Targeted Apoptosis of Senescent Cells Restores Tissue Homeostasis in Response to Chemotoxicity and Aging.

The accumulation of irreparable cellular damage restricts healthspan after acute stress or natural aging. Senescent cells are thought to impair tissue function, and their genetic clearance can delay features of aging. Identifying how senescent cells avoid apoptosis allows for the prospective design of anti-senescence compounds to address whether homeostasis can also be restored. Here, we identify FOXO4 as a pivot in senescent cell viability. We designed a FOXO4 peptide that perturbs the FOXO4 interaction with p53. In senescent cells, this selectively causes p53 nuclear exclusion and cell-intrinsic apoptosis. Under conditions where it was well tolerated in vivo, this FOXO4 peptide neutralized doxorubicin-induced chemotoxicity. Moreover, it restored fitness, fur density, and renal function in both fast aging XpdTTD/TTD and naturally aged mice. Thus, therapeutic targeting of senescent cells is feasible under conditions where loss of health has already occurred, and in doing so tissue homeostasis can effectively be restored.

FOXP3 recognizes microsatellites and bridges DNA through multimerization.

FOXP3 is a transcription factor that is essential for the development of regulatory T cells, a branch of T cells that suppress excessive inflammation and autoimmunity1-5. However, the molecular mechanisms of FOXP3 remain unclear. Here we here show that FOXP3 uses the forkhead domain-a DNA-binding domain that is commonly thought to function as a monomer or dimer-to form a higher-order multimer after binding to TnG repeat microsatellites. The cryo-electron microscopy structure of FOXP3 in a complex with T3G repeats reveals a ladder-like architecture, whereby two double-stranded DNA molecules form the two 'side rails' bridged by five pairs of FOXP3 molecules, with each pair forming a 'rung'. Each FOXP3 subunit occupies TGTTTGT within the repeats in a manner that is indistinguishable from that of FOXP3 bound to the forkhead consensus motif (TGTTTAC). Mutations in the intra-rung interface impair TnG repeat recognition, DNA bridging and the cellular functions of FOXP3, all without affecting binding to the forkhead consensus motif. FOXP3 can tolerate variable inter-rung spacings, explaining its broad specificity for TnG-repeat-like sequences in vivo and in vitro. Both FOXP3 orthologues and paralogues show similar TnG repeat recognition and DNA bridging. These findings therefore reveal a mode of DNA recognition that involves transcription factor homomultimerization and DNA bridging, and further implicates microsatellites in transcriptional regulation and diseases.

FOXOs: signalling integrators for homeostasis maintenance.

Forkhead box O (FOXO) transcription factors are involved in the regulation of the cell cycle, apoptosis and metabolism. In model organisms, FOXO activity also affects stem cell maintenance and lifespan as well as age-related diseases, such as cancer and diabetes. Multiple upstream pathways regulate FOXO activity through post-translational modifications and nuclear-cytoplasmic shuttling of both FOXO and its regulators. The diversity of this upstream regulation and the downstream effects of FOXOs suggest that they function as homeostasis regulators to maintain tissue homeostasis over time and coordinate a response to environmental changes, including growth factor deprivation, metabolic stress (starvation) and oxidative stress.

Forkhead Transcription Factors in Health and Disease.

Forkhead box (FOX) proteins belong to an evolutionarily conserved family of transcription factors that has evolved by gene/genome duplication. FOX family members have undergone sequence and regulatory diversification. However, they have retained some degree of functional redundancy, in addition to playing specific roles, both during development and in the adult. Genetic alterations or misregulation of FOX genes underlie human genetic diseases, cancer, and/or aging. In this review, we provide an updated overview of the main characteristics of the members of this family, in terms of breadth of expression, protein domain composition, evolution, and function.

Domain tethering impacts dimerization and DNA-mediated allostery in the human transcription factor FoxP1.

Transcription factors are multidomain proteins with specific DNA binding and regulatory domains. In the human FoxP subfamily (FoxP1, FoxP2, FoxP3, and FoxP4) of transcription factors, a 90 residue-long disordered region links a Leucine Zipper (ZIP)-known to form coiled-coil dimers-and a Forkhead (FKH) domain-known to form domain swapping dimers. We used replica exchange discrete molecular dynamics simulations, single-molecule fluorescence experiments, and other biophysical tools to understand how domain tethering in FoxP1 impacts dimerization at ZIP and FKH domains and how DNA binding allosterically regulates their dimerization. We found that domain tethering promotes FoxP1 dimerization but inhibits a FKH domain-swapped structure. Furthermore, our findings indicate that the linker mediates the mutual organization and dynamics of ZIP and FKH domains, forming closed and open states with and without interdomain contacts, thus highlighting the role of the linkers in multidomain proteins. Finally, we found that DNA allosterically promotes structural changes that decrease the dimerization propensity of FoxP1. We postulate that, upon DNA binding, the interdomain linker plays a crucial role in the gene regulatory function of FoxP1.

Mechanism of forkhead transcription factors binding to a novel palindromic DNA site.

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.

Reduced frequencies of Foxp3+GARP+ regulatory T cells in COPD patients are associated with multi-organ loss of tissue phenotype.

BACKGROUND: Expression of glycoprotein A dominant repeat (GARP) has been reported to occur only in activated human naturally occurring regulatory T cells (Tregs) and their clones, and not in activated effector T cells, indicating that GARP is a marker for bona fide Tregs. A different phenotype of chronic obstructive pulmonary disease (COPD) may have a different immunologic mechanism. OBJECTIVE: To investigate whether the distribution of Tregs defined by GARP is related to the multi-organ loss of tissue phenotype in COPD. METHODS: GARP expression on T cells from peripheral blood and bronchoalveolar lavage (BAL) collected from patients with COPD was examined by flow cytometry. The correlation of GARP expression to clinical outcomes and clinical phenotype, including the body mass index, lung function and quantitative computed tomography (CT) scoring of emphysema, was analyzed. RESULTS: Patients with more baseline emphysema had lower forced expiratory volume, body mass index (BMI), worse functional capacity, and more osteoporosis, thus, resembling the multiple organ loss of tissue (MOLT) phenotype. Peripheral Foxp3+GARP+ Tregs are reduced in COPD patients, and this reduction reversely correlates with quartiles of CT emphysema severity in COPD. Meanwhile, the frequencies of Foxp3+GARP- Tregs, which are characteristic of pro-inflammatory cytokine production, are significantly increased in COPD patients, and correlated with increasing quartiles of CT emphysema severity in COPD. Tregs in BAL show a similar pattern of variation in peripheral blood. CONCLUSION: Decreased GARP expression reflects more advanced disease in MOLT phenotype of COPD. Our results have potential implications for better understanding of the immunological nature of COPD and the pathogenic events leading to lung damage.

[Research progress on the role of FOXOs family in cancer].

The Forkhead box class O proteins (FOXOs) family consists of highly conserved transcription factors, including FOXO1, FOXO3, FOXO4 and FOXO6. Each member of the FOXOs family is ubiquitously expressed and involved in regulating many biological activities such as tumor cell proliferation, apoptosis, migration and oxidative stress. The activity of FOXOs is mainly regulated by post-translational modification, and its inactivation is mainly mediated by the over-activation of its upstream modifying enzymes, which provides a possibility to use drugs to recover its activity. It is worth noting that FOXOs can not only inhibit, but also promote the occurrence and development of human tumors due to the complex effects of FOXOs. This review will summarize the structure and activity regulation of FOXOs, and discuss their tumor inhibiting effects by limiting cell proliferation and inducing apoptosis, as well as their tumor promoting effects by maintaining cell homeostasis, promoting metastasis and inducing drug resistance, so as to provide new ideas for the pathological research of related diseases and open up new ways to promote broader prevention and treatment strategies.

The crystal structure of human forkhead box N1 in complex with DNA reveals the structural basis for forkhead box family specificity.

Forkhead box N1 (FOXN1) is a member of the forkhead box family of transcription factors and plays an important role in thymic epithelial cell differentiation and development. FOXN1 mutations in humans and mice give rise to the "nude" phenotype, which is marked by athymia. FOXN1 belongs to a subset of the FOX family that recognizes an alternative forkhead-like (FHL) consensus sequence (GACGC) that is different from the more widely recognized forkhead (FKH) sequence RYAAAYA (where R is purine, and Y is pyrimidine). Here, we present the FOXN1 structure in complex with DNA containing an FHL motif at 1.6 Å resolution, in which the DNA sequence is recognized by a mixture of direct and water-mediated contacts provided by residues in an α-helix inserted in the DNA major groove (the recognition helix). Comparisons with the structure of other FOX family members revealed that the FKH and FHL DNA sequences are bound in two distinct modes, with partially different registers for the protein DNA contacts. We identified a single alternative rotamer within the recognition helix itself as an important determinant of DNA specificity and found protein sequence features in the recognition helix that could be used to predict the specificity of other FOX family members. Finally, we demonstrate that the C-terminal region of FOXN1 is required for high-affinity DNA binding and that FOXN1 has a significantly reduced affinity for DNA that contains 5'-methylcytosine, which may have implications for the role of FOXN1 in thymic involution.

The FKH domain in FOXP3 mRNA frequently contains mutations in hepatocellular carcinoma that influence the subcellular localization and functions of FOXP3.

The transcription factor forkhead box P3 (FOXP3) is a biomarker for regulatory T cells and can also be expressed in cancer cells, but its function in cancer appears to be divergent. The role of hepatocyte-expressed FOXP3 in hepatocellular carcinoma (HCC) is unknown. Here, we collected tumor samples and clinical information from 115 HCC patients and used five human cancer cell lines. We examined FOXP3 mRNA sequences for mutations, used a luciferase assay to assess promoter activities of FOXP3's target genes, and employed mouse tumor models to confirm in vitro results. We detected mutations in the FKH domain of FOXP3 mRNAs in 33% of the HCC tumor tissues, but in none of the adjacent nontumor tissues. None of the mutations occurred at high frequency, indicating that they occurred randomly. Notably, the mutations were not detected in the corresponding regions of FOXP3 genomic DNA, and many of them resulted in amino acid substitutions in the FKH region, altering FOXP3's subcellular localization. FOXP3 delocalization from the nucleus to the cytoplasm caused loss of transcriptional regulation of its target genes, inactivated its tumor-inhibitory capability, and changed cellular responses to histone deacetylase (HDAC) inhibitors. More complex FKH mutations appeared to be associated with worse prognosis in HCC patients. We conclude that mutations in the FKH domain of FOXP3 mRNA frequently occur in HCC and that these mutations are caused by errors in transcription and are not derived from genomic DNA mutations. Our results suggest that transcriptional mutagenesis of FOXP3 plays a role in HCC.

Expanding the Nude SCID/CID Phenotype Associated with FOXN1 Homozygous, Compound Heterozygous, or Heterozygous Mutations.

Human nude SCID is a rare autosomal recessive inborn error of immunity (IEI) characterized by congenital athymia, alopecia, and nail dystrophy. Few cases have been reported to date. However, the recent introduction of newborn screening for IEIs and high-throughput sequencing has led to the identification of novel and atypical cases. Moreover, immunological alterations have been recently described in patients carrying heterozygous mutations. The aim of this paper is to describe the extended phenotype associated with FOXN1 homozygous, compound heterozygous, or heterozygous mutations. We collected clinical and laboratory information of a cohort of 11 homozygous, 2 compound heterozygous, and 5 heterozygous patients with recurrent severe infections. All, except one heterozygous patient, had signs of CID or SCID. Nail dystrophy and alopecia, that represent the hallmarks of the syndrome, were not always present, while almost 50% of the patients developed Omenn syndrome. One patient with hypomorphic compound heterozygous mutations had a late-onset atypical phenotype. A SCID-like phenotype was observed in 4 heterozygous patients coming from the same family. A spectrum of clinical manifestations may be associated with different mutations. The severity of the clinical phenotype likely depends on the amount of residual activity of the gene product, as previously observed for other SCID-related genes. The severity of the manifestations in this heterozygous family may suggest a mechanism of negative dominance of the specific mutation or the presence of additional mutations in noncoding regions.

E3 ubiquitin ligase PJA1 regulates lung adenocarcinoma apoptosis and invasion through promoting FOXR2 degradation.

Among all lung cancer cases, lung adenocarcinoma (LAC) represents nearly 40% and remains the leading cause of cancer deaths worldwide. Although the combination therapy of surgical treatment with radiotherapy, chemotherapy, and immunotherapy, has been used to treat LAC, unfortunately, high recurrence rates and poor survival remain. Therefore, novel prognostic markers and new targets for molecular targeted therapy in LAC is urgently needed. Fork-head box R2 (FOXR2) plays a key role in a wide range of cellular processes, including cellular proliferation, invasion, differentiation, and apoptosis, and it has been reported to be implicated in progression of LAC, thus inhibition of FOXR2 may be a novel targeting therapy for lung cancer. This current study found that E3 ligase PJA1 regulates ubiquitin-mediated degradation of FOXR2 and predicts good outcome of patients with LAC. In addition, it was showed force expression of PJA1 significantly inhibited LAC cells invasion and induced apoptosis in vitro through inactivating Wnt/ß-catenin signaling pathway. In short, our findings reveal that PJA1 could be a potential diagnostic and prognostic biomarkers and the PJA1- FOXR2 axis could be served as a promising target for LAC therapy.

Evaluation of urinary FOXP3 mRNA as a biomarker of lupus nephritis in Egyptian patients with systemic lupus erythematosus.

AIM: Lupus nephritis (LN) is one of the most serious complications of SLE. Tregs (Regulatory T lymphocytes) are thought to play a part in the pathogenesis of SLE. According to recent research, Foxp3, a Treg identification marker, plays a significant role in the pathogenesis of SLE. This study aimed to compare the urinary Foxp3 mRNA levels of patients with active and inactive forms of LN and healthy control subjects to see whether it played a role in disease activity. METHODS: We measured FOXP3 messenger RNA (mRNA) expression in the urine of 50 people with active LN, 50 people with inactive lupus, and 50 healthy people. RESULTS: We found that the expression level of FOXP3 was significantly higher in urine from patients with active LN than from subjects with inactive lupus and healthy controls (22.93 ± 4.13 vs 5.66 ± 0.47 vs 0.57 ± 0.15copy; P < 0.001).Urinary FOXP3 mRNA level significantly correlated with SLEDAI (0.000057) In the active group, urinary FOXP3 mRNA level also significantly correlated with histological activity index (< 0.00001). CONCLUSION: We concluded that urinary FOXP3 mRNA is elevated in patients with active LN and that it is linked to the SLEDAI and the severity of the disease. FOXP3 mRNA in urine sediment may be used as a non-invasive biomarker for evaluating the severity of LN and risk stratification.

Structural basis for DNA recognition by FOXC2.

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.

Coiled-coil structure-dependent interactions between polyQ proteins and Foxo lead to dendrite pathology and behavioral defects.

Neurodegenerative disorders, such as Huntington's diseases and spinocerebellar ataxias (SCAs), are driven by proteins with expanded polyglutamine (polyQ) tracts. Recently, coiled-coil structures in polyQ regions of such proteins were shown to facilitate aggregate formation and ultimately lead to cell death. However, the molecular mechanism linking these structural domains to neuronal toxicity of polyQ proteins remains elusive. Here, we demonstrate that coiled-coil structures in the Q repeat region of SCA type 3 (SCA3) polyQ proteins confer protein toxicity in Drosophila neurons. To functionally characterize coiled-coil structures in the Q repeat regions, we generated three structural variants of SCA3 polyQ proteins: (i) MJDtr-76Q, containing both α-helical coiled-coil and ß-sheet hairpin structures in the Q repeat region; (ii) MJDtr-70Q_cc0, possessing only α-helical coiled-coil structures due to the incorporation of ß-sheet-breaking residues (Q-to-N or Q-to-E mutations); and (iii) MJDtr-70Q_pQp, with no secondary structure due to the introduced proline residues (Q-to-P mutations). Through comparative analysis of these variants, we found that coiled-coil structures facilitated nuclear localization of SCA3 polyQ proteins and induced dendrite defects in Drosophila dendritic arborization neurons. Furthermore, genetic and functional screening identified the transcription factor Foxo as a target of polyQ proteins, and coiled-coil-mediated interactions of Foxo and polyQ proteins in the nucleus resulted in the observed dendrite and behavioral defects in Drosophila These results demonstrate that coiled-coil structures of polyQ proteins are crucial for their neuronal toxicity, which is conferred through coiled-coil to coiled-coil interactions with the nuclear targets of these proteins.

FoxP3 in Treg cell biology: a molecular and structural perspective.

Regulatory T cells (Tregs ) are specialized in immune suppression and play a dominant role in peripheral immune tolerance. Treg  cell lineage development and function maintenance is determined by the forkhead box protein 3 (FoxP3) transcriptional factor, whose activity is fine-tuned by its post-translational modifications (PTMs) and interaction partners. In this review, we summarize current studies in the crystal structures, the PTMs and interaction partners of FoxP3 protein, and discuss how these insights may provide a roadmap for new approaches to modulate Treg  suppression, and new therapies to enhance immune tolerance in autoimmune diseases.

SLP-65 regulates immunoglobulin light chain gene recombination through the PI(3)K-PKB-Foxo pathway.

Although the essential role of the adaptor protein SLP-65 in pre-B cell differentiation is established, the molecular mechanism underlying its function is poorly understood. In this study, we uncover a link between SLP-65-dependent signaling and the phosphoinositide-3-OH kinase (PI(3)K)-protein kinase B (PKB)-Foxo pathway. We show that the forkhead box transcription factor Foxo3a promotes light chain rearrangement in pre-B cells. Our data suggest that PKB suppresses light chain recombination by phosphorylating Foxo proteins, whereas reconstitution of SLP-65 function counteracts PKB activation and promotes Foxo3a and Foxo1 activity in pre-B cells. Together, these data illuminate a molecular function of SLP-65 and identify a key role for Foxo proteins in the regulation of light chain recombination, receptor editing and B cell selection.

Structure of a domain-swapped FOXP3 dimer on DNA and its function in regulatory T cells.

The transcription factor FOXP3 is essential for the suppressive function of regulatory T cells that are required for maintaining self-tolerance. We have solved the crystal structure of the FOXP3 forkhead domain as a ternary complex with the DNA-binding domain of the transcription factor NFAT1 and a DNA oligonucleotide from the interleukin-2 promoter. A striking feature of this structure is that FOXP3 forms a domain-swapped dimer that bridges two molecules of DNA. Structure-guided or autoimmune disease (IPEX)-associated mutations in the domain-swap interface diminished dimer formation by the FOXP3 forkhead domain without compromising FOXP3 DNA binding. These mutations also eliminated T cell-suppressive activity conferred by FOXP3, both in vitro and in a murine model of autoimmune diabetes in vivo. We conclude that FOXP3-mediated suppressor function requires dimerization through the forkhead domain and that mutations in the dimer interface can lead to the systemic autoimmunity observed in IPEX patients.

Recruitment of Fkh1 to replication origins requires precisely positioned Fkh1/2 binding sites and concurrent assembly of the pre-replicative complex.

In budding yeast, activation of many DNA replication origins is regulated by their chromatin environment, whereas others fire in early S phase regardless of their chromosomal location. Several location-independent origins contain at least two divergently oriented binding sites for Forkhead (Fkh) transcription factors in close proximity to their ARS consensus sequence. To explore whether recruitment of Forkhead proteins to replication origins is dependent on the spatial arrangement of Fkh1/2 binding sites, we changed the spacing and orientation of the sites in early replication origins ARS305 and ARS607. We followed recruitment of the Fkh1 protein to origins by chromatin immunoprecipitation and tested the ability of these origins to fire in early S phase. Our results demonstrate that precise spatial and directional arrangement of Fkh1/2 sites is crucial for efficient binding of the Fkh1 protein and for early firing of the origins. We also show that recruitment of Fkh1 to the origins depends on formation of the pre-replicative complex (pre-RC) and loading of the Mcm2-7 helicase, indicating that the origins are regulated by cooperative action of Fkh1 and the pre-RC. These results reveal that DNA binding of Forkhead factors does not depend merely on the presence of its binding sites but on their precise arrangement and is strongly influenced by other protein complexes in the vicinity.

Conserved forkhead dimerization motif controls DNA replication timing and spatial organization of chromosomes in S. cerevisiae.

Forkhead Box (Fox) proteins share the Forkhead domain, a winged-helix DNA binding module, which is conserved among eukaryotes from yeast to humans. These sequence-specific DNA binding proteins have been primarily characterized as transcription factors regulating diverse cellular processes from cell cycle control to developmental fate, deregulation of which contributes to developmental defects, cancer, and aging. We recently identified Saccharomyces cerevisiae Forkhead 1 (Fkh1) and Forkhead 2 (Fkh2) as required for the clustering of a subset of replication origins in G1 phase and for the early initiation of these origins in the ensuing S phase, suggesting a mechanistic role linking the spatial organization of the origins and their activity. Here, we show that Fkh1 and Fkh2 share a unique structural feature of human FoxP proteins that enables FoxP2 and FoxP3 to form domain-swapped dimers capable of bridging two DNA molecules in vitro. Accordingly, Fkh1 self-associates in vitro and in vivo in a manner dependent on the conserved domain-swapping region, strongly suggestive of homodimer formation. Fkh1- and Fkh2-domain-swap-minus (dsm) mutations are functional as transcription factors yet are defective in replication origin timing control. Fkh1-dsm binds replication origins in vivo but fails to cluster them, supporting the conclusion that Fkh1 and Fkh2 dimers perform a structural role in the spatial organization of chromosomal elements with functional importance.