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Novel Mechanism of Bispecificity in Forkhead Transcription Factors

ABSTRACT: 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.

ORGANISM(S): Homo sapiens

PROVIDER: GSE112672 | GEO | 2019/02/27

REPOSITORIES: GEO

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Project description:The forkhead box (FOX) family of proteins is composed of more than 50 members that are phylogenetically classified into 19 subclasses (A to S). These transcription factors all share a homology in their DNA-binding domain, the forkhead domain, also known as winged helix due to its butterfly-like structural appearance.FOX proteins have been implicated in a broad spectrum of cellular processes including cell proliferation, differentiation, DNA repair, metabolism, and aging. However, the biological function of the mammalian forkhead transcription factors of the N class including FOXN3 remains to be investigated. The SIN3A complex is a large protein assembly consisting of over a dozen of proteins including SIN3A, HDAC1/2, RBBP4/7 and etc. This complex is recruited by multiple transcription repressors and impacts on multifaceted biological functions ranging from development, differentiation, and senescence to oncogenic transformation. Long noncoding RNAs (lncRNAs) are a class of noncoding RNAs that are over 200 nucleotides in length. In the past years, these molecules have emerged as important components of the epigenetic regulatory network to influence transcription as well as other nuclear activities, and their dysregulation underlies several pathologic states including cancer. One of the lncRNAs, NEAT1 (nuclear paraspeckle assembly transcript 1), is a highly abundant lncRNA initially described as a structural component of nuclear paraspeckles. Subsequent studies suggest that NEAT1 also influences transcription through either an indirect mechanism or a direct way. In this study, we investigated the function and the underlying mechanism of FOXN3 in breast cancer cells. We showed that FOXN3 is physically associated with the SIN3A complex and identified that NEAT1, which is induced by estrogen in breast cancer cells, is required for this interaction. We analyzed the genomic targets of the FOXN3/SIN3A/NEAT1 complex and demonstrated that estrogen receptor (ER) itself is regulated, through negative feedback, by the FOXN3/SIN3A/NEAT1 complex. We investigated the role of the FOXN3/SIN3A/NEAT1 complex in breast cancer metastasis and explored the clinicopathological significance of the ER-NEAT1-FOXN3/NEAT1/SIN3A-GATA3 axis in breast cancer.

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Novel Mechanism of Bispecificity in Forkhead Transcription Factors

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