Assembly of Transcriptional Regulatory Factors, c-Myb and C/EBP β from Separated Sites on a Promoter Assembly of stereospecific, multiprotein complexes on enhancers and promoters is a key step in transcriptional activation. Recent X-ray analyses of high-order complexes comprised of transcription factors bound to DNA have concentrated exclusively on cases in which interactions between transcription factors enhance their cooperative binding to adjacent sites on a promoter. In many eukaryotic genes, however, transcription factors bind to promoters at sites distant from one another, yet act synergistically to activate transcription. It has been proposed that DNA looping mediated by their interaction brings transcription factors scattered along the DNA into sufficiently close proximity to enable them to form nucleoprotein complexes. Here we address this problem in the case of the synergistic trans -activation of myeloid genes by c- M yb and C/EBP β bound distantly from each other to the DNA . T he c- M yb transcriptional regulator is involved in the proliferation and differentiation of hematopoietic cells. S pecifically, it cooperates with a C/EBP (C AA T/ enhancer-binding protein ) family member to induce myeloid cell differentiation. c- M yb has three Fig. 1. Overview of three closely packed c-Myb–C/ EBP β –DNA complex molecules [3]. α - helical regions within the proteins are shown as ribbons; the remaining parts are shown as tubes. c-Myb R1, R2 and R3 and C/EBP β chains A and B are respectively in dark yellow, pink, blue, yellow and green. The DNA molecules are shown in a stick representation. and atomic force microscopy ( A FM) analyses were carried out [3] . X-ray diffraction data were collected on a RI KE N beamline BL45XU [4] . In the c- M yb ( R 1 R 2 R 3)–C /EBP β ( b Z ip )– DNA complex (F ig. 1) , c- M yb –C/EBP β intercomplex interactions were observed between the R 2 subdomain of c- M yb bound to one DNA fragment and the C -terminal leucine- z ipper part of bound to another DNA fragment. O n the c- M yb side, a hydrogen bond between a backbone amide of c- M yb R 2 and a DNA minor groove phosphate, which plays an important role in the c- M yb – DNA interaction [5 , 6] , becomes stabili z ed by a hydrogen-bonding network provided by binding. T hese findings led us to assume an interaction between c- M yb and separately bound to the native promoter DNA, with an intervening DNA loop, as shown in F ig. 2 , where the mim-1 promoter was used as a native promoter. T he oncogenic mutation points noted in the A MV v- M yb map near the interaction site with C/EBP β , and the c- M yb –C/EBP β interactions were disrupted in the crystal of the A MV v- M yb ( truncated R 1 R 2 R 3)–C/ EBP β ( b Z ip )– DNA complex [3] . T he data on M yb –C/EBP β tandem subdomains ( R 1 , R 2 and R 3) , each bearing a helix-turn-helix ( H T H ) -related motif, in its DNA-binding domain [1 , 2] , whereas C/EBP family members contain a b Z ip-type motif for DNA binding. T he mim-1 promoter is a well-characteri z ed c- M yb target gene promoter and this gene is a marker for granulocyte differentiation. Its promoter region includes the binding sites for c- M yb and C/EBP family members, which are critical for transcriptional regulation, separated by an intervening se q uence of ~ 80 base pairs. Avian myeloblastosis virus ( A MV) v- M yb, an oncogenic mutant of c- M yb, has no synergistic capacity with C/EBP for trans -activation of the mim-1 gene, resulting in the inhibition of granulocyte differentiation and induction of leukemia. T o establish the structural basis of the trans - activational synergy between c- M yb and C/EBP family members, we analy z ed the complex structures including c- M yb or A MV v- M yb, C/EBP β and the promoter DNA. B ecause the X-ray crystallographic analysis of a DNA-loop-containing multiprotein – DNA complex seemed to be q uite difficult, combinational X-ray crystallography experiments C/EBP β C/EBP β C/EBP β 10 Tahir H. Tahirov a,c , Ko Sato a,b and Kazuhiro Ogata a,b,c (a) Kanagawa Academy of Science and Technology (KAST) (b) Yokohama City University School of Medicine (c) SPring-8/ RIKEN E-mail: ogata@med.yokohama-cu.ac.jp Fig. 2. A modeled structure of the complex composed of c-Myb, C/EBP β and the mim-1 promoter DNA, showing DNA loop formation [3,6]. interactions in crystals were consistent with those from the GST pull-down experiments in solution using GST-fused c-Myb or AMV v-Myb, and radioisotope- labeled C/EBP β or its mutants [3]. The proposed DNA loop formation in the c-Myb–C/EBP β – mim-1 DNA complex was confirmed by using the A F M method. The A F M observations indicated that about 75% of the c-Myb and C/EBP β -bound mim-1 promoter DNA exhibited DNA looping, whereas the AMV v-Myb and C/EBP β -bound mim-1 promoter DNA had no DNA looping ( F ig. 3). F rom the luciferase trans -activation assays, the aforementioned c-Myb–C/EBP β interaction was shown to be critical for the cooperative trans - activation of the mim-1 gene by c-Myb and C/EBP β [3]. In transcriptional regulation, two factors are considered to be involved: the stabilization of the regulatory factor–DNA complex and the stereospecific assembly of these proteins on the looped or deformed D N A . F o r t h e s t a b i l i z a t i o n o f p r o t e i n – D N A interactions, a direction-sensitive protein backbone amide–DNA phosphate hydrogen bond, whose formation depends on the protein conformation and which is surrounded by the sidechains of adjacent residues interacting with DNA minor groove atoms and/or partner protein atoms, seems to be critical [ 5 , 6 ]. F or the stereospecific assembly of regulatory factors, particularly protein-induced DNA looping, there are few examples for which structure/function relationships have been analyzed. The present study p r o v i d e s i m p o r t a n t c l u e s t h a t i n c r e a s e o u r understanding of the regulation of trans -activation mediated by distantly bound transcription factors and the dysregulation caused by oncogenic mutations. It is expected that extensive studies in the future will elucidate the entire molecular mechanism of enhanceosome or repressosome formation. Fig. 3. Representative AFM images of mim-1 promoter DNA complexes with c-Myb and C/EBP β (left), and with AMV v-Myb and C/EBP β (right) [3,6]. The bottom scheme shows the DNA construct used for the AFM experiments. The mim-1 promoter with the separated c-Myb and C/EBP β - binding sites was fused to the pGL3 vector. References [ 1 ] K. Ogata et al. : Cell 79 ( 1994 ) 6 3 9 . [ 2 ] K. Ogata et al. : Nature Struct. Biol. 2 ( 1995 ) 3 09 . [3] T.H. Tahirov et al. : Cell 108 ( 2002 ) 57 . [ 4 ] T.H. Tahirov et al. : Acta Cryst. D 57 ( 2001 ) 1655 . [ 5 ] T.H. Tahirov et al. : Cell 104 ( 2001 ) 755 . [ 6 ] K. Ogata, K. Sato and T.H. Tahirov: Curr. Opin. Struct. Biol. 13 ( 200 3) 40 . 11
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