Forkhead box P3 (FOXP3), a key marker of regulatory T cells (Tregs), is crucial for Treg differentiation and development. Emerging evidence suggests that FOXP3 is also expressed in various tumor cells; however, its role in tumor progression remains controversial. This study aimed to elucidate the impact of FOXP3 on breast cancer development.

Breast cancer cell lines, including HCC1937, HCC1806, Hs 578T, MDA-MB-231, and MCF-7, along with xenograft mouse models, to assess the effects of FOXP3 on cell proliferation and tumor growth. FOXP3 expression in human breast cancer samples was analyzed using quantitative PCR and immunohistochemistry analyses. Cell proliferation and invasion were evaluated through MTS and transwell assays, respectively. Chromatin immunoprecipitation (ChIP) assays were performed to determine FOXP3 binding to the β-catenin gene promoter.

FOXP3 expression was elevated in advanced breast cancer and correlates with poor clinical outcomes. FOXP3 directly binds to β-catenin gene promoter - 986 to - 1168 region to facilitate β-catenin transcription, consequently resulting in increased breast cancer cell proliferation, migration, and invasion in vitro and tumor growth in vivo. Furthermore, IGF1R activated FOXP3-β-catenin signaling to promote breast tumor growth. Moreover, elesclomol, a potent copper ionophore, significantly inhibited FOXP3 expression to suppress breast tumor growth.

This study indicates that FOXP3 plays an oncogenic role in breast cancer development and suggests that targeting IGF1R-FOXP3-β-catenin signaling may be a putative therapeutic strategy for human breast cancer treatment.

The forkhead family of transcription factors of class O (FOXOs) consisting of four functionally related proteins, FOXO1, FOXO3, FOXO4, and FOXO6, are mammalian homologs of daf-16 in Caenorhabditis elegans and were previously identified as tumor suppressors, oxidative stress sensors, and cell survival modulators. Under normal physiological conditions, FOXO protein activities are negatively regulated by phosphorylation via the phosphoinositide 3-kinase (PI3K)-Akt pathway, a well-known cell survival pathway: Akt phosphorylates FOXOs to inactivate their transcriptional activity by relocalizing FOXOs from the nucleus to the cytoplasm for degradation. However, under oxidative stress or absent the cellular survival drive of growth factors, FOXO proteins translocate to the nucleus and upregulate a series of target genes, thereby promoting cell growth arrest and cell death and altering mitochondrial homeostasis. FOXO gene expression is also regulated by other transcriptional factors such as p53 or autoregulation by their activities and end products. Here we summarize the structure, posttranslational modifications, and translocation of FOXOs linking to their transcriptional control of cellular functions, survival, and death, emphasizing their role in regulating the cellular response to some acute insults and chronic neurological disorders. This review will conclude with a brief section on potential therapeutic interventions that can be used to modulate FOXOs' activities when treating acute and chronic neurological disorders.

Forkhead transcription factors regulate several important biological processes in many eukaryotic species including fungi. Bioinformatic analysis of the Aspergillus flavus genome revealed four putative forkhead transcription factor genes. Genetic disruption of (AFLA_005634), a homolog of the Aspergillus nidulans fhpA/fkhA gene (AN4521), revealed that the fhpA gene is a negative regulator of both asexual spore production and aflatoxin B1 production in A. flavus. Furthermore, disruption of the fhpA gene caused a complete loss of sclerotial formation. Overexpression of the fhpA gene caused A. flavus to become more sensitive to sodium chloride whereas disruption of the fhpA gene did not change the ability of A. flavus to respond to any osmotic stress agent tested. Interestingly, both disruption and overexpression of the fhpA gene led to increases in sensitivity to the oxidative stress agent menadione. Overall, these results suggest that fhpA is an important regulator of morphological and chemical development in addition to stress response in A. flavus.

The Forkhead box M1 (FOXM1) gene-mediated Wnt signaling pathway plays a significant role in the development and growth of glioblastoma multiforme (GBM), an exceptionally aggressive form of brain cancer. Our research explores the crucial involvement of the FOXM1 gene, a key transcription factor within the Wnt signaling pathway using bioinformatics techniques in both GBM and glioma stem cells (GSCs). Elevated FOXM1 gene expression is strongly associated with poor patient survival in GBM. Furthermore, FOXM1 gene expression is correlated with stemness-related factors, such as SOX2 and SOX9, which act as key drivers in the progression of cancer stem cells. Moreover, we specifically look into the direct associations of the FOXM1 gene with angiogenetic-related factors, metabolic genes, metastatic genes, pluripotency-related factors, immune cell infiltration, transcriptional networks, and functional category enrichment analysis, shedding light on the intricate molecular mechanisms involved in GBM initiation and progression. Additionally, our research identifies FOXM1-targeting miRNAs, revealing their potential as therapeutic candidates with implications for patient survival rates and DNA methylation patterns of the FOXM1 gene, uncovering insights into its epigenetic regulation. This knowledge contributes to a comprehensive understanding of the molecular landscape and potential avenues for developing more effective therapeutic approaches against GBM and GSCs.

This study investigates the role of SPINK1 in liver cancer and its regulatory relationship with FOXM1. Using differential gene analysis in the GEO database, SPINK1 was identified as overexpressed in liver cancer tissues and associated with poor prognosis, confirmed via PCR. Functional assays demonstrated that SPINK1 knockdown reduced proliferation, migration, and invasion in liver cancer cells, while promoting apoptosis. In vivo experiments revealed that SPINK1 knockdown inhibited tumor growth, decreased Ki-67 and N-cadherin levels, increased E-cadherin levels, and suppressed lung metastasis. Analysis of upstream factors indicated that FOXM1 binds to the SPINK1 promoter, as validated by dual-luciferase and ChIP assays, thereby promoting SPINK1 transcription. TCGA database analysis and clinical tissue validation showed that FOXM1 expression correlates with poor prognosis in liver cancer. Functional studies demonstrated that FOXM1 knockdown suppressed liver cancer progression, while SPINK1 overexpression reversed these effects. KEGG enrichment analysis identified the p53 pathway as a key downstream target of SPINK1, and Western blotting confirmed its role in modulating p53 pathway activity. These findings reveal a critical FOXM1-SPINK1 axis in liver cancer progression. FOXM1 directly promotes SPINK1 transcription, enhancing tumor cell proliferation and metastasis while regulating the p53 pathway. Targeting this axis could provide a potential therapeutic approach for liver cancer.

Forkhead box (FOX) genes are a family of transcription factors that participate in many biological activities, from early embryogenesis to the formation of organs, and from regulation of glucose metabolism to regulation of longevity. Given the extensive influence in the multicellular process, FOX family proteins are responsible for the progression of many types of cancers, especially lung cancer, breast cancer, prostate cancer, and other cancers. Breast cancer is the most common cancer among women, and 2.3 million women were diagnosed in 2020. So, various drugs targeting the FOX signaling pathway have been developed to inhibit breast cancer progression. While the role of the FOX family gene in cancer development has not received enough attention, discovering more potential drugs targeting the FOX signaling pathway is urgently demanded. Here, we review the main members in the FOX gene family and summarize their signaling pathway, including the regulation of the FOX genes and their effects on breast cancer progression. We hope this review will emphasize the understanding of the role of the FOX gene in breast cancer and inspire the discovery of effective anti-breast cancer medicines targeting the FOX gene in the future.

Eukaryotic genomes are organized into distinct chromatin compartments, some of which exhibit properties of biomolecular condensates. These condensates primarily form due to chromatin-associated proteins/complexes (CAPs). CAPs play a crucial role in gene expression, functioning as either transcriptional repressors or activators. Phase separation, a well-established biophysical phenomenon, is a key driver of chromatin condensate formation by CAPs. Notably, multivalent CAPs with the ability to engage in diverse interactions promote chromatin compaction, leading to the formation of transcriptionally repressed compartments. Conversely, interactions between intrinsically disordered region (IDR)-containing transcriptional regulators, mediated by their multivalent IDRs, lead to the formation of protein-rich, transcriptionally active droplets on decondensed genomic regions. Interestingly, both repressive heterochromatin and activating euchromatin condensates exhibit spontaneous phase separation and selectively enrich components with concordant transcriptional functions. This review delves into the mechanisms by which transcriptionally repressive CAPs orchestrate the formation of repressed chromatin domains. We further explore how a diverse array of transcription-related CAPs or core histone variants, via phase separation, influence gene expression by inducing erroneous transcription events, regulating expression levels, and facilitating the interconversion of transcriptionally repressed and active regions.

FoxO is a downstream target gene of cellular nutrient and growth factors, oxidative stress responses, and insulin signaling pathways. It play a crucial role in insect growth, development, and reproduction. Locusta migratoria is a significant agricultural pest; therefore, the identification of novel control targets for its management is of significant importance. After injecting dsRNA to interfere with FoxO expression, we observed changes in the reproduction-related gene expression and ovary development through RT-qPCR and morphological observation. Simultaneously, the trehalose and glycogen contents were measured following RNAi. The results demonstrate that interference with FoxO significantly downregulates key genes in the Hippo pathway and Notch gene expression. In terms of carbohydrate metabolism, the trehalose content decreases significantly while the glycogen content increases markedly after FoxO silencing. Additionally, FoxO silencing considerably inhibits reproductive-related gene expression, resulting in delayed ovarian development. These findings indicate that FoxO regulates L. migratoria reproduction through the Hippo signaling pathway: when impaired, the reproductive capacity function declines. In addition, FoxO-mediated energy mobilization is involved in the regulation of egg production. These results indicate that the RNAi of FoxO may be a useful control strategy against L. migratoria.

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.

The spinal cord is a crucial part of the vertebrate CNS, controlling movements and receiving and processing sensory information from the trunk and limbs. However, there is much we do not know about how this essential organ develops. Here, we describe expression of 21 transcription factors and one transcriptional regulator in zebrafish spinal cord.

We analyzed the expression of aurkb, foxb1a, foxb1b, her8a, homeza, ivns1abpb, mybl2b, myt1a, nr2f1b, onecut1, sall1a, sall3a, sall3b, sall4, sox2, sox19b, sp8b, tsc22d1, wdhd1, zfhx3b, znf804a, and znf1032 in wild-type and MIB E3 ubiquitin protein ligase 1 zebrafish embryos. While all of these genes are broadly expressed in spinal cord, they have distinct expression patterns from one another. Some are predominantly expressed in progenitor domains, and others in subsets of post-mitotic cells. Given the conservation of spinal cord development, and the transcription factors and transcriptional regulators that orchestrate it, we expect that these genes will have similar spinal cord expression patterns in other vertebrates, including mammals and humans.

Our data identify 22 different transcriptional regulators that are strong candidates for playing different roles in spinal cord development. For several of these genes, this is the first published description of their spinal cord expression.

FOXA1 serves as a crucial pioneer transcription factor during developmental processes and plays a pivotal role as a mitotic bookmarking factor to perpetuate gene expression profiles and maintain cellular identity. During mitosis, the majority of FOXA1 dissociates from specific DNA binding sites and redistributes to non-specific binding sites; however, the regulatory mechanisms governing molecular dynamics and activity of FOXA1 remain elusive. Here, we show that mitotic kinase Aurora B specifies the different DNA binding modes of FOXA1 and guides FOXA1 biomolecular condensation in mitosis. Mechanistically, Aurora B kinase phosphorylates FOXA1 at Serine 221 (S221) to liberate the specific, but not the non-specific, DNA binding. Interestingly, the phosphorylation of S221 attenuates the FOXA1 condensation that requires specific DNA binding. Importantly, perturbation of the dynamic phosphorylation impairs accurate gene reactivation and cell proliferation, suggesting that reversible mitotic protein phosphorylation emerges as a fundamental mechanism for the spatiotemporal control of mitotic bookmarking.

Mammalian gene expression is controlled by transcription factors (TFs) that engage sequence motifs in a chromatinized genome, where nucleosomes can restrict DNA access. Yet, how nucleosomes affect individual TFs remains unclear. Here, we measure the ability of over one hundred TF motifs to recruit TFs in a defined chromosomal locus in mouse embryonic stem cells. This identifies a set sufficient to enable the binding of TFs with diverse tissue specificities, functions, and DNA-binding domains. These chromatin-competent factors are further classified when challenged to engage motifs within a highly phased nucleosome. The pluripotency factors OCT4-SOX2 preferentially engage non-nucleosomal and entry-exit motifs, but not nucleosome-internal sites, a preference that also guides binding genome wide. By contrast, factors such as BANP, REST, or CTCF engage throughout, causing nucleosomal displacement. This supports that TFs vary widely in their sensitivity to nucleosomes and that genome access is TF specific and influenced by nucleosome position in the cell.

Mechanosensory hair cells located in the inner ear mediate the sensations of hearing and balance. If damaged, mammalian inner ear hair cells are unable to regenerate, resulting in permanent sensory deficits. Aquatic vertebrates like zebrafish (Danio rerio) have a specialized class of mechanosensory hair cells found in the lateral line system, allowing them to sense changes in water current. Unlike mammalian inner ear hair cells, lateral line hair cells can robustly regenerate following damage. In mammals, the transcription factor Foxg1 functions to promote normal development of the inner ear. Foxg1a is expressed in lateral line sensory organs in zebrafish larvae, but its function during lateral line development and regeneration has not been investigated. Our study demonstrates that mutation of foxg1a results in slower posterior lateral line primordium migration and delayed neuromast formation. In developing and regenerating neuromasts, we find that loss of Foxg1a function results in reduced hair cell numbers, as well as decreased proliferation of neuromast cells. Foxg1a specifically regulates the development and regeneration of Islet1-labeled hair cells. These data suggest that Foxg1 may be a valuable target for investigation of clinical hair cell regeneration.

The Mediator complex is essential for eukaryotic transcription, yet its role and the function of its individual subunits in plants, especially in rice, remain poorly understood. Here, we investigate the function of OsMED14_2, a subunit of the Mediator tail module, in rice development. Overexpression and knockout of OsMED14_2 resulted in notable changes in panicle morphology and grain size. Microscopic analysis revealed impact of overexpression on pollen maturation, reflected by reduced viability, irregular shapes, and aberrant intine development. OsMED14_2 was found to interact with proteins involved in pollen development, namely, OsMADS62, OsMADS63 and OsMADS68, and its overexpression negatively affected the expression of OsMADS68 and the expression of other genes involved in intine development, including OsCAP1, OsGCD1, OsRIP1, and OsCPK29. Additionally, we found that OsMED14_2 overexpression influences jasmonic acid (JA) homeostasis, affecting bioactive JA levels, and expression of OsJAZ genes. Our data suggest OsMED14_2 may act as a regulator of JA-responsive genes through its interactions with OsHDAC6 and OsJAZ repressors. These findings contribute to better understanding of the Mediator complex's role in plant traits regulation.

FOXM1 is a key transcriptional regulator involved in various biological processes in mammals, including carbohydrate and lipid metabolism, aging, immune regulation, development, and disease. Early studies have shown that FOXM1 acts as an oncogene by regulating cell proliferation, cell cycle, migration, metastasis, and apoptosis, as well as genes related to diagnosis, treatment, chemotherapy resistance, and prognosis. Researchers are increasingly focusing on FOXM1 functions in tumor microenvironment, epigenetics, and immune infiltration. However, researchers have not comprehensively described FOXM1's involvement in tumor microenvironment shaping, epigenetics, and immune cell infiltration. Here we review the role of FOXM1 in the formation and development of malignant tumors, and we will provide a comprehensive summary of the role of FOXM1 in transcriptional regulation, interacting proteins, tumor microenvironment, epigenetics, and immune infiltration, and suggest areas for further research.

Mouse FOXA1 and GATA4 are prototypes of pioneer factors, initiating liver cell development by binding to the N1 nucleosome in the enhancer of the ALB1 gene. Using cryoelectron microscopy (cryo-EM), we determined the structures of the free N1 nucleosome and its complexes with FOXA1 and GATA4, both individually and in combination. We found that the DNA-binding domains of FOXA1 and GATA4 mainly recognize the linker DNA and an internal site in the nucleosome, respectively, whereas their intrinsically disordered regions interact with the acidic patch on histone H2A-H2B. FOXA1 efficiently enhances GATA4 binding by repositioning the N1 nucleosome. In vivo DNA editing and bioinformatics analyses suggest that the co-binding mode of FOXA1 and GATA4 plays important roles in regulating genes involved in liver cell functions. Our results reveal the mechanism whereby FOXA1 and GATA4 cooperatively bind to the nucleosome through nucleosome repositioning, opening chromatin by bending linker DNA and obstructing nucleosome packing.

Chromatin is densely packed with nucleosomes, which limits the accessibility of many chromatin-associated proteins. Pioneer factors (PFs) are usually viewed as a special group of sequence-specific transcription factors (TFs) that can recognize nucleosome-embedded motifs, invade compact chromatin, and generate open chromatin regions. Through this process, PFs initiate a cascade of events that play key roles in gene regulation and cell differentiation. A current debate in the field is if PFs belong to a unique subset of TFs with intrinsic "pioneering activity", or if all TFs have the potential to function as PFs within certain cellular contexts. There are also different views regarding the key feature(s) that define pioneering activity. In this review, we present evidence from the literature related to these alternative views and discuss how to potentially reconcile them. It is possible that both intrinsic properties, like tight nucleosome binding and structural compatibility, and cellular conditions, like concentration and co-factor availability, are important for PF function.

Cancer cells re-program normal lung endothelial cells (EC) into tumor-associated endothelial cells (TEC) that form leaky vessels supporting carcinogenesis. Transcriptional regulators that control the reprogramming of EC into TEC are poorly understood. We identified Forkhead box F1 (FOXF1) as a critical regulator of EC-to-TEC transition. FOXF1 was highly expressed in normal lung vasculature but was decreased in TEC within non-small cell lung cancers (NSCLC). Low FOXF1 correlated with poor overall survival of NSCLC patients. In mice, endothelial-specific deletion of FOXF1 decreased pericyte coverage, increased vessel permeability and hypoxia, and promoted lung tumor growth and metastasis. Endothelial-specific overexpression of FOXF1 normalized tumor vessels and inhibited the progression of lung cancer. FOXF1 deficiency decreased Wnt/β-catenin signaling in TECs through direct transcriptional activation of Fzd4. Restoring FZD4 expression in FOXF1-deficient TECs through endothelial-specific nanoparticle delivery of Fzd4 cDNA rescued Wnt/β-catenin signaling in TECs, normalized tumor vessels and inhibited the progression of lung cancer. Altogether, FOXF1 increases tumor vessel stability, and inhibits lung cancer progression by stimulating FZD4/Wnt/β-catenin signaling in TECs. Nanoparticle delivery of FZD4 cDNA has promise for future therapies in NSCLC.

Mechanosensory hair cells located in the inner ear mediate the sensations of hearing and balance. If damaged, mammalian inner ear hair cells are unable to regenerate, resulting in permanent sensory deficits. Aquatic vertebrates like zebrafish (Danio rerio) have a specialized class of mechanosensory hair cells found in the lateral line system, allowing them to sense changes in water current. Unlike mammalian inner ear hair cells, lateral line hair cells can robustly regenerate following damage. In mammalian models, the transcription factor Foxg1 functions to promote normal development of the inner ear. Foxg1a is expressed in lateral line sensory organs in zebrafish larvae, but its function during lateral line development and regeneration has not been investigated. We find that loss of Foxg1a function results in reduced hair cell development and regeneration, as well as decreased cellular proliferation in the lateral line system. These data suggest that Foxg1 may be a valuable target for investigation of clinical hair cell regeneration.

Our work demonstrates a role for Foxg1a in developing and regenerating new sensory cells through proliferation.

FOXA1 is a pioneer transcription factor that is frequently mutated in prostate, breast, bladder, and salivary gland malignancies. Indeed, metastatic castration-resistant prostate cancer (mCRPC) commonly harbour FOXA1 mutations with a prevalence of 35%. However, despite the frequent recurrence of FOXA1 mutations in prostate cancer, the mechanisms by which FOXA1 variants drive its oncogenic effects are still unclear. Semaphorin 3C (SEMA3C) is a secreted autocrine growth factor that drives growth and treatment resistance of prostate and other cancers and is known to be regulated by both AR and FOXA1. In the present study, we characterize FOXA1 alterations with respect to its regulation of SEMA3C. Our findings reveal that FOXA1 alterations lead to elevated levels of SEMA3C both in prostate cancer specimens and in vitro. We further show that FOXA1 negatively regulates SEMA3C via intronic cis elements, and that mutations in FOXA1 forkhead domain attenuate its inhibitory function in reporter assays, presumably by disrupting DNA binding of FOXA1. Our findings underscore the key role of FOXA1 in prostate cancer progression and treatment resistance by regulating SEMA3C expression and suggest that SEMA3C may be a driver of growth and tumor vulnerability of mCRPC harboring FOXA1 alterations.

Pioneer transcription factors (TFs) exhibit a specialized ability to 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 small molecules that stereoselectively and site-specifically bind the pioneer TF, 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 covalent ligands relax the canonical DNA binding preference of FOXA1 by strengthening interactions with suboptimal ancillary 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.

Forkhead box M1 (FOXM1) is a transcription factor in the forkhead (FOX) family, which is required for cellular proliferation in normal and neoplastic cells. FOXM1 is highly expressed in many different cancers, and its expression is associated with a higher tumor stage and worse patient-related outcomes. Abnormally high expression of FOXM1 in cancers compared to normal tissue makes FOXM1 an attractive target for pharmacological inhibition. FOXM1-inhibiting agents and specific FOXM1-targeted small-molecule inhibitors have been developed in the lab and some of them have shown promising efficacy and safety profiles in mouse models. While the future goal is to translate FOXM1 inhibitors to clinical trials, potential synergistic drug combinations can maximize anti-tumor efficacy while minimizing off-target side effects. Hence, we discuss the rationale and efficacy of all previously studied drug combinations with FOXM1 inhibitors for cancer therapies.

Eukaryotic DNA is packaged into chromatin in the nucleus, restricting the binding of transcription factors (TFs) to their target DNA sites. FOXA1 functions as a pioneer TF to bind condensed chromatin and initiate the opening of local chromatin for gene expression. However, the principles of FOXA1 recruitment and how it subsequently unpacks the condensed chromatin remain elusive. Here, we revealed that FOXA1 intrinsically forms submicron-sized condensates through its N- and C-terminal intrinsically disordered regions (IDRs). Notably, both IDRs enable FOXA1 to dissolve the condensed chromatin. In addition, the DNA-binding capacity of FOXA1 contributes to its ability to both form condensates and dissolve condensed chromatin. Further genome-wide investigation showed that IDRs enable FOXA1 to bind and unpack the condensed chromatin to regulate the proliferation and migration of breast cancer cells. This work provides a principle of how pioneer TFs function to initiate competent chromatin states using their IDRs.

Renal cell carcinoma (RCC) is a frequent malignancy of the urinary system with high mortality and morbidity. However, the molecular mechanisms underlying RCC progression are still largely unknown. In this study, we identified FOXA2, a pioneer transcription factor, as a driver oncogene for RCC. We show that FOXA2 was commonly upregulated in human RCC samples and promoted RCC proliferation, as evidenced by assays of cell viability, colony formation, migratory and invasive capabilities, and stemness properties. Mechanistically, we found that FOXA2 promoted RCC cell proliferation by transcriptionally activating HIF2α expression in vitro and in vivo. Furthermore, we found that FOXA2 could interact with VHL (von Hippel‒Lindau), which ubiquitinated FOXA2 and controlled its protein stability in RCC cells. We showed that mutation of lysine at position 264 to arginine in FOXA2 could mostly abrogate its ubiquitination, augment its activation effect on HIF2α expression, and promote RCC proliferation in vitro and RCC progression in vivo. Importantly, elevated expression of FOXA2 in patients with RCC positively correlated with the expression of HIF2α and was associated with shorter overall and disease-free survival. Together, these findings reveal a novel role of FOXA2 in RCC development and provide insights into the underlying molecular mechanisms of FOXA2-driven pathological processes in RCC.

The urothelium is a stratified epithelium composed of basal cells, one or more layers of intermediate cells, and an upper layer of differentiated umbrella cells. Most bladder cancers (BLCA) are urothelial carcinomas. Loss of urothelial lineage fidelity results in altered differentiation, highlighted by the taxonomic classification into basal and luminal tumors. There is a need to better understand the urothelial transcriptional networks. To systematically identify transcription factors (TFs) relevant for urothelial identity, we defined highly expressed TFs in normal human bladder using RNA-Seq data and inferred their genomic binding using ATAC-Seq data. To focus on epithelial TFs, we analyzed RNA-Seq data from patient-derived organoids recapitulating features of basal/luminal tumors. We classified TFs as "luminal-enriched", "basal-enriched" or "common" according to expression in organoids. We validated our classification by differential gene expression analysis in Luminal Papillary vs. Basal/Squamous tumors. Genomic analyses revealed well-known TFs associated with luminal (e.g., PPARG, GATA3, FOXA1) and basal (e.g., TP63, TFAP2) phenotypes and novel candidates to play a role in urothelial differentiation or BLCA (e.g., MECOM, TBX3). We also identified TF families (e.g., KLFs, AP1, circadian clock, sex hormone receptors) for which there is suggestive evidence of their involvement in urothelial differentiation and/or BLCA. Genomic alterations in these TFs are associated with BLCA. We uncover a TF network involved in urothelial cell identity and BLCA. We identify novel candidate TFs involved in differentiation and cancer that provide opportunities for a better understanding of the underlying biology and therapeutic intervention.

The forkhead box protein O (FOXO, consisting of FOXO1, FOXO3, FOXO4 and FOXO6) transcription factors are the mammalian orthologues of Caenorhabditis elegans DAF-16, which gained notoriety for its capability to double lifespan in the absence of daf-2 (the gene encoding the worm insulin receptor homologue). Since then, research has provided many mechanistic details on FOXO regulation and FOXO activity. Furthermore, conditional knockout experiments have provided a wealth of data as to how FOXOs control development and homeostasis at the organ and organism levels. The lifespan-extending capabilities of DAF-16/FOXO are highly correlated with their ability to induce stress response pathways. Exogenous and endogenous stress, such as cellular redox stress, are considered the main drivers of the functional decline that characterizes ageing. Functional decline often manifests as disease, and decrease in FOXO activity indeed negatively impacts on major age-related diseases such as cancer and diabetes. In this context, the main function of FOXOs is considered to preserve cellular and organismal homeostasis, through regulation of stress response pathways. Paradoxically, the same FOXO-mediated responses can also aid the survival of dysfunctional cells once these eventually emerge. This general property to control stress responses may underlie the complex and less-evident roles of FOXOs in human lifespan as opposed to model organisms such as C. elegans.

Stapled peptides have rapidly established themselves as a powerful technique to mimic α-helical interactions with a short peptide sequence. There are many examples of stapled peptides that successfully disrupt α-helix-mediated protein-protein interactions, with an example currently in clinical trials. DNA-protein interactions are also often mediated by α-helices and are involved in all transcriptional regulation processes. Unlike DNA-binding small molecules, which typically lack DNA sequence selectivity, DNA-binding proteins bind with high affinity and high selectivity. These are ideal candidates for the design DNA-binding stapled peptides. Despite the parallel to protein-protein interaction disrupting stapled peptides and the need for sequence specific DNA binders, there are very few DNA-binding stapled peptides. In this review we examine all the known DNA-binding stapled peptides. Their design concepts are compared to stapled peptides that disrupt protein-protein interactions and based on the few examples in the literature, DNA-binding stapled peptide trends are discussed.

Forkhead box gene R2 (FOXR2) belongs to the family of FOX genes which codes for highly conserved transcription factors (TFs) with critical roles in biological processes ranging from development to organogenesis to metabolic and immune regulation to cellular homeostasis. A number of FOX genes are associated with cancer development and progression and poor prognosis. A growing body of evidence suggests that FOXR2 is an oncogene. Studies suggested important roles for FOXR2 in cancer cell growth, metastasis, and drug resistance. Recent studies showed that FOXR2 is overexpressed by a subset of newly identified entities of embryonal tumors. This review discusses the role(s) FOXR2 plays in the pathology of pediatric brain cancers and its potential as a therapeutic target.

Lung adenocarcinoma (LUAD) is a clinically challenging disease due to its poor prognosis and limited therapeutic methods. The aim of the present study was to identify prognosis-related genes and therapeutic targets for LUAD. Raw data from the GSE32863, GSE41271 and GSE42127 datasets were downloaded from the Gene Expression Omnibus database. Following normalization, the data were merged into a matrix, which was first used to identify differentially expressed genes (DEGs). Weighted gene co-expression network analysis (WGCNA) and survival analysis were performed to screen potential prognosis-related genes. Gene overlaps among DEGs, survival-related genes and WGCNA genes were finally constructed to obtain candidate genes. An analysis with the STRING database was performed to construct a protein-protein interaction network and hub genes were selected using Cytoscape. The candidate genes were finally identified by univariate and multivariate Cox regression analysis. Furthermore, in vivo and in vitro experiments, including immunohistochemistry, immunofluorescence, Cell Counting Kit-8, colony-formation and migration assays, were performed to validate the potential mechanism of these genes in LUAD. Two genes, namely forkhead box M1 (FOXM1) and centromere protein F (CENPF), were identified as unfavorable indicators of prognosis in patients with LUAD. High expression of FOXM1 and CENPF were associated with poor survival. Furthermore, LUAD cells with FOXM1 and CENPF knockdown showed a significant reduction in proliferation and migration (P<0.05). FOXM1 and CENPF may have an essential role in the prognosis of patients with LUAD by influencing cell proliferation and migration, and they provide potential molecular targets for LUAD therapy.

The FOXL2 (forkhead box L2) gene is located on chromosome 3 and encodes for forkhead box (FOX) family of transcription factors which play a critical role in various biological processes. Germline FOXL2 mutations have been identified in blepharophimosis/ptosis/epicanthus inversus syndrome. The somatic missense mutation in FOXL2 (FOXL2 C134W) is now known to be the defining molecular feature of adult-type granulosa cell tumour of the ovary, present in over 90% of cases of this tumour type. Immunohistochemistry for FOXL2 is used as a marker of sex cord-stromal differentiation. However, expression is not restricted to lesions harbouring FOXL2 mutations, and it is positive in a variety of sex cord-stromal proliferations other than adult-type granulosa cell tumour.

Psoriasis is a common, chronic, and relapsing inflammatory skin disease characterized by hyperproliferation of keratinocytes (KCs) and infiltration of immune cells. The pathogenesis of psoriasis is complex, and the exact mechanism remains partially understood. In this study, we showed that the forkhead box family protein, FOXE1, had increased expression in lesional skins compared with nonlesional skin from patients with psoriasis. FOXE1 expression was also increased in an imiquimod-induced psoriatic mouse model as well as in M5-stimulated KCs. Using combinational approaches of knockdown and overexpression of FOXE1, we demonstrated that FOXE1 may promote the proliferation of KCs by facilitating G1/S transition and activating extracellular signal-regulated kinase 1/2 signaling pathway. In addition, knockdown of FOXE1 reduced the production of IL-1β, IL-6, and TNF-α by KCs. RNA-sequencing profiling identified WNT5A as a potential downstream effector of FOXE1. Knockdown of WNT5A inhibited the proliferation of KCs; reduced the production of IL-1β, IL-6, and TNF-α by KCs; and mitigated the growth-promoting effect of FOXE1 in FOXE1-overexpressed KCs. Finally, depletion of FOXE1 by lentiviral delivery of small hairpin RNAs or genetic approach ameliorated dermatitis symptoms in imiquimod-induced psoriasis-like mouse models. Taken together, our results indicated that FOXE1 participates in the pathogenesis of psoriasis and can serve as a target of psoriasis treatment.

As a transcriptional factor, the Forkhead box (FOX) gene family is closely connected with apoptosis, proliferation, and other cellular processes. FOXD2, as one descendant of the FOX gene family, has been mentioned in many articles to show a high expression in several cancers. However, whether FOXD2 has a connection with gastric adenocarcinoma remains an unanswered question. Expression of FOXD2 and IQGAP3 in gastric adenocarcinoma was evaluated by bioinformatics analysis, which was further detected by real-time quantitative PCR (qRT-PCR) and western blot. The downstream target genes of FOXD2 were also mined by bioinformatics analysis. Pathway enrichment analysis was then performed on the target genes. Chromatin immunoprecipitation assay (ChIP) and dual-luciferase reporter assay were conducted to validate the regulatory relationship between FOXD2 and its downstream target gene IQGAP3. Methyl thiazolyl tetrazolium assay (MTT), combined with cell colony formation assay, was employed to assess the effect of FOXD2 and IQGAP3 on the proliferation of gastric adenocarcinoma cells. Intracytoplasmic Ca2+ concentration was measured by Fluo-3 fluorescence staining. FOXD2 showed a high expression in gastric adenocarcinoma tissues and cells, and FOXD2 silencing considerably attenuated gastric adenocarcinoma cell proliferation. IQGAP3, a downstream target gene of FOXD2, had a positive connection with the expression of FOXD2. The binding relationship between FOXD2 and the promoter region of IQGAP3 was further verified by ChIP and dual-luciferase reporter assays. The results of cell function experiments indicated that FOXD2 could promote gastric adenocarcinoma cell proliferation by transcriptionally activating IQGAP3 to induce an increase in intracellular Ca2+ level. This study confirmed that FOXD2 increased intracellular Ca2+ level through transcriptional activation of IQGAP3, which in turn propelled the proliferation of gastric adenocarcinoma cells, revealing the considerable significance of FOXD2 in the development of gastric adenocarcinoma.

Covalent chemistry represents an attractive strategy for expanding the ligandability of the proteome, and chemical proteomics has revealed numerous electrophile-reactive cysteines on diverse human proteins. Determining which of these covalent binding events affect protein function, however, remains challenging. Here we describe a base-editing strategy to infer the functionality of cysteines by quantifying the impact of their missense mutation on cancer cell proliferation. The resulting atlas, which covers more than 13,800 cysteines on more than 1,750 cancer dependency proteins, confirms the essentiality of cysteines targeted by covalent drugs and, when integrated with chemical proteomic data, identifies essential, ligandable cysteines in more than 160 cancer dependency proteins. We further show that a stereoselective and site-specific ligand targeting an essential cysteine in TOE1 inhibits the nuclease activity of this protein through an apparent allosteric mechanism. Our findings thus describe a versatile method and valuable resource to prioritize the pursuit of small-molecule probes with high function-perturbing potential.

The DNA-binding activities of transcription factors (TFs) are influenced by both intrinsic sequence preferences and extrinsic interactions with cell-specific chromatin landscapes and other regulatory proteins. Disentangling the roles of these binding determinants remains challenging. For example, the FoxA subfamily of Forkhead domain (Fox) TFs are known pioneer factors that can bind to relatively inaccessible sites during development. Yet FoxA TF binding also varies across cell types, pointing to a combination of intrinsic and extrinsic forces guiding their binding. While other Forkhead domain TFs are often assumed to have pioneering abilities, how sequence and chromatin features influence the binding of related Fox TFs has not been systematically characterized. Here, we present a principled approach to compare the relative contributions of intrinsic DNA sequence preference and cell-specific chromatin environments to a TF's DNA-binding activities. We apply our approach to investigate how a selection of Fox TFs (FoxA1, FoxC1, FoxG1, FoxL2, and FoxP3) vary in their binding specificity. We over-express the selected Fox TFs in mouse embryonic stem cells, which offer a platform to contrast each TF's binding activity within the same preexisting chromatin background. By applying a convolutional neural network to interpret the Fox TF binding patterns, we evaluate how sequence and preexisting chromatin features jointly contribute to induced TF binding. We demonstrate that Fox TFs bind different DNA targets, and drive differential gene expression patterns, even when induced in identical chromatin settings. Despite the association between Forkhead domains and pioneering activities, the selected Fox TFs display a wide range of affinities for preexiting chromatin states. Using sequence and chromatin feature attribution techniques to interpret the neural network predictions, we show that differential sequence preferences combined with differential abilities to engage relatively inaccessible chromatin together explain Fox TF binding patterns at individual sites and genome-wide.

Gynecological and breast tumors are one of the main causes of cancer-related mortalities among women. Despite recent advances in diagnostic and therapeutic methods, tumor relapse is observed in a high percentage of these patients due to the treatment failure. Late diagnosis in advanced tumor stages is one of the main reasons for the treatment failure and recurrence in these tumors. Therefore, it is necessary to assess the molecular mechanisms involved in progression of these tumors to introduce the efficient early diagnostic markers. Fokhead Box (FOX) is a family of transcription factors with a key role in regulation of a wide variety of cellular mechanisms. Deregulation of FOX proteins has been observed in different cancers. MicroRNAs (miRNAs) as a group of non-coding RNAs have important roles in post-transcriptional regulation of the genes involved in cellular mechanisms. They are also the non-invasive diagnostic markers due to their high stability in body fluids. Considering the importance of FOX proteins in the progression of breast and gynecological tumors, we investigated the role of miRNAs in regulation of the FOX proteins in these tumors. MicroRNAs were mainly involved in progression of these tumors through FOXM, FOXP, and FOXO. The present review paves the way to suggest a non-invasive diagnostic panel marker based on the miRNAs/FOX axis in breast and gynecological cancers.

FOXM1 is an oncogenic transcriptional factor and includes several isoforms generated by alternative splicing. Inclusion of alternative exon 9 produces FOXM1a, a transcriptionally inactive isoform. However, the role of FOXM1a in tumorigenesis remains unknown. In addition, the regulatory mechanisms of exon 9 splicing are also unclear. In the present study, we found that overexpression of FOXM1a significantly reduced cell proliferation and colony formation of oral squamous cell carcinoma (OSCC) cell proliferation in vitro. Importantly, OSCC cells with FOXM1a overexpression showed significantly slower tumor formation in nude mice. Moreover, we identified a U-rich exonic splicing suppressor (ESS) which is responsible for exon 9 skipping. Splicing factor heterogeneous nuclear ribonucleoprotein C (hnRNP C) can bind to the ESS and suppress exon 9 inclusion and FOXM1a expression. Silence of hnRNP C also significantly suppresses OSCC cell proliferation. HnRNP C is significantly co-expressed with FOXM1 in cancers. Our study uncovered a novel regulatory mechanism of oncogene FOXM1 expression in OSCC.

Aging is a major risk factor of high incidence and increased mortality of acute respiratory distress syndrome (ARDS). Here, we demonstrated that persistent lung injury and high mortality in aged mice after sepsis challenge were attributable to impaired endothelial regeneration and vascular repair. Genetic lineage tracing study showed that endothelial regeneration after sepsis-induced vascular injury was mediated by lung resident endothelial proliferation in young adult mice, whereas this intrinsic regenerative program was impaired in aged mice. Expression of forkhead box M1 (FoxM1), an important mediator of endothelial regeneration in young mice, was not induced in lungs of aged mice. Transgenic FOXM1 expression or in vivo endothelium-targeted nanoparticle delivery of the FOXM1 gene driven by an endothelial cell (EC)-specific promoter reactivated endothelial regeneration, normalized vascular repair and resolution of inflammation, and promoted survival in aged mice after sepsis challenge. In addition, treatment with the FDA-approved DNA demethylating agent decitabine was sufficient to reactivate FoxM1-dependent endothelial regeneration in aged mice, reverse aging-impaired resolution of inflammatory injury, and promote survival. Mechanistically, aging-induced Foxm1 promoter hypermethylation in mice, which could be inhibited by decitabine treatment, inhibited Foxm1 induction after sepsis challenge. In COVID-19 lung autopsy samples, FOXM1 was not induced in vascular ECs of elderly patients in their 80s, in contrast with middle-aged patients (aged 50 to 60 years). Thus, reactivation of FoxM1-mediated endothelial regeneration and vascular repair may represent a potential therapy for elderly patients with ARDS.

Pioneer factors are transcription factors (TFs) that have the unique ability to recognise their target DNA sequences within closed chromatin. Whereas their interactions with cognate DNA is similar to other TFs, their ability to interact with chromatin remains poorly understood. Having previously defined the modalities of DNA interactions for the pioneer factor Pax7, we have now used natural isoforms of this pioneer as well as deletion and replacement mutants to investigate the Pax7 structural requirements for chromatin interaction and opening. We show that the GL+ natural isoform of Pax7 that has two extra amino acids within the DNA binding paired domain is unable to activate the melanotrope transcriptome and to fully activate a large subset of melanotrope-specific enhancers targeted for Pax7 pioneer action. This enhancer subset remains in the primed state rather than being fully activated, despite the GL+ isoform having similar intrinsic transcriptional activity as the GL- isoform. C-terminal deletions of Pax7 lead to the same loss of pioneer ability, with similar reduced recruitments of the cooperating TF Tpit and of the co-regulators Ash2 and BRG1. This suggests complex interrelations between the DNA binding and C-terminal domains of Pax7 that are crucial for its chromatin opening pioneer ability.

Overexpression of Forkhead box protein C2 (FOXC2) has been associated with different types of carcinomas. FOXC2 plays an important role in the initiation and maintenance of the epithelial-mesenchymal transition (EMT) process, which is essential for the development of higher-grade tumors with an enhanced ability for metastasis. Thus, FOXC2 has become a therapeutic target for the development of anticancer drugs. MC-1-F2, the only identified experimental inhibitor of FOXC2, interacts with the full length of FOXC2. However, only the DNA-binding domain (DBD) of FOXC2 has resolved crystal structure. In this work, a three-dimensional (3D) structure of the full-length FOXC2 using homology modeling was developed and used for structure-based drug design (SBDD). The quality of this 3D model of the full-length FOXC2 was evaluated using MolProbity, ERRAT, and ProSA modules. Molecular dynamics (MD) simulation was also carried out to verify its stability. Ligand-based drug design (LBDD) was carried out to identify similar analogues for MC-1-F2 against 15 million compounds from ChEMBL and ZINC databases. 792 molecules were retrieved from this similarity search. De novo SBDD was performed against the full-length 3D structure of FOXC2 through homology modeling to identify novel inhibitors. The combination of LBDD and SBDD helped in gaining a better insight into the binding of MC-1-F2 and its analogues against the full length of the FOXC2. The binding free energy of the top hits was further investigated using MD simulations and MM/GBSA calculations to result in eight promising hits as lead compounds targeting FOXC2.