Dynamics of Fos-Jun-NFAT1 complexes

Abstract

Transcription initiation in eukaryotes is controlled by nucleoprotein complexes formed through cooperative interactions among multiple transcription regulatory proteins. These complexes may be assembled via stochastic collisions or defined pathways. We investigated the dynamics of Fos-Jun-NFAT1 complexes by using a multicolor fluorescence resonance energy transfer assay. Fos-Jun heterodimers can bind to AP-1 sites in two opposite orientations, only one of which is populated in mature Fos-Jun-NFAT1 complexes. We studied the reversal of Fos-Jun binding orientation in response to NFAT1 by measuring the efficiencies of energy transfer from donor fluorophores linked to opposite ends of an oligonucleotide to an acceptor fluorophore linked to one subunit of the heterodimer. The reorientation of Fos-Jun by NFAT1 was not inhibited by competitor oligonucleotides or heterodimers. The rate of Fos-Jun reorientation was faster than the rate of heterodimer dissociation at some binding sites. The facilitated reorientation of Fos-Jun heterodimers therefore can enhance the efficiency of Fos-Jun-NFAT1 complex formation. We also examined the influence of the preferred orientation of Fos-Jun binding on the stability and transcriptional activity of Fos-Jun-NFAT1 complexes. Complexes formed at sites where Fos-Jun favored the same binding orientation in the presence and absence of NFAT1 exhibited an 8-fold slower dissociation rate than complexes formed at sites where Fos-Jun favored the opposite binding orientation. Fos-Jun-NFAT1 complexes also exhibited greater transcription activation at promoter elements that favored the same orientation of Fos-Jun binding in the presence and absence of NFAT1. Thus, the orientation of heterodimer binding can influence both the dynamics and promoter selectivity of multiprotein transcription regulatory complexes.M any transcription regulatory proteins form heterodimers that bind to palindromic DNA sequences. Such heterodimers can potentially bind their recognition sites in either of two opposite orientations (1-5). Heterodimers that bind in opposite orientations present different protein surfaces for interactions with adjacent DNA binding proteins. Thus, the orientation of heterodimer binding may influence interactions with other transcription factors. Opposite orientations of heterodimer binding can result in different transcriptional activities (6-9). The mechanisms whereby the orientation of heterodimer binding controls transcriptional activity have not been defined.Fos and Jun family basic region-leucine zipper (bZIP) proteins regulate the expression of different genes in different cell types through cooperative interactions with other transcription factor families (10). Complexes formed by Fos and Jun with members of the NFAT family at composite regulatory elements induce cytokine gene expression in T cells activated by antigen presentation (11,12). The x-ray crystal structures of DNA-bound complexes formed by the bZIP domains of Fos and Jun alone as well as by the DNA binding domains of Fos, Jun...

The NFATc2 Gene Is Involved in a Novel Cloned Translocation in a Ewing Sarcoma Variant That Couples Its Function in Immunology to Oncology

“…This cooperative action has been well documented both for ETS-AP1 (8), ERG-AP1 (9), and more interestingly for EWSR1/ FLI1-AP1 (10) and NFAT-AP1 (25). It has been shown that both NFAT and ETS transcription factors interact with the bZIP motifs of the Fos-Jun complex (9,10,25,26). Intriguingly, when comparing the sequence recognition patterns of these two gene families (ETS and NFAT), an overlap of the core consensus sequence was observed.…”

Asymmetric Recognition of Nonconsensus AP-1 Sites by Fos-Jun and Jun-Jun Influences Transcriptional Cooperativity with NFAT1

“…Assembly of these nucleoprotein complexes can be affected by the orientations of the individual regulatory elements (26,27,39,40). Some heterodimers that recognize palindromic DNA sequences also exhibit orientation-dependent transcriptional activities at regulatory elements that contain asymmetric base pairs either within the recognition site or in flanking sequences (6,10,35,36). This orientation dependence can be mediated by cooperative DNA binding with transcription factors that recognize adjacent binding sites within composite regulatory elements (10,35,36).…”

“…Some heterodimers that recognize palindromic DNA sequences also exhibit orientation-dependent transcriptional activities at regulatory elements that contain asymmetric base pairs either within the recognition site or in flanking sequences (6,10,35,36). This orientation dependence can be mediated by cooperative DNA binding with transcription factors that recognize adjacent binding sites within composite regulatory elements (10,35,36).Fos and Jun family bZIP proteins form homo-and heterodimers via a leucine zipper dimerization interface and bind to palindromic AP-1 recognition elements [TGA(C/G)TCA] via a basic DNA contact region. Fos and Jun make essentially identical contacts with the two half-sites in the X-ray crystal structure of the Fos-Jun-AP-1 complex (13).…”

“…The orientation of heterodimer binding can influence cooperative interactions with transcription factors that bind to adjacent regulatory elements (8,10,35,36). Fos-Jun heterodimers can form cooperative complexes with NFAT1 at composite NFAT-AP-1 regulatory elements that activate cytokine gene expression in response to antigen presentation (17).…”

Opposite Orientations of a Transcription Factor Heterodimer Bind DNA Cooperatively with Interaction Partners but Have Different Effects on Interferon-β Gene Transcription

“…Interactions between such heterodimers and transcription factors that bind adjoining sequences are generally thought to require a specific orientation of heterodimer binding. For instance, cooperative DNA binding by Fos-Jun, Fos-Activating Transcription Factor 2 (ATF2), 2 or ATF2-Jun heterodimers with Nuclear Factor of Activated T cells 1 (NFAT1) at composite regulatory elements imposes a fixed orientation of heterodimer binding (3)(4)(5)(6)(7). Differences in the preferred orientation of Fos-Jun heterodimer binding at different regulatory elements affect cooperative DNA binding and synergistic transcription activation with NFAT1 (6 -8).…”