Structural basis for the FOXM1 DNA binding domain to specific dsDNA substrate

Abstract

Forkhead box protein M1 (FOXM1) is a key transcription factor that regulates cell cycle progression and is frequently overexpressed in human cancers, driving tumor proliferation and therapy resistance. FOXM1 recognizes the canonical forkhead response element (FKH motif, RYAAAYA) through its conserved DNA-binding domain (DBD). Here, we report the high-resolution crystal structure of the FOXM1-DBD in complex with a double-stranded DNA substrate containing two FKH motifs. The structure reveals that FOXM1-DBD adopts the canonical winged-helix fold, with the third α-helix (α3) inserted into the DNA major groove to mediate sequence-specific recognition. Within this helix, Asn283, Arg286, and His287 form an essential triad that engages DNA bases through specific hydrogen bonds and hydrophobic interactions. Using structure-guided mutagenesis of key DNA-interacting residues combined with biophysical validation by isothermal titration calorimetry (ITC) and DNA binding assessment via electrophoretic mobility shift assay (EMSA), we confirm the functional importance of these residues and uncover position-dependent tolerance to base substitutions within the FKH motif. Furthermore, we demonstrate that FOXM1 overexpression promotes cell proliferation and upregulates the transcription of target genes in a DBD-dependent manner. Our findings provide a structural basis for understanding the DNA recognition mechanism of FOXM1 and offer mechanistic insights into how FOXM1 selectively binds to its genomic targets to regulate transcription.

1. Introduction

The precise regulation of cell cycle progression is fundamental to cellular homeostasis, and its dysregulation underlies the pathogenesis of numerous human diseases [1,2]. In eukaryotes, this process is governed by an integrated network of cyclins, cyclin-dependent kinases (CDKs), checkpoint signaling pathways, and ubiquitin-mediated regulatory mechanisms [3,4]. When this sophisticated control system is compromised, it contributes not only to cancer development but also to a spectrum of other pathological conditions, including cardiovascular disorders, inflammatory diseases, and neurodegenerative processes [2]. Understanding the molecular mechanisms that maintain cell cycle-metabolic coordination is therefore essential for elucidating fundamental biological processes such as differentiation and aging while also providing critical insights for addressing disease mechanisms.

Within this regulatory framework, transcription factors (TFs) serve as crucial intermediaries that translate cell cycle signals into specific gene expression programs [5,6]. As sequence-specific DNA-binding proteins, TFs recognize distinct regulatory elements to establish phase-dependent gene expression patterns essential for orderly cell cycle progression [7,8]. This regulatory system features intricate feedback mechanisms, as many TFs themselves exhibit cell cycle-dependent expression or activity, creating sophisticated regulatory circuits with CDK/cyclin complexes [9,10]. Through these reciprocal interactions, TFs coordinate the sequential activation of genes governing critical cell cycle transitions while simultaneously integrating extracellular signals to ensure that division occurs only under appropriate conditions. The crucial role of TFs in cell cycle control is frequently exploited in cancer pathogenesis [11]. Malignant cells commonly overexpress specific TFs to drive proliferation and increase survival [12]. Notably, many cancers are dependent on lineage-specific TFs that normally regulate cell fate decisions and differentiation programs [5,13]. This appropriation of developmental regulatory mechanisms represents a fundamental strategy by which cancer cells maintain their proliferative advantage and survival capacity.