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

ABSTRACT: 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.

SUBMITTER: Li J

PROVIDER: S-EPMC8034652 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Novel Mechanism of Bispecificity in Forkhead Transcription Factors

Project description:The forkhead transcription factor (TF) FoxN3 recognizes both the canonical forkhead DNA motif (RYAAAYA), as well as a novel alternate motif (GACGC). These two motifs are more distinct than the typically observed primary and secondary motifs of other TFs, as the two motifs cannot be aligned and are not clearly related to each other. Amino acids at the canonical base-contacting positions in the forkhead DNA binding domain are largely conserved throughout the forkhead family, so the ability of FoxN3 and other bispecific forkhead factors to recognize the alternate site cannot be explained by amino acid substitutions at these positions. Neither the mechanism of this bispecific DNA recognition nor its regulatory function are understood. Here we show that human FoxN3 does recognize both motifs in cells. We also report the crystal structures of the FoxN3 DNA binding domain bound to the canonical forkhead binding site, and separately to the alternate DNA binding site. FoxN3 adopts a remarkably similar conformation to recognize both motifs, making contacts with different DNA bases using the same amino acids. However, the DNA structure is significantly altered between the two motifs, allowing the same domain to recognize two motifs of different lengths. Changes between the two structures in the conformation of segments of the forkhead domain outside of the direct DNA-contacting positions contribute to the ability to bind the alternate site. Swapping these segments of the domain between different forkhead proteins changes the ability of proteins to recognize the alternate site, highlighting the importance of non-base-contacting positions in determining TF-DNA binding specificity. The structures present a new mechanism by which a single DBD can recognize two divergent sequences.

2019-02-27 | GSE112672 | GEO

DNA-binding specificity changes in the evolution of forkhead transcription factors.

Project description:The evolution of transcriptional regulatory networks entails the expansion and diversification of transcription factor (TF) families. The forkhead family of TFs, defined by a highly conserved winged helix DNA-binding domain (DBD), has diverged into dozens of subfamilies in animals, fungi, and related protists. We have used a combination of maximum-likelihood phylogenetic inference and independent, comprehensive functional assays of DNA-binding capacity to explore the evolution of DNA-binding specificity within the forkhead family. We present converging evidence that similar alternative sequence preferences have arisen repeatedly and independently in the course of forkhead evolution. The vast majority of DNA-binding specificity changes we observed are not explained by alterations in the known DNA-contacting amino acid residues conferring specificity for canonical forkhead binding sites. Intriguingly, we have found forkhead DBDs that retain the ability to bind very specifically to two completely distinct DNA sequence motifs. We propose an alternate specificity-determining mechanism whereby conformational rearrangements of the DBD broaden the spectrum of sequence motifs that a TF can recognize. DNA-binding bispecificity suggests a previously undescribed source of modularity and flexibility in gene regulation and may play an important role in the evolution of transcriptional regulatory networks.

| S-EPMC3725104 | biostudies-literature

DNA binding specificity of all four Saccharomyces cerevisiae forkhead transcription factors.

Project description:Quantifying the nucleotide preferences of DNA binding proteins is essential to understanding how transcription factors (TFs) interact with their targets in the genome. High-throughput in vitro binding assays have been used to identify the inherent DNA binding preferences of TFs in a controlled environment isolated from confounding factors such as genome accessibility, DNA methylation, and TF binding cooperativity. Unfortunately, many of the most common approaches for measuring binding preferences are not sensitive enough for the study of moderate-to-low affinity binding sites, and are unable to detect small-scale differences between closely related homologs. The Forkhead box (FOX) family of TFs is known to play a crucial role in regulating a variety of key processes from proliferation and development to tumor suppression and aging. By using the high-sequencing depth SELEX-seq approach to study all four FOX homologs in Saccharomyces cerevisiae, we have been able to precisely quantify the contribution and importance of nucleotide positions all along an extended binding site. Essential to this process was the alignment of our SELEX-seq reads to a set of candidate core sequences determined using a recently developed tool for the alignment of enriched k-mers and a newly developed approach for the reprioritization of candidate cores.

| S-EPMC10287902 | biostudies-literature

Novel Mechanism of Bispecificity in Forkhead Transcription Factors

Project description:Novel Mechanism of Bispecificity in Forkhead Transcription Factors

| PRJNA448691 | ENA

Genome-wide binding studies reveal DNA binding specificity mechanisms and functional interplay amongst Forkhead transcription factors.

Project description:Transcription factors belonging to the same transcription factor families contain very similar DNA binding domains and hence have the potential to bind to related DNA sequences. However, subtle differences in binding specificities can be detected in vitro with the potential to direct specific responses in vivo. Here, we have examined the binding properties of three Forkhead (FOX) transcription factors, FOXK2, FOXO3 and FOXJ3 in vivo. Extensive overlap in chromatin binding is observed, although underlying differential DNA binding specificity can dictate the recruitment of FOXK2 and FOXJ3 to chromatin. However, functionally, FOXO3-dependent gene regulation is generally mediated not through uniquely bound regions but through regions occupied by both FOXK2 and FOXO3 where both factors play a regulatory role. Our data point to a model whereby FOX transcription factors control gene expression through dynamically binding and generating partial occupancy of the same site rather than mutually exclusive binding derived by stable binding of individual FOX proteins.

| S-EPMC4770209 | biostudies-literature

Control of cyclin G2 mRNA expression by forkhead transcription factors: novel mechanism for cell cycle control by phosphoinositide 3-kinase and forkhead.

Project description:Cyclin G2 is an unconventional cyclin highly expressed in postmitotic cells. Unlike classical cyclins that promote cell cycle progression, cyclin G2 blocks cell cycle entry. Here we studied the mechanisms that regulate cyclin G2 mRNA expression during the cell cycle. Analysis of synchronized NIH 3T3 cell cultures showed elevated cyclin G2 mRNA expression levels at G(0), with a considerable reduction as cells enter cell cycle. Downregulation of cyclin G2 mRNA levels requires activation of phosphoinositide 3-kinase, suggesting that this enzyme controls cyclin G2 mRNA expression. Because the phosphoinositide 3-kinase pathway inhibits the FoxO family of forkhead transcription factors, we examined the involvement of these factors in the regulation of cyclin G2 expression. We show that active forms of the forkhead transcription factor FoxO3a (FKHRL1) increase cyclin G2 mRNA levels. Cyclin G2 has forkhead consensus motifs in its promoter, which are transactivated by constitutive active FoxO3a forms. Finally, interference with forkhead-mediated transcription by overexpression of an inactive form decreases cyclin G2 mRNA expression levels. These results show that FoxO genes regulate cyclin G2 expression, illustrating a new role for phosphoinositide 3-kinase and FoxO transcription factors in the control of cell cycle entry.

| S-EPMC350549 | biostudies-literature

Toward a mechanistic understanding of DNA binding by forkhead transcription factors and its perturbation by pathogenic mutations.

Project description:Forkhead box (FOX) proteins are an evolutionarily conserved family of transcription factors that play numerous regulatory roles in eukaryotes during developmental and adult life. Dysfunction of FOX proteins has been implicated in a variety of human diseases, including cancer, neurodevelopment disorders and genetic diseases. The FOX family members share a highly conserved DNA-binding domain (DBD), which is essential for DNA recognition, binding and function. Since the first FOX structure was resolved in 1993, >30 FOX structures have been reported to date. It is clear now that the structure and DNA recognition mechanisms vary among FOX members; however, a systematic review on this aspect is lacking. In this manuscript, we present an overview of the mechanisms by which FOX transcription factors bind DNA, including protein structures, DNA binding properties and disease-causing mutations. This review should enable a better understanding of FOX family transcription factors for basic researchers and clinicians.

| S-EPMC8501956 | biostudies-literature

Forkhead transcription factors regulate mosquito reproduction.

Project description:Forkhead-box (Fox) genes encode a family of transcription factors defined by a 'winged helix' DNA-binding domain. In this study we aimed to identify Fox factors that are expressed within the fat body of the yellow fever mosquito Aedes aegypti, and determine whether any of these are involved in the regulation of mosquito yolk protein gene expression. The Ae. aegypti genome contains 18 loci that encode putative Fox factors. Our stringent cladistic analysis has profound implications for the use of Fox genes as phylogenetic markers. Twelve Ae. aegypti Fox genes are expressed within various tissues of adult females, six of which are expressed within the fat body. All six Fox genes expressed in the fat body displayed dynamic expression profiles following a blood meal. We knocked down the 'fat body Foxes' through RNAi to determine whether these 'knockdowns' hindered amino acid-induced vitellogenin gene expression. We also determined the effect of these knockdowns on the number of eggs deposited following a blood meal. Knockdown of FoxN1, FoxN2, FoxL, and FoxO, had a negative effect on amino acid-induced vitellogenin gene expression and resulted in significantly fewer eggs laid. Our analysis stresses the importance of Fox transcription factors in regulating mosquito reproduction.

| S-EPMC2441594 | biostudies-literature

Forkhead transcription factors and cardiovascular biology.

Project description:The Forkhead family of transcription factors modulates a wide variety of cellular functions in cardiovascular tissues. In this review article, we discuss recent advances in our understanding of regulation provided by the forkhead factors in cardiac myocytes and vascular cells.

| S-EPMC2752022 | biostudies-literature

A screening system to identify transcription factors that induce binding site-directed DNA demethylation.

Project description:BackgroundDNA methylation is a fundamental epigenetic modification that is involved in many biological systems such as differentiation and disease. We and others recently showed that some transcription factors (TFs) are involved in the site-specific determination of DNA demethylation in a binding site-directed manner, although the reports of such TFs are limited.ResultsHere, we develop a screening system to identify TFs that induce binding site-directed DNA methylation changes. The system involves the ectopic expression of target TFs in model cells followed by DNA methylome analysis and overrepresentation analysis of the corresponding TF binding motif at differentially methylated regions. It successfully identified binding site-directed demethylation of SPI1, which is known to promote DNA demethylation in a binding site-directed manner. We extended our screening system to 15 master TFs involved in cellular differentiation and identified eight novel binding site-directed DNA demethylation-inducing TFs (RUNX3, GATA2, CEBPB, MAFB, NR4A2, MYOD1, CEBPA, and TBX5). Gene ontology and tissue enrichment analysis revealed that these TFs demethylate genomic regions associated with corresponding biological roles. We also describe the characteristics of binding site-directed DNA demethylation induced by these TFs, including the targeting of highly methylated CpGs, local DNA demethylation, and the overlap of demethylated regions between TFs of the same family.ConclusionsOur results show the usefulness of the developed screening system for the identification of TFs that induce DNA demethylation in a site-directed manner.

| S-EPMC5723091 | biostudies-literature

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