The long-term goal is to understand how activators stimulate transcription in mammalian cells. We focus on adenovirus (Ad) E1A-conserved region 3 (CR3) as a model activation domain, capitalizing on recent work showing that it activates by binding the Sur2 subunit of the mammalian Mediator complex. We will determine if the E1A-CR3--Sur2 interaction stimulates promoter binding by RNA polymerase II (Pol II) or other general transcription factors (GTFs) by performing promoter binding assays using matrix-bound templates and nuclear extract from wild-type (wt) and sur2-/- embryonic stem (ES) cells. We will determine if the E1A-CR3-- Sur2 interaction retains Mediator at the promoter following initiation, thereby stimulating re-initiation. This will be done by hybridizing a complementary biotinylated 2'-O-methyl oligoribonucleotide to nascent RNA of Pol II elongation complexes stalled at the end of a G-less cassette to isolate templates from which transcription has initiated. We will analyze the effect of the E1A-CR3--Sur2 interaction on the binding of Pol II, GTFs, histones, chromatin remodeling complexes, and histone acetylases to early Ad E2 and E3 promoters in vivo by performing chromatin-immunoprecipitation (ChIP) assays. Recent work has shown that Elk-1, a MAP Kinase-regulated cellular TF, activates the egr-1 and egr-2 promoters by binding Mediator via a Sur2-dependent interaction. ChIP will be used to assess the interaction of Pol II, GTFs, and co-activators with the egr-1 and -2 promoters in response to serum in wt and sur2-/- ES cells. The binding sites of E1A-CR3 and Erk2- phosphorylated Elk-1 on Sur2 will be determined by analyzing Drosophila-human Sur2 chimeras. Recombinant Sur2, Med100, and Med95 will be co-expressed to prepare a soluble Mediator subcomplex for physical-chemical studies of the interaction with E1A-CR3 and Elk-1. Additional cellular TFs that activate by binding Sur2 will be identified by systematic screens of TFs regulated by known signal transduction pathways, through comparison of expressed genes in wt and sur2-/- ES cells and embryo fibroblasts, by analysis of developmental defects in sur2-/- embryos, and of expressed genes in sur2-/- embryos when developmental defects become apparent (E7.5-8.5). Transformation of sur2-/- fibroblasts by oncogenes that activate MAP Kinase pathways will be quantitated. Since many oncogenes inappropriately activate MAP Kinase pathways, these studies may lead to the design of therapeutic strategies that interfere with the interaction between the patholoqically activated TFs and their tarqet in the qeneral transcription machinery.

National Institute of Health (NIH)
National Cancer Institute (NCI)
Method to Extend Research in Time (MERIT) Award (R37)
Project #
Application #
Study Section
Virology Study Section (VR)
Program Officer
Wong, May
Project Start
Project End
Budget Start
Budget End
Support Year
Fiscal Year
Total Cost
Indirect Cost
University of California Los Angeles
Schools of Medicine
Los Angeles
United States
Zip Code
Hsu, Emily; Pennella, Mario A; Zemke, Nathan R et al. (2018) Adenovirus E1A Activation Domain Regulates H3 Acetylation Affecting Varied Steps in Transcription at Different Viral Promoters. J Virol 92:
Ferrari, Roberto; Gou, Dawei; Jawdekar, Gauri et al. (2014) Adenovirus small E1A employs the lysine acetylases p300/CBP and tumor suppressor Rb to repress select host genes and promote productive virus infection. Cell Host Microbe 16:663-76
Gallaher, Sean D; Berk, Arnold J (2013) A rapid Q-PCR titration protocol for adenovirus and helper-dependent adenovirus vectors that produces biologically relevant results. J Virol Methods 192:28-38
Ferrari, Roberto; Su, Trent; Li, Bing et al. (2012) Reorganization of the host epigenome by a viral oncogene. Genome Res 22:1212-21
Kawamata, N; Pennella, M A; Woo, J L et al. (2012) Dominant-negative mechanism of leukemogenic PAX5 fusions. Oncogene 31:966-77
Gil, J S; Gallaher, S D; Berk, A J (2010) Delivery of an EBV episome by a self-circularizing helper-dependent adenovirus: long-term transgene expression in immunocompetent mice. Gene Ther 17:1288-93
Ferrari, Roberto; Berk, Arnold J; Kurdistani, Siavash K (2009) Viral manipulation of the host epigenome for oncogenic transformation. Nat Rev Genet 10:290-4
Gallaher, Sean D; Gil, Jose S; Dorigo, Oliver et al. (2009) Robust in vivo transduction of a genetically stable Epstein-Barr virus episome to hepatocytes in mice by a hybrid viral vector. J Virol 83:3249-57
Balamotis, Michael A; Pennella, Mario A; Stevens, Jennitte L et al. (2009) Complexity in transcription control at the activation domain-mediator interface. Sci Signal 2:ra20
Wang, Wei; Huang, Lu; Huang, Yan et al. (2009) Mediator MED23 links insulin signaling to the adipogenesis transcription cascade. Dev Cell 16:764-71

Showing the most recent 10 out of 54 publications