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| Howard Hughes Medical Institute | and | The University of Michigan | |||||||||||||||||||||||||||||
We are interested in understanding how transcription factors assemble at promoters and enhancers and act cooperatively to regulate transcription initiation. The laboratory investigates both the structural basis of transcription complex assembly as well as the functional significance of the organization of transcription regulatory proteins on DNA. In these studies, we make use of a wide variety of experimental approaches, including several novel methods developed in the laboratory. Some of the interests of the laboratory include:
Interactions between transcription factors that bind to separate sites on DNA are likely to require bending of the sequences separating the binding sites. Thus, proteins that induce DNA bending may facilitate transcription factor interactions. We have discovered a novel mechanism of transcription factor induced DNA bending based on electrostatic interactions between charged amino acid residues and the phosphodiester backbone of DNA. This mechanism causes the opposite directions of DNA bending induced by different bZIP family proteins. For further information, see references 56, 55, 54, 33, 26, 17 and 16.
Many eukaryotic transcription factors bind to palindromic recognition sites as heterodimeric complexes. Such complexes could in principle bind in either orientation. The opposite sides of heterodimeric transcription complexes are likely to differ in their interactions with transcription factors that bind to adjacent sites. Thus, opposite orientations of heterodimer binding may result in distinct transcriptional activities. We have found that Fos-Jun heterodimers bind to many AP-1 sites in a preferred orientation. The orientation of heterodimer binding is determined by direct contacts to the central asymmetric base pair and the bendability of flanking DNA sequences. For further information, see references 60, 59, 58, 56, 55, and 54.
Transcription regulatory proteins frequently act in concert to regulate gene expression. One mechanisms for synergistic transcription activation is mediated by cooperative DNA binding by transcription factors that bind to adjacent regulatory elements. The nuclear factor of activated T cells (NFAT) transcription factor complex is composed of a member of the NFAT family in association with a dimer of Fos and Jun family proteins. We have discovered that the interaction between NFAT1 and Fos-Jun can reverse the orientation of Fos-Jun heterodimer binding and induce DNA bending toward the interaction interface. We have also identified the amino acid residues in Fos and Jun that are energetically most important for their asymmetric interaction with NFAT1. For further information, see references 60, 59, 52, 49, 48, 43, 39, 30 and 28.
Members of the bZIP family bind to a variety of DNA recognition sequences. Nevertheless, virtually all of the proteins in this family share the amino acid residues that contact the AP-1 site in the X-ray crystal structure of the Fos-Jun-AP1 complex. We have found that members of the Maf subfamily of bZIP proteins bind to a longer DNA recognition element. These proteins also contain an auxiliary DNA binding domain adjacent to the basic region. Thus, DNA recognition by some bZIP family members involves protein regions in addition to the bZIP domain. For further information, see references 58, 56, 55, 52, 44, 40, 36 and 13.
Heterodimers formed between different bZIP family members differ in both their DNA binding selectivity and in their interactions with other transcription factors. We have shown that the glucocorticoid receptor inhibits DNA binding and transcription activation by Fos-Jun heterodimers, but does not block the activity of Jun homodimers. The inhibition of DNA binding and transcription activation results from a direct interaction between the zinc finger DNA binding domain of GR and the transcription activation domain of Fos. Thus, different heterodimeric complexes have distinct regulatory functions. For further information, see references 58, 52, 49, 39, 34, 26, 24 and 13.
We pioneered the use of gel electrophoretic phasing analysis for quantitation of the direction and magnitude of protein induced DNA bending. This approach enables us to distinguish protein induced DNA bending from other stuctural distortions caused by protein binding to DNA. We have measured DNA bending by over 500 different protein-DNA complexes using this method, and developed quantitative models for DNA bending by a variety of protein-DNA complexes. We have extended the phasing analysis approach in multifactorial phasing analysis to exclude the possibility that factors other than DNA bending influence the results. Thus, gel electrophoretic phasing analysis provides a straightforward and reliable method for quantitation of protein induced DNA bending. For further information, see references 59, 57, 56, 55, 54, 53, 33, 26, 17 and 16.
We have developed a novel approach for analysis of the structural organization of multiprotein transcription factor complexes. This approach is based on comparison of the relative efficiencies of fluorescence resonance energy transfer between fluorophores placed at different positions on the DNA and fluorphores linked to different subunits of a multiprotein complex. We have applied this method to the analysis of the architectures of Fos-Jun-AP1 and NFAT1-Fos-Jun-ARRE2 transcription factor complexes. This powerful approach has broad applicability in studies of the structures and dynamics of nucleoprotein complexes. For further information, see references 60, 59, 58, 52, and 38.
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