Tom K. W. Kerppola, Ph.D.—Assistant Investigator
Dr. Kerppola is also Assistant Professor of Biological
Chemistry at the University of Michigan Medical School. He was
born in Finland and received his B.S. and M.S. degrees in
biochemistry and biophysics from Washington State University and
his Ph.D. degree in biochemistry from the University of
California, Berkeley, where he studied with Michael Chamberlin
and Caroline Kane. He was a Helen Hay Whitney postdoctoral fellow
at the Roche Institute of Molecular Biology with Tom Curran,
where he studied gene regulation by the Fos and Jun
proto-oncogene transcription factors.
COOPERATION among transcription factors is a hallmark of eukaryotic gene regulation. A typical eukaryotic promoter contains binding sites for multiple transcription factors, and enhancers frequently consist of complex arrays of control elements. This profusion of regulatory information is required for the finely tuned response of each promoter to diverse extracellular signals. The activation of each gene requires the coordinated action of several regulatory proteins that bind to separate control elements. This cooperative function of different transcription factors in seemingly limitless combinations forms the basis for the regulatory network necessary for the development and function of complex multicellular organisms.
We are investigating the regulatory cooperativity of transcription factors on several different levels. On the most basic level, we are studying interactions among proteins that bind to DNA as dimeric complexes and the functional properties of different monomer combinations. On a more complex level, we are investigating the assembly of multiprotein transcription factor complexes at composite regulatory elements to understand how transcription factors modify each other's properties to extend the function of the complex beyond those of the composite subunits. In addition, we are exploring the role of transcription factor-induced changes in DNA structure, particularly DNA bending, in DNA sequence recognition and in transcription complex assembly. The methods used for these studies range from the biophysical characterization of protein-DNA complex structures to analyses of transcription factor function in cultured cells and in animals.
The protein products of the c-fos and c-jun proto-oncogenes form a heterodimeric complex that can bind and activate transcription of promoters that contain AP-1 sites. Fos and Jun are members of gene families whose protein products can form an extended array of homo- and heterodimeric complexes, all of which can bind to the AP-1 site. We have found that Fos and Jun family proteins can also form heterodimers with more distantly related proteins, including members of the Maf/Nrl protein family. Maf and Nrl are prototypical members of a rapidly expanding subfamily of transcription factors containing the basic region and leucine zipper (bZIP) motif. Members of this subfamily include genes that display exquisite tissue specificity in their expression, such as the retina-specific Nrl; genes expressed during specific stages in development, such as the gene that is defective in the mouse mutant Kreisler; and genes that are ubiquitously expressed.
To identify potential target genes for members of this family, we determined the DNA-binding specificities of both the homodimers formed by Maf and Nrl, as well as the heterodimers they form with Fos and Jun. The DNA recognition sites for these complexes are unexpectedly large, extending over at least 13 base pairs for the Maf and Nrl homodimers. To recognize these large binding sites, these proteins contain an ancillary DNA-binding domain that is adjacent to the basic region conserved among all bZIP family proteins. The DNA-binding specificities of the Maf and Nrl homodimers and the heterodimers they form with Fos and Jun are distinct, and they differ from those of heterodimers formed among Fos and Jun family members. Thus each family of complexes may have a distinct set of target genes.
Activation of T cells by antigen presentation results in the rapid induction of a spectrum of cytokine genes that regulate the subsequent immune response. The signal elicited by the T cell receptor is mediated in part by an elevation in intracellular calcium, which activates the serine-threonine phosphatase calcineurin. The signal can be intercepted, and cytokine gene induction blocked, by the clinically important immunosuppressive drugs cyclosporin A and FK506, which inhibit calcineurin phosphatase activity.
One substrate of calcineurin is a subunit of the nuclear factor of activated T cells, known as NFATp. The dephosphorylation of NFATp results in its translocation into the nucleus, where it binds to regulatory elements in cytokine gene promoters. In the interleukin-2 promoter, NFATp binding to a composite regulatory element leads to the recruitment of Fos and Jun family proteins to a ternary complex. We have investigated the assembly and functional properties of this ternary complex, in collaboration with the laboratory of Anjana Rao (Dana-Farber Cancer Institute). The DNA-binding specificity of the complex is primarily contributed by NFATp, which can bind to the element in the absence of Fos and Jun. In contrast, Fos and Jun do not bind to the element with high affinity in the absence of NFATp.
The regions of Fos and Jun required for ternary complex formation coincide with the leucine zipper and basic regions required for binding to the AP-1 site. However, these regions are not sufficient to confer transcriptional activity, as the transcription activation domains of Fos and Jun provide at least part of the transcription activation potential of this complex. Thus there is an apparent separation of functions in this complex, with NFATp providing the DNA recognition specificity and Fos and Jun providing the transcriptional activity.
Regulation of transcription initiation requires that proteins that are bound to upstream regulatory elements interact with components of the general initiation complex assembled at the site of transcription initiation. Since the distance separating these sites is generally longer than can be spanned by individual proteins, the intervening DNA must be distorted to form a loop. Such looping is energetically unfavorable and may become kinetically limiting at short distances. Factors that induce DNA bending can reduce the unfavorable free energy and increase the rate of complex formation. This provides a potential mechanism for transcription factor cooperativity.
We investigated DNA bending by members of the bZIP family and found that different members of this family induce DNA bends of distinct orientations and magnitudes. This family also includes members that do not induce significant DNA bending. The opposite DNA bend orientations induced by different family members correlate with the presence of oppositely charged amino acid residues in a region immediately adjacent to the basic domain, suggesting that these residues influence the DNA bend orientation. Thus electrostatic interactions between residues flanking the basic region and the phosphodiester backbone are likely to contribute to DNA bending by the bZIP motif.
No DNA bending was observed in x-ray crystallographic analysis of Fos and Jun peptides bound to an AP-1 site. However, electrostatic interactions can be shielded by the high concentrations of divalent cations used during crystallization of the Fos-Jun-DNA complex. In collaboration with Jack Griffith's laboratory (Lineberger Cancer Center), we used electron microscopy to visualize directly the DNA bend induced by Fos and Jun, using an intrinsic bend as an internal standard. Comparison of the DNA bends induced by a series of Fos and Jun deletion derivatives indicates that regions outside of their DNA-binding domains modulate the extent of DNA bending. These regions coincide with the transcription activation domains of Fos and Jun. Thus DNA bending may contribute to the functional activities of this family of transcription-regulatory proteins.
A full understanding of the parameters governing transcription complex assembly requires determination of the rate and affinity of transcription factor interactions under conditions approximating the physiological environment. We used the Fos-Jun model system to develop an approach based on fluorescence energy transfer for the detection of transcription factor interactions in solution. The proteins are labeled at unique positions with fluorescein and rhodamine. We can then examine their association in real time by monitoring the energy transfer that occurs when the fluorophores are brought into close proximity. This approach allows measurement of the dissociation coefficient of the Fos-Jun heterodimer, as well as the half-times for Fos-Jun association, DNA binding, and subunit exchange. We are in the process of applying these methods to the analysis of higher-order transcription factor complexes. Through the analysis of the structure and dynamics of these complexes, we hope to gain an understanding of the mechanisms of transcription factor cooperativity.
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