Research in the Kerppola Lab

Outline:

Molecular Architecture and Dynamics of Transcription Regulatory Complexes
DNA bending by Fos-Jun family proteins
Orientation of Fos-Jun heterodimer binding
Dynamics of Fos-Jun-NFAT1 complexes
Functional consequences of Fos-Jun binding orientation
Conformational divergence between Maf-and other bZIP family proteins
Summary

Molecular Architecture and Dynamics of Transcription Regulatory Complexes

Transcription factor interactions control gene expression.
The appropriate growth and differentiation of mammalian cells requires selective regulation of the expression of every gene in the genome. The independent control of individual transcription units is achieved through combinatorial interactions among multiple transcription regulatory proteins at each promoter and enhancer. The participation of multiple transcription regulatory proteins is necessary for the specification of unique sites within the genome whether the regulation occurs at the level of nuclear compartmentalization, chromatin remodeling, coactivator recruitment or assembly of the transcription machinery. Transcription factors can interact either prior to DNA binding or upon binding to the same regulatory region. Interactions between transcription factors can be controlled both independent of DNA binding as well as depending on the DNA regulatory region at which they function. The research in the laboratory is directed toward understanding the assembly and structural organization of transcription regulatory protein complexes.
[Publications on transcription factor interactions]

DNA bending by Fos-Jun family proteins

Interactions among transcription factors require juxtaposition of complementary protein surfaces. These protein surfaces are frequently part of or directly linked to the DNA binding domain, restricting the conformational flexibility of the proteins bound to their recognition sites. Interactions between transcription factors that bind to separate regulatory elements therefore often require bending of the DNA sequences separating the binding sites. Regulatory elements within promoter and enhancer regions are typically separated by DNA segments less than 100 base pairs in length that are relatively inflexible. Proteins that induce bending in the intervening DNA can therefore facilitate transcription factor interactions. Protein-induced DNA bending can also influence the structural organization of transcription factor complexes. We discovered that Fos and Jun induce opposite directions of DNA bending upon binding to the AP-1 site. Different members of the Fos and Jun related protein family induce DNA bends of distinct directions and magnitudes. Substitution of single charged amino acid residues adjacent to the bZIP domains of Fos and Jun alters DNA bending in proportion to the change in net charge. Regions outside the bZIP dimerization and DNA binding domains influence the extent of DNA bending. The opposite DNA bending properties of Fos and Jun are caused by the opposite electrostatic charge distributions of the proteins. Electrostatic interactions also cause the transcription activation domains of Fos and Jun to be located on opposite sides of the DNA helix. The structural organization of the Fos-Jun-AP-1 nucleoprotein complex is likely to determine the functional specificity of the complex at different promoter and enhancer regions.
[Publications on DNA bending]

Orientation of Fos-Jun heterodimer binding

Many mammalian transcription regulatory proteins bind DNA as heterodimeric complexes. Such heterodimers frequently recognize palindromic DNA sequence elements with symmetrical half-sites. These heterodimers can potentially bind to their recognition elements in either of two opposite orientations. Opposite orientations of heterodimer binding can alter interactions with transcription factors that bind to adjacent regulatory elements. We have used heterodimers formed by the proto-oncogene transcription factors Fos and Jun as a model system to investigate the structural basis and functional significance of the orientation of heterodimer binding. We developed a novel gel-based fluorescence resonance energy transfer assay (gelFRET) for quantification of the fraction of heterodimers bound in each orientation. We found that Fos-Jun heterodimers bind in opposite orientations to different AP-1 recognition sequences. Remarkably, amino acid residues and base pairs outside the DNA contact interface observed in the Fos-Jun-AP-1 crystal structure influence the orientation of heterodimer binding. Amino acid residues of opposite charge on Fos and on Jun induce opposite directions of DNA bending, and differences in the bending propensities of sequences flanking the AP-1 site determine the preferred orientation of heterodimer binding. Long-range electrostatic interactions are therefore an important determinant of the architecture of nucleoprotein complexes.
[Publications on Fos-Jun orientation]

Dynamics of Fos-Jun-NFAT1 complexes

The assembly of multiprotein transcription regulatory complexes by proteins that bind to the same regulatory region may occur through random collisions or through facilitated pathways. We have used the Fos-Jun-NFAT1 complex as a model system to investigate the pathway for complex assembly at composite regulatory elements. We developed a multi-color fluorescence resonance energy transfer assay for investigation of the dynamics of transcription complex assembly and conformational isomerization. Cooperative DNA binding by Fos-Jun and NFAT1 requires a specific orientation of heterodimer binding. We found that the orientation of Fos-Jun binding could be reversed in response to the interaction with NFAT1. Remarkably, the orientation of Fos-Jun binding could also be reversed in the presence of protein or DNA competitors. Fos-Jun therefore remain associated with DNA during heterodimer reorientation. The rate of Fos-Jun reorientation was faster than the rate of heterodimer dissociation at some binding sites. Fos-Jun heterodimer reorientation in association with DNA can therefore facilitate Fos-Jun-NFAT1 complex assembly.
[Publications on Fos-Jun-NFAT1 complexes]

Functional consequences of Fos-Jun binding orientation

The preferred orientation of heterodimer binding can influence interactions with transcription factors that bind to adjacent DNA sequences. We have investigated the functional consequences of Fos-Jun orientation preference on cooperative interactions with NFAT1 at composite regulatory elements. We developed a multi-template in vitro transcription assay to compare the transcriptional activities of heterodimers on templates that favor opposite binding orientations. The preferred orientation of Fos-Jun binding affected the stability of Fos-Jun-NFAT1 complexes. Single base pair substitutions that shifted the orientation of heterodimer binding in opposite directions had opposite effects on Fos-Jun-NFAT1 complex stability. Mutations in Fos and Jun that reversed the effects of these base substitutions on heterodimer orientation also reversed their effects on Fos-Jun-NFAT1 complex stability. The preferred orientation of Fos-Jun binding also affected the transcriptional activity of Fos-Jun-NFAT1 complexes. Heterodimers that favored the orientation of binding required for interaction with NFAT1 exhibited higher transcriptional activity in cooperation with NFAT1 than heterodimers that favored the opposite binding orientation. Relocation of the NFAT recognition element to the opposite side of the AP-1 site reversed the transcriptional activities of the heterodimers. The preferred orientation of heterodimer binding can therefore influence the transcriptional activity and promoter selectivity of Fos-Jun-NFAT1 complexes.
[Publications on Fos-Jun orientation]

Conformational divergence between Maf-and other bZIP family proteins

Members of a DNA binding protein family generally adopt a common secondary structure and contact DNA in a similar manner. We have investigated the conformations of an unusual group of bZIP family proteins related to the proto-oncogene c-maf. These proteins contain a bZIP region, an ancillary DNA binding region, and recognize DNA sequence elements that are almost twice as long as elements recognized by other bZIP proteins. We have combined circular dichroism, protease sensitivity, fluorescence anisotropy and DNA contact mapping studies with bioinformatics structural predictions to compare Maf conformations at different DNA binding sites. The results of these studies demonstrate that DNA binding by Maf is coupled to conformational changes that vary at different DNA sequence elements. In contrast to other bZIP family proteins, the basic region of Maf does not adopt a fully a-helical conformation upon DNA binding. The atypical structure of the Maf basic region facilitates the conformational change required for recognition of the extended DNA binding site. The DNA contacts of Maf also differ from those observed in complexes formed by other bZIP proteins. Molecular modeling and threading analysis suggest that the ancillary DNA binding region forms a helical bundle that can contact the extended DNA recognition sequence. Thus, both the secondary structure and the DNA contact interface of Maf differ from those of other bZIP family proteins. DNA binding proteins that are members of the same protein family based on sequence similarity can therefore differ in the structural basis of DNA recognition.
[Publications on Maf family proteins]

Summary

In summary, the conformational variability of transcription factor complexes at different DNA regulatory elements can contribute to the specificity of gene regulation in different cell types and in response to different extracellular signals. Cooperative interactions among transcription factors that bind to the same regulatory region can be affected both by the relative positions, conformations and rotational orientations of the individual transcription factors. The pathway for transcription complex assembly can contribute to the dynamics of transcription activation and repression. Future studies of the architecture and dynamics of transcription factor complexes will identify mechanisms that contribute to the unique temporal and spatial regulation of each gene in the genome.
[Reviews]