that MYC rewires cell physiology in a manner designed to provide the tumor cell with greater biosynthetic properties. However, the specific details that have emerged from these studies are likely to guide the development of new clinical tools and strategies. This raises the second concept that instills renewed optimism regarding MYC target genes. It is now clear that not all MYC target genes are of equal functional relevance. Thus, it may be possible to discern, from among the thousands of potential MYC target genes, those whose inhibition will truly debilitate the tumor cell. In short, targeting the targets may ultimately be a realistic approach after all.

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.

Epstein Barr virus (EBV) can establish distinct latency types with different growth-transforming properties. Type I latency and type III latency can be distinguished by the expression of EBNA2, which has been shown to be regulated, in part, by the EBNA1-dependent enhancer activity of the origin of replication (OriP). Here, we report that CTCF, a chromatin boundary factor with well-established enhancer-blocking activity, binds to EBV sequences between the OriP and the RBP-Jκ response elements of the C promoter (Cp) and regulates transcription levels of EBNA2 mRNA. Using DNA affinity, electrophoretic mobility shift assay, DNase I footprinting, and chromatin immunoprecipitation (ChIP), we found that CTCF binds both in vitro and in vivo to the EBV genome between OriP and Cp, with an ∼50-bp footprint at EBV coordinates 10515 to 10560. Deletion of this CTCF binding site in a recombinant EBV bacterial artificial chromosome (BAC) increased EBNA2 transcription by 3.5-fold compared to a wild-type EBV BAC. DNA affinity and ChIP showed more CTCF binding at this site in type I latency cell lines (MutuI and KemI) than in type III latency cell lines (LCL3456 and Raji). CTCF protein and mRNA expression levels were higher in type I than type III cell lines. Short interfering RNA depletion of CTCF in type I MutuI cells stimulated EBNA2 mRNA levels, while overexpression of CTCF in type III Raji cells inhibited EBNA2 mRNA levels. These results indicate that increased CTCF can repress EBNA2 transcription. We also show that c-MYC, as well as EBNA2, can stimulate CTCF mRNA levels, suggesting that CTCF levels may contribute to B-cell differentiation as well as EBV latency type determination.

The family of myc proto-oncogenes encodes transcription factors (c-, N-, and L-Myc) that regulate cell growth and proliferation and are involved in the etiology of diverse cancers. Myc proteins are thought to function by binding and regulating specific target genes. Here we report that Myc proteins are required for the widespread maintenance of active chromatin. Disruption of N-myc in neuronal progenitors and other cell types leads to nuclear condensation accompanied by large-scale changes in histone modifications associated with chromatin inactivation, including hypoacetylation and altered methylation. These effects are largely reversed by exogenous Myc as well as by differentiation and are mimicked by the Myc antagonist Mad1. The first chromatin changes are evident within 6 h of Myc loss and lead to changes in chromatin structure. Myc widely influences chromatin in part through upregulation of the histone acetyltransferase GCN5. This study provides the first evidence for regulation of global chromatin structure by an oncoprotein and may explain the broad effects of Myc on cell behavior and tumorigenesis.

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.