Kinetic analysis of the interaction of b/HLH/Z transcription factors Myc, Max, and Mad with cognate DNA.

Myc, Max and Mad are members of the basic helix-loop-helix leucine zipper family of transcription factors. Myc was first discovered as the protooncogene of avian retroviruses inducing lymphoid tumors (1). It is believed to regulate 15% of all genes (2). Deregulation of Myc has been implicated in the development of many human cancers, including Burkitt lymphoma, neuroblastomas, small cell lung cancers, breast cancers, oesophageal, adenocarcinoma and meduloblastomas (3, 4). c-Myc is over expressed in neoplasia by a number of different mechanisms, including gene amplification, translocation, retroviral insertion, and activation of pathways upstream of c-Myc expression (5). The Myc family proteins, c-Myc, n-Myc and l-Myc, have been implicated almost exclusively in cell proliferation, differentiation and neoplasia (6–10). However, recent advances have shown that c-Myc is involved in a wide catalog of cellular activity, which includes recruitment to the transcriptosome of a variety of transcriptional effectors including TRRAP (transformation/transcription) domain associated protein, Miz1 (transcriptional repressor), and the E2 ubbiquin ligase Skp2; and activates the cad gene that encodes the trifunctional enzyme carbamoyl-phosphate synthase/aspartate transcarbamoylase/dehydroorotase, which is required for the first three rate-limiting steps of pyrimidine biosynthesis (11–13). Since neither dimerization nor DNA specific binding could be readily demonstrated for Myc protein, a search for Myc interacting proteins led to the identification of Max protein. The Mycobligate factor X, Max, is a b/HLH/Z1 family protein similar to Myc, but lacks the transactivation domain. Max can also form homodimers. Studies showed that it may act as a transcription repressor in a homodimer form (14). All known oncogene functions of Myc require dimerization with Max. The Myc·Max transcription activator is involved in the transcriptional regulation of target genes associated with cellular growth, proliferation, metabolism and differentiation (7, 15). The fact that Max is expressed in the absence of Myc lead to searches for other Max-interacting partners. Mad family proteins were all identified in expression cloning screens by their ability to bind specifically to Max (16–18). Similarly to Myc, Mad homodimerizes poorly but interacts with Max, forming a sequence-specific DNA binding complex similar to Myc·Max heterodimer. Overexpression of Mad in tissue culture and mice interferes with cell proliferation and inhibits transformation (15). Mad and Myc compete for binding to Max. The x-ray crystal structures of the b/HLH/Z domains of Myc·Max and Mad·Max revealed that both heterodimers bind to their common DNA target, the enhancer box (E box) hexanucleotide (5′-CACGTG-3′) (8, 19). E-boxes are located in the proximal region of class II nuclear gene promoters, between 50 and 200 base pairs upstream of the transcription sites. Binding of these structurally similar transcription factor dimers to the E-Box determines whether a cell will divide and proliferate (Myc·Max) or differentiate and become quiescent (Mad·Max). Many transcriptional factors form dimeric complexes with DNA (20, 21). In the absence of DNA, they may be found either as monomers (22) or dimers (23). Homodimer/heterodimer of Max-Max, Myc-Max, and Mad-Max transcription factors recognized DNA E-box (24). The discrimination between cognate and nonspecific Max b/HLH/Z/DNA complex have been reported using EMSA, CD, NMR techniques (25, 26) and crystal structures using X-ray crystallography (27). Mass spectrometry/proteolysis has also been employed to characterize Max binding to specific and non-specific DNA (28). Given the ever expanding catalog of c-Myc functions as a transcription factor, we in this laboratory ascertained that little quantitative were available regarding the thermodynamic cycle of how the Myc/Max/Mad network behaves in relation to theirprotein-protein interactions and to the E box. In this study, as in our previous work, the b/HLH/Z domains of Myc and Max and full length Mad1 were studied (see materials). In our first study, Hu et al. (2005) using fluorescenceanisotropy showed that specific binding between MLP DNA and Max was about 10-fold higher affinity than LCR DNA and about 100 fold higher than for non-specific DNA. USF had a similar binding affinity as Max to MLP DNA (29, 30) but Max bound more tightly to LCR and non-specific DNA. A series of E-Box alterations showed for Max binding that most of the energy involved could be attributed to the two halves of the E-box showing virtually no cooperativity. Measurement of binding for the entire thermodynamic cycle of monomer-dimer-DNA interactions showed that monomer-Max-DNA complex showed a significantly reduced affinity for the second monomer of Max. Our work showed that Myc-Max formed the most stable heterodimer. Further, Banerjee et al. (2006) also from this laboratory showed that formation of a dimer complex might be affected by a general environment with respect to charge. Thus by fluorescence anisotropy it was shown that poly-L-lysine had an insignificant effects, poly-L-glutamic acid stabilized both homodimers and heterodimers. These results showed that dimerization of all three monomers in a negatively charged milieu were driven by both negative enthalpy and entropic contributions. van der Waals and H-bonding interactions are most likely at play in the formation of these complexes. Kinetic analysis has shown that assembly of the dimeric transcription factors (bZIP and bHLHZip) and specific DNA sites follows a pathway in which two protein monomers bind DNA sequentially and form their dimerization interface while bound to DNA (monomer pathway) (31). The individual steps of this process provide kinetic control which will lead to different intermediates that can interact at different rates with other proteins and non-specific DNA and ultimately allow for discrimination between specific and non-specific DNA. Therefore, it is important to determine the rate limiting steps and kinetics of the individual steps in order to understand regulation of transcription by cellular and viral proteins and other pharmacological agents. In this work, stopped-flow anisotropy techniques were utilized to study the dimerization kinetics of Max, Myc and Mad in the absence and presence of DNA E-box. The rate constants for Myc·Max, Mad·Max and Max·Max dimerization and the temperature dependence of these reactions were measured and compared. Our kinetic data showed that Myc·Max dimerization is faster than Max·Max or Mad·Max. The Myc, Max and Mad dimerization rates were found to be concentration independent suggesting conformational changes are rate limiting. In order to understand the assembly of transcription complex, we analyzed whether Myc or Mad competes to form a heterodimer with Max at the DNA E-box. Further, we measured and compared the dimer-DNA and monomer-DNA binding rate of the individual steps in the assembly pathway (monomer/dimer pathway) and determined the activation energies of the reactions.