Formation of a structurally-stable conformation by the intrinsically disordered MYC:TRRAP complex.
ABSTRACT: Our primary goal is to therapeutically target the oncogenic transcription factor MYC to stop tumor growth and cancer progression. Here, we report aspects of the biophysical states of the MYC protein and its interaction with one of the best-characterized MYC cofactors, TRansactivation/tRansformation-domain Associated Protein (TRRAP). The MYC:TRRAP interaction is critical for MYC function in promoting cancer. The interaction between MYC and TRRAP occurs at a precise region in the MYC protein, called MYC Homology Box 2 (MB2), which is central to the MYC transactivation domain (TAD). Although the MYC TAD is inherently disordered, this report suggests that MB2 may acquire a defined structure when complexed with TRRAP which could be exploited for the investigation of inhibitors of MYC function by preventing this protein-protein interaction (PPI). The MYC TAD, and in particular the MB2 motif, is unique and invariant in evolution, suggesting that MB2 is an ideal site for inhibiting MYC function.
Project description:MYC is an oncogenic DNA-binding transcription activator of many genes and is often upregulated in human cancers. MYC has an N-terminal transcription activation domain (TAD) that is also required for cell transformation. Various MYC TAD-interacting coactivators have been identified, including the transcription/transformation-associated protein (TRRAP), a subunit of different histone acetyltransferase (HAT) complexes such as the human "SPT3-TAF9-GCN5 Acetyltransferase" (STAGA) complex involved in MYC transactivation of the TERT gene. However, it remains unclear whether TRRAP and/or other subunits are directly contacted by MYC within these macromolecular complexes. Here, we characterize the interactions of MYC TAD with the STAGA complex. By protein crosslinking we identify both TRRAP and the GCN5 acetyltransferase as MYC TAD-interacting subunits within native STAGA. We show that purified GCN5 binds to an N-terminal sub-domain of MYC TAD (residues 21-108) and that the interaction of GCN5 and STAGA with this sub-domain is dependent on two related sequence motifs: M2 within the conserved MYC homology box I (MBI), and M3 located between residues 100-106. Interestingly, specific substitutions within the M2/3 motifs that only moderately reduce the intracellular MYC-STAGA interaction and do not influence dimerization of MYC with its DNA-binding partner MAX, strongly inhibit MYC acetylation by GCN5 and reduce MYC binding and transactivation of the GCN5-dependent TERT promoter in vivo. Hence, we propose that MYC associates with STAGA through extended interactions of the TAD with both TRRAP and GCN5 and that the TAD-GCN5 interaction is important for MYC acetylation and MYC binding to certain chromatin loci.
Project description:Transactivation-transformation domain-associated protein (TRRAP) is a component of several multiprotein histone acetyltransferase (HAT) complexes implicated in transcriptional regulation. TRRAP was shown to be required for the mitotic checkpoint and normal cell cycle progression. MRE11, RAD50, and NBS1 (product of the Nijmegan breakage syndrome gene) form the MRN complex that is involved in the detection, signaling, and repair of DNA double-strand breaks (DSBs). By using double immunopurification, mass spectrometry, and gel filtration, we describe the stable association of TRRAP with the MRN complex. The TRRAP-MRN complex is not associated with any detectable HAT activity, while the isolated other TRRAP complexes, containing either GCN5 or TIP60, are. TRRAP-depleted extracts show a reduced nonhomologous DNA end-joining activity in vitro. Importantly, small interfering RNA knockdown of TRRAP in HeLa cells or TRRAP knockout in mouse embryonic stem cells inhibit the DSB end-joining efficiency and the precise nonhomologous end-joining process, further suggesting a functional involvement of TRRAP in the DSB repair processes. Thus, TRRAP may function as a molecular link between DSB signaling, repair, and chromatin remodeling.
Project description:Transactivation/transformation-domain associated protein (TRRAP) is a component of several multi-protein HAT complexes implicated in both transcriptional regulation and DNA repair. We recently identified Trrap, the murine ortholog of TRRAP, as an essential protein implicated in mitotic progression control, although its target genes are not known. In the present study, we analyzed the expression profiles of Trrap-responsive genes, using cDNA microarray in mitotic cells. From a panel of 17 664 transcript elements, we found that loss of Trrap leads to expression alteration of a large fraction of genes at mitotic stage. Functional classification of these genes indicates that Trrap influences a variety of cellular processes including cell cycle progression, cytoskeleton and cell adhesion, protein turnover, metabolism and signal transduction. The majority (71%) of differentially expressed genes was down-regulated in Trrap- deficient cells, whereas the rest were up-regulated, suggesting that Trrap may also play a role in transcriptional silencing. ChIP analysis revealed that Trrap might regulate gene expression by participating in acetylation of histone H4 and/or H3 depending on target genes and cell cycle stage. Our study indicates that Trrap regulates the expression of a wide range of genes in both quiescence and mitotic stages. Removal of the Trrap protein is associated with both increased and decreased gene expression.
Project description:Activation of RNA polymerase (pol) II transcription by c-Myc generally involves recruitment of histone acetyltransferases and acetylation of histones H3 and H4. Here, we describe the mechanism used by c-Myc to activate pol III transcription of tRNA and 5S rRNA genes. Within 2 h of its induction, c-Myc appears at these genes along with the histone acetyltransferase GCN5 and the cofactor TRRAP. At the same time, occupancy of the pol III-specific factor TFIIIB increases and histone H3 becomes hyperacetylated, but increased histone H4 acetylation is not detected at these genes. The rapid acetylation of histone H3 and promoter assembly of TFIIIB, c-Myc, GCN5, and TRRAP are followed by recruitment of pol III and transcriptional induction. The selective acetylation of histone H3 distinguishes pol III activation by c-Myc from mechanisms observed in other systems.
Project description:Transcriptional activation of histone subtypes is coordinately regulated and tightly coupled with the onset of DNA replication during S-phase entry. The underlying molecular mechanisms for such coordination and coupling are not well understood. The cyclin E-Cdk2 substrate NPAT has been shown to play an essential role in the transcriptional activation of histone genes at the G(1)/S-phase transition. Here, we show that NPAT interacts with components of the Tip60 histone acetyltransferase complex through a novel amino acid motif, which is functionally conserved in E2F and adenovirus E1A proteins. In addition, we demonstrate that transformation/transactivation domain-associated protein (TRRAP) and Tip60, two components of the Tip60 complex, associate with histone gene promoters at the G(1)/S-phase boundary in an NPAT-dependent manner. In correlation with the association of the TRRAP-Tip60 complex, histone H4 acetylation at histone gene promoters increases at the G(1)/S-phase transition, and this increase involves NPAT function. Suppression of TRRAP or Tip60 expression by RNA interference inhibits histone gene activation. Thus, our data support a model in which NPAT recruits the TRRAP-Tip60 complex to histone gene promoters to coordinate the transcriptional activation of multiple histone genes during the G(1)/S-phase transition.
Project description:Cellular transformation by adenovirus E1A requires targeting TRRAP, a scaffold protein which helps assemble histone acetyltransferase complexes, including the NuA4 complex. We recently reported that E1A and E1A 1-80 (N-terminal 80 aa) promote association of the proto-oncogene product MYC with the NuA4 complex. The E1A N-terminal TRRAP-targeting (ET) domain is required for E1A 1-80 to interact with the NuA4 complex. We demonstrate that an ET-MYC fusion associates with the NuA4 complex more efficiently than does MYC alone. Because MYC regulates genes for multiple cellular pathways, we performed global RNA-sequence analysis of cells expressing MYC or ET-MYC, and identified a panel of genes (262) preferentially activated by ET-MYC and significantly enriched in genes involved in gene expression and ribosome biogenesis, suggesting that E1A enhances MYC association with the NuA4 complex to activate a set of MYC target genes likely involved in cellular proliferation and cellular transformation by E1A and by MYC.
Project description:Epigenetic control of neural stem/progenitor cell fate is fundamental to achieve a fully brain architecture. Two intrinsic programs regulate neurogenesis, one by epigenetic-mediated gene transcription and another by cell cycle control. Whether and how these two are coordinated to determine temporally and spatially neural development remains unknown. Here we show that deletion of Trrap (Transcription translation associated protein), an essential cofactor for HAT (histone acetyltransferase), leads to severe brain atrophy due to a combination of cell death and a blockade of neuron production. Specifically, Trrap deletion forces differentiation of apical progenitor (AP) fate into basal progenitors (BP) and neurons thereby limiting the total neurogenic production. Despite TrrapM-bM-^@M-^Ys general role in transcriptional regulation, a genome-wide transcriptome analysis of neuroprogenitors identified the cell cycle regulators that are specifically affected by Trrap deletion. Furthermore, E2F-dependent recruitment of HAT and transcription factors to the promoter of cell cycle regulators is impaired in Trrap-deleted neuroprogenitors. Consistent with these molecular changes, Trrap deletion lengthens particularly G1 and S phases in APs in vivo. Therefore, our study reveals an essential and a distinct function of Trrap-HAT in regulation of cell cycle progression that is required for proper determination of neuroprogenitor fate. Determine gene transcriptions by comparing Trrap-deleted and wild type samples
Project description:The c-Myc oncoprotein functions as a transcription factor that can transform normal cells into tumor cells, as well as playing a direct role in normal cell proliferation. The c-Myc protein transactivates cellular promoters by recruiting nuclear cofactors to chromosomal sites through an N-terminal transactivation domain. We have previously reported the identification and functional characterization of four different c-Myc cofactors: TRRAP, hGCN5, TIP49, and TIP48. Here we present the identification and characterization of the actin-related protein BAF53 as a c-Myc-interacting nuclear cofactor that forms distinct nuclear complexes. In addition to the human SWI/SNF-related BAF complex, BAF53 forms a complex with TIP49 and TIP48 and a separate biochemically distinct complex containing TRRAP and a histone acetyltransferase which does not contain TIP60. Using deletion mutants of BAF53, we show that BAF53 is critical for c-Myc oncogenic activity. Our results indicate that BAF53 plays a functional role in c-Myc-interacting nuclear complexes.
Project description:Human cancers frequently arise from increased expression of proto-oncogenes, such as MYC and HER2. Understanding the cellular pathways regulating the transcription and expression of proto-oncogenes is important for targeted therapies for cancer treatment. Adenoviral (Ad) E1A 243R (243 aa residues) is a viral oncoprotein that interacts with key regulators of gene transcription and cell proliferation. We have shown previously that the 80 amino acid N-terminal transcriptional repression domain of E1A 243R (E1A 1-80) can target the histone acetyltransferase (HAT) p300 and repress HER2 in the HER2-overexpressing human breast cancer cell line SKBR3. Expression of E1A 1-80 induces death of SKBR3 and other cancer cell lines. In this study, we performed total cell RNA sequence analysis and identified MYC as the regulatory gene for cellular proliferation most strongly repressed by E1A 1-80. By RT-quantitative PCR analysis we show that repression of MYC in SKBR3 cells occurs early after expression of E1A 1-80, suggesting that MYC may be an early responder of E1A 1-80-mediated transcriptional repression. Of interest, while E1A 1-80 repression of MYC occurs in all eight human cancer cell lines examined, repression of HER2 is cell-type dependent. We demonstrate by ChIP analysis that MYC transcriptional repression by E1A 1-80 is associated with inhibition of acetylation of H3K18 and H4K16 on the MYC promoter, as well as inhibition of RNA Pol II binding to the MYC promoter. Deletion mutant analysis of E1A 1-80 suggests that both p300/CBP and TRRAP are involved in E1A 1-80 repression of MYC transcription. Further, E1A 1-80 interaction with p300/CBP and TRRAP is correlated with inhibition of H3K18 and H4K16 acetylation on the MYC promoter, respectively. Our results indicate that E1A 1-80 may target two important pathways for histone modification to repress transcription in human cancer cells.
Project description:As a component of chromatin-modifying complexes with histone acetyltransferase (HAT) activity, TRRAP has been shown to be involved in various cellular processes including gene transcription and oncogenic transformation. Inactivation of Trrap, the murine ortholog of TRRAP, in mice revealed its function in development and cell cycle progression. However, the underlying mechanism is unknown. Here, we show that the loss of Trrap in mammalian cells leads to chromosome missegregation, mitotic exit failure and compromised mitotic checkpoint. These mitotic checkpoint defects are caused by defective Trrap-mediated transcription of the mitotic checkpoint proteins Mad1 and Mad2. The mode of regulation by Trrap involves acetylation of histones H4 and H3 at the gene promoter of these mitotic players. Trrap associated with the HAT Tip60 and PCAF at the Mad1 and Mad2 promoters in a cell cycle-dependent manner and Trrap depletion abolished recruitment of these HATs. Finally, ectopic expression of Mad1 and Mad2 fully restores the mitotic checkpoint in Trrap-deficient cells. These results demonstrate that Trrap controls the mitotic checkpoint integrity by specifically regulating Mad1 and Mad2 genes.