Biophysical investigation of the dual binding surfaces of human transcription factors FOXO4 and p53

AbstractCellular senescence is protective against external oncogenic stress, but its accumulation causes aging-related diseases. Forkhead box O4 (FOXO4) and p53 are human transcription factors known to promote senescence by interacting in the promyelocytic leukemia bodies. Inhibiting their binding is a strategy for inducing apoptosis of senescent cells, but the binding surfaces that mediate the interaction of FOXO4 and p53 remain elusive. Here, we investigated two binding sites involved in the interaction between FOXO4 and p53 by using NMR spectroscopy. NMR chemical shift perturbation analysis showed that the binding between FOXO4’s forkhead domain (FHD) and p53’s transactivation domain (TAD), and between FOXO4’s C-terminal transactivation domain (CR3) and p53’s DNA binding domain (DBD), mediate the FOXO4-p53 interaction. Also, we showed that the CR3-binding surface of FOXO4 FHD interacts with p53 TAD2, and four residues of FOXO4 CR3 interact with the DNA-binding surface of p53 DBD. Further isothermal titration calorimetry experiments showed that the FOXO4 FHD-p53 TAD interaction takes precedence with high affinity and that the FOXO4 CR3-p53 DBD interaction follows. This work provides structural information at the molecular level that is key to understanding the interplay of two proteins responsible for cellular senescence.

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The forkhead domain hinge-loop plays a pivotal role in DNA binding and transcriptional activity of FOXP2

Biological Chemistry ◽

10.1515/hsz-2018-0185 ◽

2018 ◽

Vol 399 (8) ◽

pp. 881-893 ◽

Cited By ~ 2

Author(s):

Gavin Morris ◽

Stoyan Stoychev ◽

Previn Naicker ◽

Heini W. Dirr ◽

Sylvia Fanucchi

Keyword(s):

Transcription Factors ◽

Dna Binding ◽

Transcriptional Activity ◽

Critical Role ◽

Backbone Dynamics ◽

Luciferase Reporter ◽

Titration Calorimetry ◽

Loop Region ◽

Forkhead Domain ◽

Wide Range

Abstract Forkhead box (FOX) proteins are a ubiquitously expressed family of transcription factors that regulate the development and differentiation of a wide range of tissues in animals. The FOXP subfamily members are the only known FOX proteins capable of forming domain-swapped forkhead domain (FHD) dimers. This is proposed to be due to an evolutionary mutation (P539A) that lies in the FHD hinge loop, a key region thought to fine-tune DNA sequence specificity in the FOX transcription factors. Considering the importance of the hinge loop in both the dimerisation mechanism of the FOXP FHD and its role in tuning DNA binding, a detailed investigation into the implications of mutations within this region could provide important insight into the evolution of the FOX family. Isothermal titration calorimetry and hydrogen exchange mass spectroscopy were used to study the thermodynamic binding signature and changes in backbone dynamics of FOXP2 FHD DNA binding. Dual luciferase reporter assays were performed to study the effect that the hinge-loop mutation has on FOXP2 transcriptional activity in vivo. We demonstrate that the change in dynamics of the hinge-loop region of FOXP2 alters the energetics and mechanism of DNA binding highlighting the critical role of hinge loop mutations in regulating DNA binding characteristics of the FOX proteins.

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Differential Contribution of N- and C-Terminal Regions of HIF1α and HIF2α to Their Target Gene Selectivity

International Journal of Molecular Sciences ◽

10.3390/ijms21249401 ◽

2020 ◽

Vol 21 (24) ◽

pp. 9401

Author(s):

Antonio Bouthelier ◽

Florinda Meléndez-Rodríguez ◽

Andrés A. Urrutia ◽

Julián Aragonés

Keyword(s):

Transcription Factors ◽

Dna Binding ◽

Cellular Response ◽

Target Gene ◽

Target Genes ◽

Transactivation Domain ◽

Transactivation Domains ◽

Relative Contribution

Cellular response to hypoxia is controlled by the hypoxia-inducible transcription factors HIF1α and HIF2α. Some genes are preferentially induced by HIF1α or HIF2α, as has been explored in some cell models and for particular sets of genes. Here we have extended this analysis to other HIF-dependent genes using in vitro WT8 renal carcinoma cells and in vivo conditional Vhl-deficient mice models. Moreover, we generated chimeric HIF1/2 transcription factors to study the contribution of the HIF1α and HIF2α DNA binding/heterodimerization and transactivation domains to HIF target specificity. We show that the induction of HIF1α-dependent genes in WT8 cells, such as CAIX (CAR9) and BNIP3, requires both halves of HIF, whereas the HIF2α transactivation domain is more relevant for the induction of HIF2 target genes like the amino acid carrier SLC7A5. The HIF selectivity for some genes in WT8 cells is conserved in Vhl-deficient lung and liver tissue, whereas other genes like Glut1 (Slc2a1) behave distinctly in these tissues. Therefore the relative contribution of the DNA binding/heterodimerization and transactivation domains for HIF target selectivity can be different when comparing HIF1α or HIF2α isoforms, and that HIF target gene specificity is conserved in human and mouse cells for some of the genes analyzed.

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BSAP Can Repress Enhancer Activity by Targeting PU.1 Function

Molecular and Cellular Biology ◽

10.1128/mcb.20.6.1911-1922.2000 ◽

2000 ◽

Vol 20 (6) ◽

pp. 1911-1922 ◽

Cited By ~ 53

Author(s):

Shanak Maitra ◽

Michael Atchison

Keyword(s):

Transcription Factors ◽

Cell Differentiation ◽

Dna Binding ◽

B Cell ◽

Transactivation Domain ◽

B Cell Differentiation ◽

Developmentally Regulated ◽

Enhancer Activity ◽

Dna Binding Domains ◽

Binding Domains

ABSTRACT PU.1 and BSAP are transcription factors crucial for proper B-cell development. Absence of PU.1 results in loss of B, T, and myeloid cells, while absence of BSAP results in an early block in B-cell differentiation. Both of these proteins bind to the immunoglobulin κ chain 3′ enhancer, which is developmentally regulated during B-cell differentiation. We find here that BSAP can repress 3′ enhancer activity. This repression can occur in plasmacytoma lines or in a non-B-cell line in which the enhancer is activated by addition of the appropriate enhancer binding transcription factors. We show that the transcription factor PU.1 is a target of the BSAP-mediated repression. Although PU.1 and BSAP can physically interact through their respective DNA binding domains, this interaction does not affect DNA binding. When PU.1 function is assayed in isolation on a multimerized PU.1 binding site, BSAP targets a portion of the PU.1 transactivation domain (residues 7 to 30) for repression. The BSAP inhibitory domain (residues 358 to 385) is needed for this repression. Interestingly, the coactivator protein p300 can eliminate this BSAP-mediated repression. We also show that PU.1 can inhibit BSAP transactivation and that this repression requires PU.1 amino acids 7 to 30. Transfection of p300 resulted in only a partial reversal of PU.1-mediated repression of BSAP. When PU.1 function is assayed in the context of the immunoglobulin κ chain 3′ enhancer and associated binding proteins, BSAP represses PU.1 function by a distinct mechanism. This repression does not require the PU.1 transactivation or PEST domains and cannot be reversed by p300 expression. The possible roles of BSAP and PU.1 antagonistic activities in hematopoietic development are discussed.

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A phosphorylation-dependent switch in the disordered p53 transactivation domain regulates DNA binding

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2021456118 ◽

2020 ◽

Vol 118 (1) ◽

pp. e2021456118

Author(s):

Xun Sun ◽

H. Jane Dyson ◽

Peter E. Wright

Keyword(s):

Dna Damage ◽

Dna Binding ◽

Posttranslational Modifications ◽

Cellular Response ◽

Transactivation Domain ◽

Tumor Suppressor P53 ◽

Rich Domain ◽

Binding Surface ◽

Cellular Dna ◽

Binding Curve

The tumor-suppressor p53 is a critical regulator of the cellular response to DNA damage and is tightly regulated by posttranslational modifications. Thr55 in the AD2 interaction motif of the N-terminal transactivation domain functions as a phosphorylation-dependent regulatory switch that modulates p53 activity. Thr55 is constitutively phosphorylated, becomes dephosphorylated upon DNA damage, and is subsequently rephosphorylated to facilitate dissociation of p53 from promoters and inactivate p53-mediated transcription. Using NMR and fluorescence spectroscopy, we show that Thr55 phosphorylation inhibits DNA-binding by enhancing competitive interactions between the disordered AD2 motif and the structured DNA-binding domain (DBD). Nonphosphorylated p53 exhibits positive cooperativity in binding DNA as a tetramer. Upon phosphorylation of Thr55, cooperativity is abolished and p53 binds initially to cognate DNA sites as a dimer. As the concentration of phosphorylated p53 is further increased, a second dimer binds and causes p53 to dissociate from the DNA, resulting in a bell-shaped binding curve. This autoinhibition is driven by favorable interactions between the DNA-binding surface of the DBD and the multiple phosphorylated AD2 motifs within the tetramer. These interactions are augmented by additional phosphorylation of Ser46 and are fine-tuned by the proline-rich domain (PRD). Removal of the PRD strengthens the AD2–DBD interaction and leads to autoinhibition of DNA binding even in the absence of Thr55 phosphorylation. This study reveals the molecular mechanism by which the phosphorylation status of Thr55 modulates DNA binding and controls both activation and termination of p53-mediated transcriptional programs at different stages of the cellular DNA damage response.

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Constitutively active FOXO1a and a DNA-binding domain mutant exhibit distinct co-regulatory functions to enhance progesterone receptor A activity

Journal of Molecular Endocrinology ◽

10.1677/jme-07-0017 ◽

2007 ◽

Vol 38 (6) ◽

pp. 673-690 ◽

Cited By ~ 12

Author(s):

Michael D Rudd ◽

Ignacio Gonzalez-Robayna ◽

Inmaculada Hernandez-Gonzalez ◽

Nancy L Weigel ◽

William E Bingman ◽

...

Keyword(s):

Transcription Factors ◽

Dna Binding ◽

Hormone Receptors ◽

Binding Domain ◽

Nuclear Hormone Receptors ◽

Dna Binding Domain ◽

Transactivation Domain ◽

Response Element ◽

Regulatory Functions ◽

Constitutively Active

FOXO (Forkhead box O1 transcription factors) factors interact with and modify the activity of other transcription factors, including nuclear hormone receptors. However, not all of the structural domains within the FOXO proteins that mediate these functional interactions have been clearly defined. To address this issue, we used a constitutively active (nuclear) mutant of FOXO1a (designated FOXOA3) and within FOXOA3 made additional mutations to alter the putative nuclear hormone interacting domain (NID), minimal activation domain (MAD), DNA-binding domain (DBD), and the N terminus. We document that FOXOA3 enhanced the hormone-dependent transcriptional activity of liganded progesterone receptors A (PGRA) on a glucocorticoid response element-responsive promoter, PGRA on the insulin-like growth factor-binding protein 1 promoter, and estrogen receptor α on an estrogen response element-responsive promoter. The effects of FOXOA3 on PGRA were dependent, in part, on an intact NID, the MAD, and N-terminal domain. In striking contrast, a FOXOA3 DNA-binding mutant (FOXOA3-mDBD) modulated PGRA, PGRB, and ESR1 activities by distinctly different mechanisms, markedly elevating ligand-independent activity of these nuclear hormone receptors even in the double mutant lacking the MAD. Furthermore, both FOXOA3 and FOXOA3-mDBD enhanced the activity of a transcriptionally defective PGRA lacking its AF1 transactivation domain, indicating that this region of the receptor is not essential in this context. Since FOXOA3, FOXOA3-mDBD, and FOXOA3-mNID all bound PGRA in a GST pull-down assay, it appears that the LXXLL (leucine–X–X–leucine–leucine) motif within the NID is not critical for FOXOA3 interactions with PGRA, but may modify the recruitment of other co-regulatory molecules. Collectively, the results show that FOXOA3 exerts co-regulatory functions independent of DNA binding and that the DNA-binding defective form of FOXO1a is transcriptionally active as a co-regulator of these nuclear hormone receptors.

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