Enhanceosome factors ATF-2/c-Jun and IRF-3 complexed with the interferon-beta enhancer

Oliver DeBarros '15 and Nate Petrou '15


I. Introduction

Enhanceosomes are complexes of proteins that bind cooperatively to a gene's enhancer region.  Enhanceosomes are necessary for transcriptional activation and important in the recruitment of basal transcription factors to the promoter. One of the best understood enhanceosome complexes is the virus-inducible enhancer of the interferon-beta (IFN-beta) gene.  In the presence of a virus, interferon proteins are made by the IFN-beta gene, which is activated by enhanceosome assembly.  The assembly of the enhanceosome of the IFN-beta gene involves the group of transcription factors including ATF-3, c-Jun, IRF-3/IRF-7, NF-kB and HMGA1. Through DNA binding to 4 different positive regulatory domains (PRDs) I-IV of the IFN-beta enhancer these transcription factors can activate the gene. A high level of specificity in the activation of the gene is achieved through the cooperative binding of these proteins.

II. General Structure

This structure focuses on the transcription factors ATF-2, c-Jun, IRF-3A and IRF-3B in complex with 31 bp of the PRDIV-PRDIII region of the IFN-beta enhancer.   ATF-2 and c-Jun are two long alpha helices that form a leucine zipper heterodimer. Both proteins contain a DNA-binding region containing several basic amino-acid residues; as well as a dimerization domain following a coiled-coil motif with heptad repeats. The heterodimer interacts in a sequence specific manner with the PRDIV enhancer within the promoter element of the gene. The IRF-3 domains are composed of a four-stranded antiparallel beta-sheets, three alpha helices, and 3 long loops. The IRF-3 domains interact with DNA and cooperatively recruit the ATF-2/c-Jun heterodimer; creating a complex that recognizes a continuous sequence of approximately 24 base pairs within the PRDIV and PRDIII enhancer elements of the IFN-beta promoter. The enhanceosome's interaction with DNA is mostly sequence specific. 

III. ATF-2 and c-Jun DNA Binding

While the C-terminus of the leucine zipper is involved in the dimerization interface between ATF-2 and c-Jun, the N-terminus interacts with DNA.  Basic amino acid residues, primarily Lysine and Arginine, make extensive contact with the major groove of the DNA. The ATF-2/c-Jun complex binds in the PRDIV region to an 8 bp sequence 5'-TGACATAG-3', similar to the high-affinity CRE recognition sequence 5'-TGACGTCA-3'.  ATF-2 binds the consensus half site (TGAC) and c-Jun binds the nonconsensus half site (ATAG), resulting in asymmetric binding favoring ATF-2.   ATF-2 and c-Jun both bind DNA through hydrogen bonding to either the bases or phosphate backbone.  Van der Waals forces are also present between DNA and both bZIP proteins.  One important interaction of ATF-2 involves the conserved Asn 344 residue.  Asn 344 hydrogen bonds with O4 of base T5 as well as N4 of base C27* (*refers to c-Jun) on the opposite strand. One residue of c-Jun critical for DNA interaction is R270, which engages in three hydrogen bonds; two with the phosphate backbone and one with the base adenine.

IV. IRF-3 DNA Binding

The interferon regulatory factor (IRF) domains are characterized by a conserved N-terminal DNA-binding domain of around 120 amino acids that recognizes conserved DNA sequences, named IFN-stimulated response elements (ISRE), in the PRDIII region.  IRF-3A and IRF-3B have essentially the same structure and make many similar DNA contacts. IRF-3A and IRF-3B molecules are bound flanking the PRDIII region on opposite faces of DNA, with the DNA duplex bending slightly around the IRF recognition alpha helix. The alpha3 recognition helix makes major groove contacts along the DNA backbone, such as hydrogen bonds between Arg 81 and the conserved G.   IRF-3 also interacts with the minor groove, primarily by the L1 Loop.  His 40, which is conserved in all IRF domains, participates in two water-mediated hydrogen bonds, both as an acceptor and donator.  Leu 42, specific to IRF-3, provides van der Waals contacts.
Also, after viral infection, IRF-3 is phosphorylated at several serine and threonine residues which stimulates DNA binding and increases transcriptional activation.

V. Cooperative Binding

Similar to other bZIP structures, the C-terminal leucine zipper of the ATF-2/c-Jun heterodimer has a parallel coiled-coil dimerization interface extending perpendicularly to the DNA.  The leucine zipper structural motif promotes dimerization by interaction of hydrophobic leucines at the
d position of the heptad repeat.    Also, long, charged residues at the e and g positions of the heptad repeat are important for dimerization, exemplified by pairs E379-R302*  and E386-K309*.   A salt bridge between E363 and R276* also contributes to stabilization.     

Binding of ATF-2/c-Jun and IRF-3 to DNA is a cooperative process, with the proteins making contacts with each other as well.  IRF-3 preferentially binds to ATF-2/c-Jun bound DNA, evidenced by the fact that IRF-3 exhibits decreased binding with free DNA.  ATF-2 is contacted by the L1 and L3 loops of IRF-3A, through hydrogen bonds between amino acids.   Extensive hydrogen bonding also occurs at the interface of ATF-2 and IRF-3A.   The bending of the DNA favors IRF-3A interaction, rather than IRF-3B, with ATF-2/c-Jun due to the lack of binding of c-Jun to the nonconsensus sequence. 

IRF-3A binds cooperatively to IRF-3B despite the fact that there are no protein-protein contacts between the two.  They both bind DNA simultaneously and the position of one IRF influences binding of the other.   

VI. References

Escalante CR, E Nistal-Villan, L Shen, A Garcia-Sastre, AK Aggarwal.  2007.  Structure of IRF-3 bound to the PRDIII-I regulatory element of the human interferon-beta enhancer.  Molecular Cell 26: 703-16.   

Panne, Daniel, Tom Maniatis, and Stephen C Harrison.  2004. Crystal structure of ATF-2/c-Jun and IRF-3 bound to the interferon-beta enhancer.  The EMBO Journal 23: 4384-4393.

Panne, Daniel, Tom Maniatis, and Stephen C Harrison.  2007.  An atomic model of the interferon-beta enhanceosome.  Cell 129: 1111-23.

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