Forkhead DNA-binding Domain Research Articles

Redirecting the pioneering function of FOXA1 with covalent small molecules.

Pioneer transcription factors (TFs) bind to and open closed chromatin, facilitating engagement by other regulatory factors involved in gene activation or repression. Chemical probes are lacking for pioneer TFs, which has hindered their mechanistic investigation in cells. Here, we report the chemical proteomic discovery of electrophilic compounds that stereoselectively and site-specifically bind the pioneer TF forkhead box protein A1 (FOXA1) at a cysteine (C258) within the forkhead DNA-binding domain. We show that these covalent ligands react with FOXA1 in a DNA-dependent manner and rapidly remodel its pioneer activity in prostate cancer cells reflected in redistribution of FOXA1 binding across the genome and directionally correlated changes in chromatin accessibility. Motif analysis supports a mechanism where the ligands relax the canonical DNA-binding preference of FOXA1 by strengthening interactions with suboptimal sequences in predicted proximity to C258. Our findings reveal a striking plasticity underpinning the pioneering function of FOXA1 that can be controlled by small molecules.

FOXK2 amplification promotes breast cancer development and chemoresistance

Oncogene activation through DNA amplification or overexpression is a crucial driver of cancer initiation and progression. The FOXK2 gene, located on chromosome 17q25, encodes a transcription factor with a forkhead DNA-binding domain. Analysis of genomic datasets reveals that FOXK2 is frequently amplified and overexpressed in breast cancer, correlating with poor patient survival. Knockdown of FOXK2 significantly inhibited breast cancer cell proliferation, migration, anchorage-independent growth, and delayed tumor growth in a xenograft mouse model. Additionally, inhibiting FOXK2 sensitized breast cancer cells to chemotherapy. Co-overexpression of FOXK2 and mutant PI3KCA transformed non-tumorigenic MCF-10A cells, suggesting a role for FOXK2 in PI3KCA-driven tumorigenesis. CCNE2, PDK1, and ESR1 were identified as transcriptional targets of FOXK2 in MCF-7 cells. Small-molecule inhibitors of CCNE2/CDK2 (dinaciclib) and PDK1 (dichloroacetate) exhibited synergistic anti-tumor effects with PI3KCA inhibitor (alpelisib) in vitro. Inhibition of FOXK2 by dinaciclib synergistically enhanced the anti-tumor effects of alpelisib in a xenograft mouse model. Collectively, these findings highlight the oncogenic function of FOXK2 and suggest that FOXK2 and its downstream genes represent potential therapeutic targets in breast cancer.

Abstract A076: The role of PARP-1-mediated FoxA1 ADP-ribosylation in breast cancer biology

Abstract Breast cancer is the most common cancer diagnosed in women. At the time of diagnosis, approximately 70% of breast cancers express Estrogen Receptor alpha (ERα), a key signature of luminal subtype tumors. Equally important in these cases is the expression of FoxA1, a subtype-specific transcription factor (TF) essential for ERα-mediated cell proliferation. FoxA1 is a key pioneer TF that assists ERα with the assembly of enhancers and the transcription of estrogen (E2)-regulated target genes. Together, the FoxA1-ERα axis is a major driver of cancer cell growth. Our recently published work implicates poly(ADP-ribose) polymerase 1 (PARP-1) and ADP-ribosylation (ADPRylation) in the regulation of E2-dependent transcriptional responses in ERα+ breast cancer cells. To determine the underlying mechanisms, we identified FoxA1 as a substrate of PARP-1-mediated ADPRylation. We hypothesize that (1) PARP-1 elicits its effects on E2 signaling and ERα enhancer function, in part, by ADPRylating FoxA1 at specific residues and (2) Loss of these regulatory ADPRylation events alters FoxA1 activity, which in turn can alter the assembly of ERα enhancers and transform the breast cancer cell transcriptome. Indeed, the sites of FoxA1 ADPRylation are recurrently mutated in breast cancers and are highly enriched in metastatic cases. Using an integrated set of biochemical, molecular, cell based, genomic and proteomic approaches, we have validated the ADPRylation of FoxA1 by PARP-1 in vitro and in vivo. We selected two FoxA1 ADPRylation sites, D226 and E255, for further analysis due to their positions in a mutation ‘hot spot’ region corresponding to the C-terminal forkhead DNA-binding domain. Mutation of these sites blocks FoxA1 ADPRylation and enhances cell proliferation in MCF-7 human breast cancer cells. Additionally, preliminary ChIP-seq analysis indicates that the binding of both ERα and FoxA1 are globally reduced at ERα enhancers in MCF-7 cells expressing the FoxA1 ADPRylation site mutants compared to wild-type but may also increase both at selected enhancers. While there is a global reduction in mutant FoxA1 binding near the canonical estrogen-regulated genes, interestingly, we observe an overall enhanced binding of FoxA1 D226N compared to wild-type outside of this canonical program. Collectively, these studies will (1) reveal new molecular endpoints modulated by PARP-1, providing new insights about the use of PARPi to treat ERα+ breast cancers, and (2) suggest new therapeutic strategies to disrupt the aberrant signaling pathways associated with mutant FoxA1 luminal breast cancers, and more importantly metastatic cases, which is of utmost clinical significance. Citation Format: Cristel V. Camacho, Aishwarya Sundaresan, Tulip Nandu, W. Lee Kraus. The role of PARP-1-mediated FoxA1 ADP-ribosylation in breast cancer biology [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Breast Cancer Research; 2023 Oct 19-22; San Diego, California. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_1):Abstract nr A076.

Identification and characterisation of a novel interaction between oestrogen receptor alpha and FOXP2

Forkhead box P2 (FOXP2) regulates expression of various genes and is associated with language, speech and neural development as well as cancer. Since there may be a putative link between sex and language and because transcription factors rarely function in isolation, this study aims to investigate whether FOXP2 directly associates with oestrogen receptor α (ER1), a nuclear receptor responsible for sexual differentiation that is also associated with cancer. Isothermal titration calorimetry and fluorescence anisotropy were used to investigate the interaction between the DNA-binding forkhead domain (FHD) of FOXP2, the N-terminal region (NT) of FOXP2, and the ligand-binding domain (LBD) of ER1. ER1 LBD does not interact with FOXP2 NT but associates with apo-FOXP2 FHD in an enthalpically favourable manner. The affinity of this interaction is inversely correlated to the salt concentration. Additionally, FOXP2 FHD that is bound to ER1 LBD, has reduced ability to interact with its cognate DNA. This research identifies a novel interaction between ER1 LBD and FOXP2 FHD and shows that the interaction is regulated by salt. Moreover, FOXP2 FHD cannot bind to both ER1 LBD and DNA simultaneously, suggesting that this interaction could be involved in regulating the transcriptional pathway of FOXP2 should the interaction be found in vivo. This study could serve as a foundation for uncovering the basis of sexual dimorphism in speech and language development and related disorders and potentially offers an alternate for targeted cancer therapies.

The forkhead DNA-binding domain binds specific G2-rich RNA sequences

Abstract Transcription factors contain a DNA-binding domain ensuring specific recognition of DNA target sequences. The family of forkhead (FOX) transcription factors is composed of dozens of paralogs in mammals. The forkhead domain (FHD) is a segment of about 100 amino acids that binds an A-rich DNA sequence. Using DNA and RNA PCR-SELEX, we show that recombinant FOXL2 proteins, either wild-type or carrying the oncogenic variant C134W, recognize similar DNA-binding sites. This suggests that the oncogenic variant does not alter the intrinsic sequence-specificity of FOXL2. Most importantly, we show that FOXL2 binds G2-rich RNA sequences whereas it virtually fails to bind similar sequences in DNA chemistry. Interestingly, a statistically significant subset of genes responding to the knock-down of FOXL2/Foxl2 harbor such G2-rich sequences and are involved in crucial signaling pathways and cellular processes. In addition, we show that FOXA1, FOXO3a and chimeric FOXL2 proteins containing the FHD of the former are also able to interact with some of the preferred FOXL2-binding sequences. Our results point to an unexpected and novel characteristic of the forkhead domain, the biological relevance of which remains to be explored.

Expanding genotype–phenotype correlations in FOXG1 syndrome: results from a patient registry

BackgroundWe refine the clinical spectrum of FOXG1 syndrome and expand genotype–phenotype correlations through evaluation of 122 individuals enrolled in an international patient registry.MethodsThe FOXG1 syndrome online patient registry allows for remote collection of caregiver-reported outcomes. Inclusion required documentation of a (likely) pathogenic variant in FOXG1. Caregivers were administered a questionnaire to evaluate clinical severity of core features of FOXG1 syndrome. Genotype–phenotype correlations were determined using nonparametric analyses.ResultsWe studied 122 registry participants with FOXG1 syndrome, aged < 12 months to 24 years. Caregivers described delayed or absent developmental milestone attainment, seizures (61%), and movement disorders (58%). Participants harbouring a missense variant had a milder phenotype. Compared to individuals with gene deletions (0%) or nonsense variants (20%), missense variants were associated with more frequent attainment of sitting (73%). Further, individuals with missense variants (41%) achieved independent walking more frequently than those with gene deletions (0%) or frameshift variants (6%). Presence of epilepsy also varied by genotype and was significantly more common in those with gene deletions (81%) compared to missense variants (47%). Individuals with gene deletions were more likely to have higher seizure burden than other genotypes with 53% reporting daily seizures, even at best control. We also observed that truncations preserving the forkhead DNA binding domain were associated with better developmental outcomes.ConclusionWe refine the phenotypic spectrum of neurodevelopmental features associated with FOXG1 syndrome. We strengthen genotype-driven outcomes, where missense variants are associated with a milder clinical course.

Pan-cancer landscape of FOXA1 alterations: Insights and clinical implications.

3090 Background: Forkhead box A1 ( FOXA1) is a pioneer transcription factor that regulates organogenesis and is associated with progression in breast and prostate cancers. To explore its role more broadly in oncogenesis, we characterize FOXA1 genomic alterations and clinical correlates in a large pan-cancer cohort from the American Association for Cancer Research project Genomics, Evidence, Neoplasia, Information, Exchange (AACR-GENIE) database. Methods: Using AACR-GENIE, FOXA1 mutations (in frame insertions/deletions (indel-inframe), frameshift insertions/deletions (indel truncating), point mutations (missense), fusions, and amplifications) were characterized across &gt;30 cancer types for primary and metastatic tumors with patient characteristics and clinical outcomes. FOXA1 alterations were queried in the MSK-MET cohort, a subset of GENIE cohort, allowing the definition of hazard ratios (HRs) and overall survival estimates based on Cox proportional hazard models. Results: Among 120,625 samples queried from the AACR GENIE data, FOXA1 was profiled in 87,706 samples: 30,268 metastases, 49,228 primary tumors, and 8,210 not specified. FOXA1 mutations were present in 1,869 samples (2.1%), mostly in the forkhead DNA-binding domain. Prostate tumors were enriched with indel-inframe alterations whereas breast cancers were enriched with missense mutations. F266, R261, and D226 mutations in the Wing2 region were common in breast and prostate cancers. In the GENIE cohort, FOXA1 copy number profiles were collected for 74,715 samples, with amplification detected in 834 (1.1%) and deletion in 96 samples (0.1%). FOXA1-amplification was most common in non-small cell lung cancer (NSCLC, 3%, n=190, in primary; 6%, n=264, in metastatic), followed by small cell lung cancer (SCLC) (4.1%, n=10, in primary; 3.5%, n=10, in metastatic), breast (2%, n=80 in primary; 1.6%, n=73, in metastatic) and prostate cancers (2.2%, n=49, in primary; 1.6%, n=27 in metastatic). In the MSK-MET cohort, FOXA1 amplifications were associated with poorer outcomes in primary breast (HR: 3.04, 95% CI: 1.89-4.89, p=4e-6), primary NSCLC (HR: 1.45, 95% CI: 1.06-1.99, p=0.02), and prostate cancer (HR: 1.94, 95% CI: 1.03-3.68, p=0.04). FOXA1 amplifications were associated with wide-spread metastases in primary breast, NSCLC, and prostate cancer. Conclusions: In this observational study of pan-cancer FOXA1 mutant specimens from the GENIE database, we found that FOXA1 alterations in prostate cancer (primary and metastatic tumors) were predominantly indels versus missense in other cancers. We also observe a consistent association between FOXA1 amplification and enhanced metastatic potential regardless of tissue of origin. Our findings suggest that FOXA1 amplifications could serve as a potential prognostic and therapeutic target, particularly for breast, prostate, and non-small cell lung cancer.

Non-canonical telomere protection role of FOXO3a of human skeletal muscle cells regulated by the TRF2-redox axis

Telomeric repeat binding factor 2 (TRF2) binds to telomeres and protects chromosome ends against the DNA damage response and senescence. Although the expression of TRF2 is downregulated upon cellular senescence and in various aging tissues, including skeletal muscle tissues, very little is known about the contribution of this decline to aging. We previously showed that TRF2 loss in myofibers does not trigger telomere deprotection but mitochondrial dysfunction leading to an increased level of reactive oxygen species. We show here that this oxidative stress triggers the binding of FOXO3a to telomeres where it protects against ATM activation, revealing a previously unrecognized telomere protective function of FOXO3a, to the best of our knowledge. We further showed in transformed fibroblasts and myotubes that the telomere properties of FOXO3a are dependent on the C-terminal segment of its CR2 domain (CR2C) but independent of its Forkhead DNA binding domain and of its CR3 transactivation domain. We propose that these non-canonical properties of FOXO3a at telomeres play a role downstream of the mitochondrial signaling induced by TRF2 downregulation to regulate skeletal muscle homeostasis and aging.

The transcription factor FoxP3 can fold into two dimerization states with divergent implications for regulatory T cell function and immune homeostasis

FoxP3 is an essential transcription factor (TF) for immunologic homeostasis, but how it utilizes the common forkhead DNA-binding domain (DBD) to perform its unique function remains poorly understood. We here demonstrated that unlike other known forkhead TFs, FoxP3 formed a head-to-head dimer using a unique linker (Runx1-binding region [RBR]) preceding the forkhead domain. Head-to-head dimerization conferred distinct DNA-binding specificity and created a docking site for the cofactor Runx1. RBR was also important for proper folding of the forkhead domain, as truncation of RBR induced domain-swap dimerization of forkhead, which was previously considered the physiological form of FoxP3. Rather, swap-dimerization impaired FoxP3 function, as demonstrated with the disease-causing mutation R337Q, whereas a swap-suppressive mutation largely rescued R337Q-mediated functional impairment. Altogether, our findings suggest that FoxP3 can fold into two distinct dimerization states: head-to-head dimerization representing functional specialization of an ancient DBD and swap dimerization associated with impaired functions.

Abstract 5730: FOXR2 is an oncogenic driver across adult and pediatric cancers

Abstract Background: Understanding how aberrant transcription factors (TFs) hijack normal development to induce oncogenesis is a critical question in oncology. Forkhead box (FOX) proteins are a superfamily of transcriptional regulators characterized by a forkhead DNA-binding domain. Within this family, Forkhead Box R2 (FOXR2) expression has been associated with a subset of cancers including CNS and peripheral neuroblastoma. While FOXR2 has been shown to stabilize MYC isoforms, the mechanistic details through which it enhances tumor formation and the true extent of its role as an oncogene across all cancers have not been systematically evaluated. Methods: We applied an integrative approach using transcriptomics, epigenetics, in vitro cancer models, and in vivo mouse models to systematically evaluate the mechanisms by which FOXR2 is activated across human cancers. Results: We performed a pan-cancer analysis of FOXR2 activation across over 10,000 adult and pediatric cancer samples, and found FOXR2 to be aberrantly upregulated in 70% of all cancer types, and 8% of all individual tumors. We identified genetic and epigenetic mechanisms that induce its expression, including hypomethylation of a novel promoter in the vast majority (78%) of FOXR2-expressing cases. We demonstrate that FOXR2 expression is both sufficient and necessary for transformation across multiple lineages, using both in vitro and in vivo models. Conclusion: Taken together, this study demonstrates the role of FOXR2 as a potent oncogene across human cancers, and highlights a novel mechanism by which its expression is activated. Citation Format: Jessica W. Tsai, Paloma Cejas, Dayle K. Wang, Smruti Patel, David W. Wu, Phonepasong Arounleut, Xin Wei, Ningxuan Zhou, Sudeepa Syamala, Frank P. Dubois, Kristine Pelton, Jayne Vogelzang, Cecilia Sousa, Audrey Baguette, Xiaolong Chen, Alexandra L. Condurat, Sarah E. Dixon-Clarke, Kevin N. Zhou, Sophie D. Lu, Elizabeth M. Gonzalez, Madison S. Chacon, Jeromy J. Digiacomo, Rushil Kumbhani, Dana Novikov, J'Ya Hunter, Maria Tsoli, David S. Ziegler, Uta Dirksen, Natalie Jager, Gnana Prakash Balasubramanian, Christof M. Kramm, Michaela Nathrath, Stefan Bielack, Suzanne J. Baker, Jinghui Zhang, James M. McFarland, Gad Getz, Francois Aguet, Nada Jabado, Olaf Witt, Stefan M. Pfister, Keith L. Ligon, Claudia Kleinman, Henry Long, David T. Jones, Pratiti Bandopadhayay, Timothy N. Phoenix. FOXR2 is an oncogenic driver across adult and pediatric cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5730.

DIPG-19. FOXR2 is an oncogenic driver across pediatric and adult cancers

BACKGROUND: Understanding how aberrant transcription factors (TFs) hijack normal development to induce oncogenesis is a critical question in oncology. Forkhead box (FOX) proteins are a superfamily of transcriptional regulators characterized by a forkhead DNA-binding domain. Within this family, Forkhead Box R2 (FOXR2) has been identified as a candidate structural variant (SV) driver in a subset of pediatric cancers including CNS embryonal tumors and peripheral neuroblastoma. While FOXR2 has been shown to stabilize MYC isoforms, the mechanistic details through which it enhances tumor formation, other non-SV mechanisms of activating aberrant expression, and the true extent of its role as an oncogene across all cancers have not been systematically evaluated. METHODS: We applied an integrative approach using transcriptomics, epigenetics, in vitro cancer models, and in vivo mouse models to systematically evaluate the mechanisms by which FOXR2 is activated across human cancers. RESULTS: We performed a pan-cancer analysis of FOXR2 activation across over 10,000 adult and pediatric cancer samples, and surprisingly found FOXR2 to be aberrantly upregulated in 70% of all cancer types (including diffuse midline gliomas), and 8% of all individual tumors. FOXR2 expression occurred predominantly in the absence of rearrangement/fusions, single nucleotide variants, or copy number aberrations at the DNA level. Transcriptomic and epigenomic analyses show the vast majority of tumors (78%) aberrantly express FOXR2 through a previously undescribed epigenetic mechanism via hypomethylation of a novel promoter. Using both in vitro and in vivo models, we demonstrate that FOXR2 expression is both sufficient and necessary for transformation across multiple lineages, including DMGs. CONCLUSION: Taken together, this study demonstrates that FOXR2 is a novel and potent oncogene across pediatric and adult cancers, and highlights a new epigenetic mechanism by which its expression is activated.

FOXO1 forkhead domain mutants in B-cell lymphoma lack transcriptional activity

Somatic point mutations of the FOXO1 transcription factor were reported in non-Hodgkin lymphoma including diffuse large B-cell lymphoma, follicular lymphoma and Burkitt lymphoma. These alterations were associated with a poor prognosis and resistance to therapy. Nearly all amino acid substitutions are localized in two major clusters, affecting either the N-terminal region (Nt mutations) or the forkhead DNA-binding domain (DBD mutations). While recent studies have focused on Nt mutations, we characterized FOXO1 DBD mutants. We analyzed their transcriptional activity, DNA binding, phosphorylation and protein–protein interaction. The majority of DBD mutants showed a decrease in activity and DNA binding, while preserving AKT phosphorylation and interaction with the cytoplasmic ATG7 protein. In addition, we investigated the importance of conserved residues of the α-helix 3 of the DBD. Amino acids I213, R214, H215 and L217 appeared to be crucial for FOXO1 activity. Our data underlined the key role of multiple amino-acid residues of the forkhead domain in FOXO1 transcriptional activity and revealed a new type of FOXO1 loss-of-function mutations in B-cell lymphoma.

Single-molecule optical tweezers reveals folding steps of the domain swapping mechanism of a protein

Domain swapping is a mechanism of protein oligomerization by which two or more subunits exchange structural elements to generate an intertwined complex. Numerous studies support a diversity of swapping mechanisms in which structural elements can be exchanged at different stages of the folding pathway of a subunit. Here, we used single-molecule optical tweezers technique to analyze the swapping mechanism of the forkhead DNA-binding domain of human transcription factor FoxP1. FoxP1 populates folded monomers in equilibrium with a swapped dimer. We generated a fusion protein linking two FoxP1 domains in tandem to obtain repetitive mechanical folding and unfolding trajectories. Thus, by stretching the same molecule several times, we detected either the independent folding of each domain or the elusive swapping step between domains. We found that a swapped dimer can be formed directly from fully or mostly folded monomer. In this situation, the interaction between the monomers in route to the domain-swapped dimer is the rate-limiting step. This approach is a useful strategy to test the different proposed domain swapping mechanisms for proteins with relevant physiological functions.

Extreme longevity variants at the FOXO3 locus may moderate FOXO3 isoform levels

The rs2802292, rs2764264 and rs13217795 variants of FOXO3 have been associated with extreme longevity in multiple human populations, but the mechanisms underpinning this remain unclear. We aimed to characterise potential effects of longevity-associated variation on the expression and mRNA processing of the FOXO3 gene. We performed a comprehensive assessment of FOXO3 isoform usage across a wide variety of human tissues and carried out a bioinformatic analysis of the potential for longevity-associated variants to disrupt regulatory regions involved in isoform choice. We then related the expression of full length and 5′ truncated FOXO3 isoforms to rs13217795 genotype in peripheral blood and skeletal muscle from individuals of different rs13217795 genotypes. FOXO3 isoforms displayed considerable tissue specificity. We determined that rs13231195 and its tightly aligned proxy variant rs9400239 may lie in regulatory regions involved in isoform choice. The longevity allele at rs13217795 was associated with increased levels of full length FOXO3 isoforms in peripheral blood and a decrease in truncated FOXO3 isoforms in skeletal muscle RNA. We suggest that the longevity effect of FOXO3 SNPs may in part derive from a shift in isoform usage in skeletal muscle away from the production of 5′ truncated FOXO3 isoforms lacking a complete forkhead DNA binding domain, which may have compromised functionality.

Multiple regulatory intrinsically disordered motifs control FOXO4 transcription factor binding and function.

Transcription factors harbor defined regulatory intrinsically disordered regions (IDRs), which raises the question of how they mediate binding to structured co-regulators and modulate their activity. Here, we present a detailed molecular regulatory mechanism of Forkhead box O4 (FOXO4) by the structured transcriptional co-regulator β-catenin. We find that the disordered FOXO4 C-terminal region, which contains its transactivation domain, binds β-catenin through two defined interaction sites, and this is regulated by combined PKB/AKT- and CK1-mediated phosphorylation. Binding of β-catenin competes with the autoinhibitory interaction of the FOXO4 disordered region with its DNA-binding Forkhead domain, and thereby enhances FOXO4 transcriptional activity. Furthermore, we show that binding of the β-catenin inhibitor protein ICAT is compatible with FOXO4 binding to β-catenin, suggesting that ICAT acts as a molecular switch between anti-proliferative FOXO and pro-proliferative Wnt/TCF/LEF signaling. These data illustrate how the interplay of IDRs, post-translational modifications, and co-factor binding contribute to transcription factor function.

Genotype-phenotype implications from three representative clinical FOXG1 variants associated with FOXG1 syndrome

FOXG1 (Forkhead Box G1) gene, as its name indicates, encodes a transcription factor with a Forkhead DNA-binding domain. FoxG1 protein is highly conserved among mammalian and reptilian species, with DNA-binding and C-terminal domains showing highest conservation, whereas its N-terminal domain contains a mammal-specific extended proline- and glutamine-rich region [1]. The neurodevelopmental disorder associated with heterozygous variants in FOXG1 gene is referred as FOXG1 syndrome (OMIM #613454), equivalent to the original designation of “congenital variant of Rett syndrome.” [2] The main clinical features of FOXG1 syndrome include, but are not limited to, impairment of postnatal growth, primary or secondary microcephaly, severe intellectual disability with absent speech development, epilepsy, stereotypies, and dyskinesia [3].

FOXO4 Transactivation Domain Interaction with Forkhead DNA Binding Domain and Effect on Selective DNA Recognition for Transcription Initiation

Forkhead box O4 (FOXO4) is a human transcription factor (TF) that participates in cell homeostasis. While the structure and DNA binding properties of the conserved forkhead domain (FHD) have been thoroughly investigated, how the transactivation domain (TAD) regulates the DNA binding properties of the protein remains elusive. Here, we investigated the role of TAD in modulating the DNA binding properties of FOXO4 using solution NMR. We found that TAD and FHD form an intramolecular complex mainly governed by hydrophobic interaction. Remarkably, TAD and DNA share the same surface of FHD for binding. While FHD did not differentiate binding to target and non-target DNA, the FHD-TAD complex showed different behaviors depending on the DNA sequence. In the presence of TAD, free and DNA-bound FHD exhibited a slow exchange with target DNA and a fast exchange with non-target DNA. The interaction of the two domains affected the kinetic function of FHD depending on the type of DNA. Based on these findings, we suggest a transcription initiation model by which TAD modulates FOXO4 recognition of its target promoter DNA sequences. This study describes the function of TAD in FOXO4 and provides a new kinetic perspective on target sequence selection by TFs.

Sensorineural hearing loss and hypoplastic cochlea in Axenfeld-Rieger syndrome with FOXC1 mutation

ObjectiveAxenfeld-Rieger syndrome (ARS) type 3 is a rare autosomal dominant disease, characterized by anterior segment dysgenesis of the eye, hearing loss, and cardiac defects. ARS type 3 is highly associated with FOXC1 mutations, which induces developmental disorders of neural crest cells. Most studies about ARS patients focused on ophthalmologic findings, but details in their hearing loss have not yet been revealed. In this report, we investigated audiological and otological manifestations in the ARS type 3 patient who had the novel heterozygous FOXC1 mutation leading deletion at the forkhead DNA-binding domain. Methods and ResultsPure tone audiometry showed bilateral sensorineural hearing loss (SNHL) and audiological examinations confirmed that major dysfunctions existed in the cochlea, rather than the spiral ganglion neurons and the cochlear nerve. CT and MRI revealed the hypoplastic cochlea at both sides. Given that the 6p25 deletion syndrome, lacking one allele of the FOXC1 gene, shows similar, but more severe cochlear malformations than the present case, the FOXC1 mutations might contribute to the hypoplasia and dysfunctions in the cochlea. ConclusionTo our knowledge, this is the first report demonstrating that the ARS type 3 patient with the FOXC1 mutation has the hypoplasia and dysfunctions in the cochlea, which results in bilateral SNHL.

Upregulation of Foxo3a protects neurons against hypoxia-ischaemia injury

IntroductionA series of transcription factors have been profiled in ischaemic stroke. Forkhead box protein O 3a (FoxO3a), which belongs to the family of transcription factors, is characterised by a forkhead DNA-binding domain. However, the role of FoxO3a and circFoxO3, which is encoded from FOXO3 gene in stroke, remains unelucidated.Material and methodsA rat model of middle cerebral artery occlusion (MCAO)/reperfusion was applied, and rat neuroblastoma B35 cells were treated with cobalt chloride to mimic cellular hypoxia in vitro. FoxO3a (or circFoxO3a) was manipulated to assess the infarction volume and apoptotic proteins.ResultsBrain infarction was enlarged in ischaemia/reperfusion compared with simple ischaemia treatment. The protein level of FoxO3a increased significantly in MCAO and subsequently decreased following reperfusion. Increased FoxO3a expression was also found in CoCl­2 treatment at 24 h and 48 h. Downregulation of FoxO3a significantly promoted apoptosis, while upregulation of FoxO3a reduced apoptosis. Further biochemical analysis demonstrated that protein expression level of caspase-3, caspase-9, and Bax were upregulated following FoxO3a inhibition but downregulated following FoxO3a overexpression. Moreover, upregulated FoxO3a level was consistent with enhanced circFoxO3 expression both in vivo and in vitro.ConclusionsBoth FoxO3a and circFoxO3 are upregulated in ischaemic stroke, which associates with the apoptosis pathway. Hence, FoxO3a and circFoxO3 might be protective factors against hypoxia/ischaemia-induced neuronal damage.

Multiple Regulatory Intrinsically Disordered Motifs Control FOXO4 Transcription Factor Binding and Function

Transcription factors harbour defined intrinsically disordered regulatory regions, which raises the question of how they mediate binding to structured co-regulators and how this regulates activity. Here, we present a detailed molecular regulatory mechanism of Forkhead box O4 (FOXO4) by the structured transcriptional co-regulator β-catenin. We find that the largely disordered FOXO4 C-terminal region, which contains its transactivation domain binds β-catenin through two defined interaction sites, and this is regulated by combined PKB/AKT- and CK1-mediated phosphorylation. Binding of β-catenin competes with the auto-inhibitory interaction of the FOXO4 disordered region with its DNA-binding forkhead domain, and thereby enhances FOXO4 transcriptional activity. Furthermore, we show that binding of the β-catenin inhibitor protein ICAT is compatible with FOXO4 binding to β-catenin, suggesting that ICAT acts as a molecular switch between anti-proliferative FOXO and pro-proliferative Wnt/TCF/LEF signalling. Together these data illustrate how the interplay of intrinsically disordered regions, post-translational modifications and co-factor binding contribute to transcription factor function.