E2F Transcriptional Activation Requires TRRAP and GCN5 Cofactors

Abstract

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 SWI⅐SNF 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 cell cycle is regulated in part by the temporal expression of specific genes. A transcription factor can control the timely induction of S phase-specific genes by either activating gene expression shortly before and during S phase or repressing expression during the remainder of the cell cycle. The E2F family of transcription factors is thought to contribute to cell cycle regulation through both mechanisms. E2F is likely to be a critical factor in cell growth regulation in vivo since most if not all human cancers contain a disruption in the E2F regulatory pathway (1).Consistent with this, enforced expression of E2F proteins drives quiescent cells into S phase and transforms cells in conjunction with activated Ras (2-6). Furthermore, E2F binding sites are found in the promoters of numerous genes whose expression is necessary for the initiation of S phase and DNA replication (7-9).DNA binding activity of the E2F transcription factor family is composed of two subunits, E2F and DP, which form heterodimeric complexes with a high affinity for the DNA sequence 5Ј-TTTCGCG-3Ј (7-9). Currently, the mammalian E2F family consists of six E2F and two DP genes. The DNA binding, heterodimerization, and marked box domains are highly conserved among all E2F family members. E2F-1, -2, and -3 have an extended N-terminal region containing a nuclear localization signal and binding sites for cyclin A, the transcription factor Sp1, and the E3 ubiquitin ligase complex SCF⅐SKP2 (10 -17). E2F-4, -5, and -6 lack these sequences. E2Fs 1-5 have an acidic transactivat...

E2F and Sp1/Sp3 Synergize but Are Not Sufficient to Activate the MYCN Gene in Neuroblastomas

“…Because E2F-1 has been shown to interact with TRRAP, a subunit of several histone acetyltransferase complexes, E2F-1 may be responsible for the recruitment of Tip60 to the MYCN promoter (35,36). In contrast, binding of E2F and Sp1/Sp3 did not correlate with the nucleosome remodeling that accompanies the switch from basal to full promoter activity.…”

“…E2F-1 has been shown to interact with the histone acetyltransferase p300/CBP and with TRRAP, a component of several histone acetyltransferase complexes that contain either Gcn5 or Tip60 as the enzymatically active subunit (35)(36)(37). However, recruitment of co-factors, histone acetylation, and loading of RNA polymerase can occur prior to the binding of a nuclear extract from the neuroblastoma cells lines SH-EP, SY5Y, and IMR-32.…”

Orchestration of chromatin-based processes: mind the TRRAP

“…These include c-Myc, p53, E2F1, E2F4, E1A, ERa, ERb, VDR, PPARg, LXR, FXR, b-catenin, Skp1 and BRCA1 Lang et al, 2001;Park et al, 2001;Ard et al, 2002;Yanagisawa et al, 2002;Lang and Hearing, 2003;Unno et al, 2005;Cheng et al, 2006;Oishi et al, 2006;Sierra et al, 2006;Finkbeiner and Herceg, unpublished results). Interestingly, the TRRAP domains that interact with diverse partners are not overlapping and it can be speculated that TRRAP may serve not only as a scaffold for HAT complexes but also as a platform for recruitment of different regulatory factors and complexes to chromatin.…”

“…The TRRAP (TRansformation/tRanscription domainAssociated Protein) and its yeast homolog Tra1, remarkably large proteins with homology to the phosphatidyl-inositol-3-kinase (PI3K) family members are common components of many HAT complexes in yeast and mammalian cells (Grant et al, 1998;Vassilev et al, 1998;Allard et al, 1999;Brand et al, 1999;Ikura et al, 2000;Pray-Grant et al, 2002;Sterner et al, 2002). TRRAP was originally identified in the Michael Cole's laboratory as an interacting partner of c-Myc , and subsequent studies showed that several other transcription factors including E2F and nuclear receptors interact with TRRAP Lang et al, 2001;Yanagisawa et al, 2002;Fan et al, 2004). Interestingly, TRRAP/Tra1 appears to be the only component through which HAT complexes (either histone H3 acetylating complexes or those with preference for histone H4) contact transcription activators (Brown et al, 2001;Bhaumik et al, 2004).…”

“…The evidence that simultaneous enrichment of TRRAP, transcription factors and HATs at chromatin is associated with promoter-specific chromatin hyperacetylation Bouchard et al, 2001;Frank et al, 2001;Herceg et al, 2003;Li et al, 2004;Taubert et al, 2004) led to the suggestion that TRRAP functions to recruit HAT complexes to transcription factors bound to their target promoters in chromatin. TRRAP was found as a target of the adenovirus E1A oncoprotein and to be important for oncogenic transformation mediated by viral oncoproteins Deleu et al, 2001;Lang et al, 2001;Lang and Hearing, 2003). We have recently shown that Trrap is involved in the repair of DNA double-strand breaks (DSBs) , indicating that the function of TRRAP and TRRAP-mediated histone acetylation is not limited to transcriptional control, but likely extents to all chromatin-based processes that use DNA as a template.…”