Search

Retinoic acid-related orphan receptor gamma (RORγ) plays critical roles in regulating various biological processes and has been linked to immunodeficiency disorders and cancers. DNA recognition is essential for RORγ to exert its functions. However, the underlying mechanism of the DNA binding by RORγ remains unclear. In this study, we present the crystal structure of the complex of RORγ1 DNA-binding domain (RORγ1-DBD)/direct repeat DNA element DR2 at 2.3 Å resolution. We demonstrate that RORγ1-DBD binds the DR2 motif as a homodimer, with the C-terminal extension (CTE) region of RORγ1-DBD contributing to the DNA recognition and the formation of dimeric interface. Further studies reveal that REV-ERB-DBD and RXR-DBD, also bind the DR2 site as a homodimer, while NR4A2-DBD binds DR2 as a monomer. Our research uncovers a binding mechanism of RORγ1 to the DR2 site and provides insights into the biological functions of RORγ1 and the broader RORs subfamily., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Class IIa Histone deacetylases (HDACs), including HDAC4, 5, 7 and 9, play key roles in multiple important developmental and differentiation processes. Recent studies have shown that class IIa HDACs exert their transcriptional repressive function by interacting with tissue-specific transcription factors, such as members of the myocyte enhancer factor 2 (MEF2) family of transcription factors. However, the molecular mechanism is not well understood. In this study, we determined the crystal structure of an HDAC4-MEF2A-DNA complex. This complex adopts a dumbbell-shaped overall architecture, with a 2:4:2 stoichiometry of HDAC4, MEF2A and DNA molecules. In the complex, two HDAC4 molecules form a dimer through the interaction of their glutamine-rich domain (GRD) to form the stem of the 'dumbbell'; while two MEF2A dimers and their cognate DNA molecules are bridged by the HDAC4 dimer. Our structural observations were then validated using biochemical and mutagenesis assays. Further cell-based luciferase reporter gene assays revealed that the dimerization of HDAC4 is crucial in its ability to repress the transcriptional activities of MEF2 proteins. Taken together, our findings not only provide the structural basis for the assembly of the HDAC4-MEF2A-DNA complex but also shed light on the molecular mechanism of HDAC4-mediated long-range gene regulation., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Forkhead transcription factors bind a canonical consensus DNA motif, RYAAAYA (R = A/G, Y = C/T), as a monomer. However, the molecular mechanisms by which forkhead transcription factors bind DNA as a dimer are not well understood. In this study, we show that FOXO1 recognizes a palindromic DNA element DIV2, and mediates transcriptional regulation. The crystal structure of FOXO1/DIV2 reveals that the FOXO1 DNA binding domain (DBD) binds the DIV2 site as a homodimer. The wing1 region of FOXO1 mediates the dimerization, which enhances FOXO1 DNA binding affinity and complex stability. Further biochemical assays show that FOXO3, FOXM1 and FOXI1 also bind the DIV2 site as homodimer, while FOXC2 can only bind this site as a monomer. Our structural, biochemical and bioinformatics analyses not only provide a novel mechanism by which FOXO1 binds DNA as a homodimer, but also shed light on the target selection of forkhead transcription factors., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Proteins of nuclear receptor subfamily 4 group A (NR4A), including NR4A1/NGFI-B, NR4A2/Nurr1, and NR4A3/NOR-1, are nuclear transcription factors that play important roles in metabolism, apoptosis, and proliferation. NR4A proteins recognize DNA response elements as monomers or dimers to regulate the transcription of a variety of genes involved in multiple biological processes. In this study, we determined two crystal structures of the NR4A2 DNA-binding domain (NR4A2-DBD) bound to two Nur-responsive elements: an inverted repeat and an everted repeat at 2.6-2.8 Å resolution. The structures revealed that two NR4A2-DBD molecules bind independently to the everted repeat, whereas two other NR4A2-DBD molecules form a novel dimer interface on the inverted repeat. Moreover, substitution of the interfacial residue valine 298 to lysine as well as mutation of DNA bases involved in the interactions abolished the dimerization. Overall, our structural, biochemical, and bioinformatics analyses provide a molecular basis for the binding of the NR4A2 protein dimers to NurREs and advance our understanding of the dimerization specificity of nuclear receptors., (© 2019 Jiang et al.)

The FOXC family of transcription factors (FOXC1 and FOXC2) plays essential roles in the regulation of embryonic, ocular, and cardiac development. Mutations and abnormal expression of FOXC proteins are implicated in genetic diseases as well as cancer. In this study, we determined two crystal structures of the DNA-binding domain (DBD) of human FOXC2 protein, in complex with different DNA sites. The FOXC2-DBD adopts the winged-helix fold with helix H3 contributing to all the base specific contacts, while the N-terminus, wing 1, and the C-terminus of FOXC2-DBD all make additional contacts with the phosphate groups of DNA. Our structural, biochemical, and bioinformatics analyses allow us to revise the previously proposed DNA recognition mechanism and provide a model of DNA binding for the FOXC proteins. In addition, our structural analysis and accompanying biochemical assays provide a molecular basis for understanding disease-causing mutations in FOXC1 and FOXC2., (© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.)

FOXP3, a lineage-specific forkhead (FKH) transcription factor, plays essential roles in the development and function of regulatory T cells. However, the DNA-binding properties of FOXP3 are not well understood. In this study, FOXP3 fragments containing different domains were purified, and their DNA-binding properties were investigated using electrophoretic mobility shift assay and isothermal titration calorimetry (ITC). Both the FKH and leucine-zipper domains were required for optimal DNA binding for FOXP3. FOXP3 protein not only binds with DNA sequences containing one FKH consensus sequence, but also binds with DNA sequences with two direct repeats of consensus sequences separated by three-nucleotides (DRE3). Our results shed lights on the mechanisms by which FOXP3 recognizes cognate DNA elements, and would facilitate further structural and functional studies of FOXP3., (© The Author 2017. Published by Oxford University Press on behalf of the Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

FOXA2, a member of the forkhead family of transcription factors, plays essential roles in liver development and bile acid homeostasis. In this study, we report a 2.8 Å co-crystal structure of the FOXA2 DNA-binding domain (FOXA2-DBD) bound to a DNA duplex containing a forkhead consensus binding site (GTAAACA). The FOXA2-DBD adopts the canonical winged-helix fold, with helix H3 and wing 1 regions mainly mediating the DNA recognition. Although the wing 2 region was not defined in the structure, isothermal titration calorimetry assays suggested that this region was required for optimal DNA binding. Structure comparison with the FOXA3-DBD bound to DNA revealed more major groove contacts and fewer minor groove contacts in the FOXA2 structure than in the FOXA3 structure. Structure comparison with the FOXO1-DBD bound to DNA showed that different forkhead proteins could induce different DNA conformations upon binding to identical DNA sequences. Our findings provide the structural basis for FOXA2 protein binding to a consensus forkhead site and elucidate how members of the forkhead protein family bind different DNA sites.