Structural basis of transcription regulation by CNC family transcription factor, Nrf2.

Several basic leucine zipper (bZIP) transcription factors have accessory motifs in their DNA-binding domains, such as the CNC motif of CNC family or the EHR motif of small Maf (sMaf) proteins. CNC family proteins heterodimerize with sMaf proteins to recognize CNC-sMaf binding DNA elements (CsMBEs) in competition with sMaf homodimers, but the functional role of the CNC motif remains elusive. In this study, we report the crystal structures of Nrf2/NFE2L2, a CNC family protein regulating anti-stress transcriptional responses, in a complex with MafG and CsMBE. The CNC motif restricts the conformations of crucial Arg residues in the basic region, which form extensive contact with the DNA backbone phosphates. Accordingly, the Nrf2-MafG heterodimer has approximately a 200-fold stronger affinity for CsMBE than canonical bZIP proteins, such as AP-1 proteins. The high DNA affinity of the CNC-sMaf heterodimer may allow it to compete with the sMaf homodimer on target genes without being perturbed by other low-affinity bZIP proteins with similar sequence specificity.

A novel allosteric mechanism on protein-DNA interactions underlying the phosphorylation-dependent regulation of Ets1 target gene expressions.

Cooperative assemblies of transcription factors (TFs) on target gene enhancers coordinate cell proliferation, fate specification, and differentiation through precise and complicated transcriptional mechanisms. Chemical modifications, such as phosphorylation, of TFs induced by cell signaling further modulate the dynamic cooperativity of TFs. In this study, we found that various Ets1-containing TF-DNA complexes respond differently to calcium-induced phosphorylation of Ets1, which is known to inhibit Ets1-DNA binding. Crystallographic analysis of a complex comprising Ets1, Runx1, and CBFß at the TCRα enhancer revealed that Ets1 acquires robust binding stability in the Runx1 and DNA-complexed state, via allosteric mechanisms. This allows phosphorylated Ets1 to be retained at the TCRα enhancer with Runx1, in contrast to other Ets1 target gene enhancers including mb-1 and stromelysin-1. This study provides a structure-based model for cell-signaling-dependent regulation of target genes, mediated via chemical modification of TFs.

Crystallization of the Ets1-Runx1-CBFß-DNA complex formed on the TCRα gene enhancer.

Gene transcription is regulated in part through the assembly of multiple transcription factors (TFs) on gene enhancers. To enable examination of the mechanism underlying the formation of these complexes and their response to a phosphorylation signal, two kinds of higher-order TF-DNA assemblies were crystallized composed of an unmodified or phosphorylated Ets1 fragment, a Runx1(L94K) fragment and a CBFß fragment on the T-cell receptor (TCR) α gene enhancer. Within these complexes, the Ets1 and Runx1 fragments contain intrinsically disordered regulatory regions as well as their DNA-binding domains. Crystals of the complex containing unmodified Ets1 belonged to space group P212121, with unit-cell parameters a = 78.7, b = 102.1, c = 195.0 Å, and diffracted X-rays to a resolution of 2.35 Å, and those containing phosphorylated Ets1 belonged to the same space group, with unit-cell parameters a = 78.6, b = 101.7, c = 194.7 Å, and diffracted X-rays to a similar resolution. To facilitate crystallization, a Runx1 residue involved in a hydrophobic patch that was predicted to be engaged in crystal packing based on the previously reported structures of Runx1-containing crystals was mutated.