Zebrafish FoxH1

Maggie Wallace '27 Pedro Araujo '27

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Contents:

I. Introduction

Zebrafish FoxH1 is a member of the forkhead box (FOX) family of transcription factors. It is a well-conserved group of proteins recognized for their ability to bind to DNA through a specialized region known as the forkhead domain. FoxH1 is recruited by phosphorylated Smad2 downstream of TGFβs, including Nodal-regulated proteins. FOX proteins, including FoxH1, are essential for regulating gene expression by interacting with specific DNA sequences and influencing various biological processes, such as development, cell differentiation, and tissue organization. Specifically, FoxH1 plays a vital role in nodal signaling during early embryonic development. Nodal signaling is a vital part of vertebrate embryonic development. FoxH1 regulates critical signaling pathways, especially those involved in mesoderm and gastrula organizer formation and the specification of the left-right axis. Basal binding of FoxH1 genes allows for mesoderm differentiation promoters to activate transcription.

FoxH1 is essential in the TGF-β signaling pathway, interacting with Smad proteins to regulate target genes critical for development. It helps establish the anterior-posterior axis and is vital for forming the primitive streak during early zebrafish development. As a FOX protein, FoxH1 binds to DNA in gene promoter regions and can activate or repress transcription based on the cellular context, playing a crucial role in mesodermal differentiation and cellular organization. 

II. General Structure

Zebrafish FoxH1, when bound to the TGTGGATT DNA sequence, adopts a specific monomeric conformation, has a molecular weight of 51,620 and consists of a single polypeptide chain around 472 amino acids in length. The structure of FoxH1 bound to DNA includes three key regions: the N-terminal domain for transcriptional activation and slight DNA bending, the forkhead domain for specific DNA binding, and the C-terminal domain which is where the smad interaction motif is located. There is a hinge region between the DNA-binding domain and the transactivation domain. 

III. Smad Protein

The "N-terminal domain" of FoxH1 contains a specialized Smad interaction motif (SIM), a short, conserved sequence that mediates direct interaction with activated Smad complexes. SIM is composed of specific amino acids that facilitate hydrophobic and electrostatic interactions between Smad proteins and the FoxH1, creating a high-affinity complex. The FoxH1 gene was the first Smad DNA-binding partner that was identified. Two specific structures have been identified that are essential for Smad function. The interaction between Smad proteins and zebrafish FoxH1 is essential for mediating transcriptional responses to the transforming growth factor, TGF-β/BMP, signaling. In this signaling pathway, Smad2 and Smad3 are phosphorylated upon receptor activation, triggering their association with Smad4, forming a trimeric Smad complex—the complex associates with FoxH1, which is already bound to DNA target sites and now FoxH1 can initiate or enhance the transcription of TGF-β/BMP-responsive genes. This complex translocates to the nucleus to target specific gene responses.

IV. DNA Binding

In order for transcription to be activated, FoxH1 needs to be bound to the Smad complex, as FoxH1 on its own is not sufficient for transcription activation. The Smad-containing DNA-binding complex can positively or negatively regulate gene expression through recruitment of co-activators and co-repressors. To initiate the complex of FoxH1 and Smad, the MH2 domain of Smad 2 and 3 bind directly to the of FoxH1 which is in the C-terminal domain. This Smad interaction motif consists of 54 amino acids. Smad 2 and 3 then recruit Smad 4. Smad 4 binding is required for transcription activation of FoxH1. FoxH1 complex can bind directly to specific elements in the promoter of target genes. The optimal consensus sequence for FoxH1 binding is TGTG/TT/GATT which is present in all FoxH1 targets. . 

V. Functional Implications

It has been proposed that FoxH1 and FoxA proteins participate in a molecular “hand-off” mechanism to maintain enhancer activation of specific cis-regulatory elements during the maternal-to-zygotic transition in Xenopus8. The structural information we gathered for the DNA binding specificities of FoxA2 and FoxH1 FH domains is compatible with the hypothesis whereby FoxA proteins substitutes for FoxH1 preferentially at TT sites, with FoxH1 remaining bound to GK sites even when the concentration of this protein is low. The presence of GK sites in many FoxH1 targets may have been optimized, together with the key differences in the FoxH1 structure, thus avoiding the misregulation of essential genes that contain GK sites by other FOX proteins optimized to select TT motifs.


VI. References

Pogoda, Hans-Martin, et al. "The zebrafish forkhead transcription factor FoxH1/Fast1 is a modulator of nodal signaling required for organizer formation." Current Biology 10.17 (2000): 1041-1049.

Pluta, Radoslaw, et al. "Molecular basis for DNA recognition by the maternal pioneer transcription factor FoxH1." Nature Communications 13.1 (2022): 7279.

Pei, Wuhong, et al. "An early requirement for maternal FoxH1 during zebrafish gastrulation." Developmental biology 310.1 (2007): 10-22.

Attisano, Liliana, et al. "The transcriptional role of Smads and FAST (FoxH1) in TGFβ and activin signalling." Molecular and cellular endocrinology 180.1-2 (2001): 3-11.

“UniProt.” Uniprot.org, 2024, www.uniprot.org/uniprotkb/Q9I9E1/entry#expression. Accessed 2 Dec. 2024.

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