We have been studying regulatory elements that are single-stranded when the human c-myc proto-oncogene is expressed and been characterizing the conformation and topology sensitive DNA binding proteins interacting with these elements. First, a cell type, differentiation specific positive cis-element, FUSE, 1.5 kb upstream of promoter P1 is single stranded when c-myc is expressed in vivo and is devoid of nucleosomes except when c-myc is repressed. This element specifically binds FBP, a sequence specific, single strand DNA binding protein. FBP's amino terminus is a potent transcriptional repressor which interacts with TFIIH while the carboxyl is a powerful transcription activator. The activation domain of FBP binds directly with the p62 subunit abd the p89 helicase subunit of TFIIH. A co-repressor with FBP, FIR, binds the central domain of FBP and interacts with TFIIH. FIR increases the affinity of FBP for binding with the FUSE element. FBP activation is defective in xeroderma pigmentosum cells mutated in TFIIH subunits. So endogenous c-myc expression is unresponsive to FBP in XP cells. In XPB cells we have discovered that tight regulation of c-myc is lost and remarkable cell-to-cell variation in MYC levels occur. Thus, the same mutation that diables nucleotide excision repair also deregulates a dangerous oncogene. Moreover we have discovered that this mutation also disturbs normal cell cycle progression and leads to an accummulation of cells in G2/M. Knockdown of FIR with siRNA provokes a similar cell cycle alteration.The central domain of FBP mediates interaction with FUSE. This portion of the protein is composed of repeated """"""""KH"""""""" motifs which comprise a bi-partite DNA binding domain. Sub-domains constituted of the amino-terminal two KH repeats or the carboxyl pair of KH motifs bind weakly and strongly, respectively with upstream and downstream contiguous sequence segments of FUSE. Recent SELEX studies have identified specific DNA sequences interacting with FBP's KH domains and so may enable a genomic analysis of prospective FBP binding sites. Upon binding with FIR, the weak binding sub-domain of FBP recognizes the upstream segment of FUSE with increased affinity. Both FBP and FIR have been shown to control levels of endogenous c-myc expression. FIR repression of c-myc also fails in XP cells. NMR studies of a complex between the strong binding sub-domain and the downstream segment of FUSE suggest that FBP is truly a DNA binding protein as particular features of the complex are unique to DNA-protein interactions. A third protein binds with activating segments in the carboxyl terminus of FBP. This portein also blunts FBP activation. All of these interactions occur both in vivo and in vitro. The mechanism of transcriptional modulation by the FBP/FIR/TFIIH/repressor/co-activator complex reveals that FBP hastens RNA polymerase movement through earliest stages of transcript elongation. In contrast FIR delays the transition of RNA polymerase into its elongation mode. mechanisms. The relevance of FBP to the regulation of c-myc has been established using defective adenoviruses directing the expression of either a dominant negative FBP or antisense FBP mRNA. Either of these viruses rapidly reduces the levels of FBP with a concomitant drop in both c-myc mRNA and protein. Whereas the dominant negative FBP augments single stranded character at FUSE in vivo, antisense FBP mRNA decreases reactivity with permanganate. Additional experiments have exposed the presence of multiple signals embedded within FBP targeting the protein to multiple nuclear compartments. Second, a complex set of factors binds to a cytidine-rich nonanucleotide sequence, repeated five times, found 100-150 bp upstream of P1. This region, required for transcription from P1 as well as augmenting expression from P2, binds several sequence specific, single strand or supercoil DNA binding proteins. hnRNP K binds to this CT-element in supercoiled DNA. In vitro transcription experiments and transfection studies indicate that hnRNP K participates in the stimulation of gene expression through the CT-element. Structural studies are underway to determine how hnRNP K binds with the CT-element. The properties of FBP and hnRNP K prompted examination of c-myc regulatory cis-elements in vivo and in vitro to determine how these sites become melted. Using actively transcribing T7 RNA polymerase to generate torque, recent experiments indicate that the dynamic transmission of mechanical stress destabilizes particular elements, such as FUSE and CT-elements, even in the absence of defined topological boundaries. Recent experiments have been devised to measure the level of superhelical stress transmitted into DNA by ongoing transcription. The level attained is close to the theoretical limit and is suprisingly high, high enough to disturb chromatin and DNA structures and so may of regulatory consequence. Because single-stranded DNA is much more flexible to torsion and flexion than is duplex, interposing CT-elements between genetically interacting sites facilitates these interactions. Therefore one function of regulated single-stranded cis-elements is to serve as protein-DNA hinge. Topological and conformational changes in chromatin structure feature prominently during metaphase when chromosomes condense, transcription ceases, and bound factors generally dissociate from DNA. DNA at transcription start sites was analyzed in vivo during mitosis and found to be conformationally distorted in genes scheduled for reactivation but undistorted for repressed genes. These protein-dependent, conformational perturbations might help to re-establish transcription after mitosis by marking genes for re-expression. Recent work suggests that establishment of this genetic bookmark is a local phenomenon.

National Institute of Health (NIH)
Division of Clinical Sciences - NCI (NCI)
Intramural Research (Z01)
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Clinical Sciences
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Levens, David (2010) You Don't Muck with MYC. Genes Cancer 1:547-554
Kouzine, Fedor; Sanford, Suzanne; Elisha-Feil, Zichrini et al. (2008) The functional response of upstream DNA to dynamic supercoiling in vivo. Nat Struct Mol Biol 15:146-54
Kouzine, Fedor; Levens, David (2007) Supercoil-driven DNA structures regulate genetic transactions. Front Biosci 12:4409-23
Liu, Juhong; Kouzine, Fedor; Nie, Zuqin et al. (2006) The FUSE/FBP/FIR/TFIIH system is a molecular machine programming a pulse of c-myc expression. EMBO J 25:2119-30
Matsushita, Kazuyuki; Tomonaga, Takeshi; Shimada, Hideaki et al. (2006) An essential role of alternative splicing of c-myc suppressor FUSE-binding protein-interacting repressor in carcinogenesis. Cancer Res 66:1409-17
Weber, Achim; Gutierrez, Marina I; Levens, David (2006) The CT-element of the c-myc gene does not predispose to chromosomal breakpoints in Burkitt's lymphoma. Cell Oncol 28:31-5
Chung, Hye-Jung; Liu, Juhong; Dundr, Miroslav et al. (2006) FBPs are calibrated molecular tools to adjust gene expression. Mol Cell Biol 26:6584-97
Liu, J; Levens, D (2006) Making myc. Curr Top Microbiol Immunol 302:1-32
Weber, Achim; Liu, Juhong; Collins, Irene et al. (2005) TFIIH operates through an expanded proximal promoter to fine-tune c-myc expression. Mol Cell Biol 25:147-61
Huth, Jeffrey R; Yu, Liping; Collins, Irene et al. (2004) NMR-driven discovery of benzoylanthranilic acid inhibitors of far upstream element binding protein binding to the human oncogene c-myc promoter. J Med Chem 47:4851-7

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