Abstract
BackgroundThe Forkhead (FKH) transcription factor FOXM1 is a key regulator of the cell cycle and is overexpressed in most types of cancer. FOXM1, similar to other FKH factors, binds to a canonical FKH motif in vitro. However, genome-wide mapping studies in different cell lines have shown a lack of enrichment of the FKH motif, suggesting an alternative mode of chromatin recruitment. We have investigated the role of direct versus indirect DNA binding in FOXM1 recruitment by performing ChIP-seq with wild-type and DNA binding deficient FOXM1.ResultsAn in vitro fluorescence polarization assay identified point mutations in the DNA binding domain of FOXM1 that inhibit binding to a FKH consensus sequence. Cell lines expressing either wild-type or DNA binding deficient GFP-tagged FOXM1 were used for genome-wide mapping studies comparing the distribution of the DNA binding deficient protein to the wild-type. This shows that interaction of the FOXM1 DNA binding domain with target DNA is essential for recruitment. Moreover, analysis of the protein interactome of wild-type versus DNA binding deficient FOXM1 shows that the reduced recruitment is not due to inhibition of protein-protein interactions.ConclusionsA functional DNA binding domain is essential for FOXM1 chromatin recruitment. Even in FOXM1 mutants with almost complete loss of binding, the protein-protein interactions and pattern of phosphorylation are largely unaffected. These results strongly support a model whereby FOXM1 is specifically recruited to chromatin through co-factor interactions by binding directly to non-canonical DNA sequences.Electronic supplementary materialThe online version of this article (doi:10.1186/s13059-015-0696-z) contains supplementary material, which is available to authorized users.
Highlights
The Forkhead (FKH) transcription factor FOXM1 is a key regulator of the cell cycle and is overexpressed in most types of cancer
To investigate the importance of direct interaction of the FOXM1 DNA binding domain (DBD) with the FKH consensus on in vitro binding, four highly conserved H3 amino acids were chosen to generate mutations that are predicted to interfere with DNA binding. (Fig. 1a; H3 residues selected for mutation are indicated with red box)
The DNA binding activity of the wild-type (WT) and mutated FOXM1 proteins was assessed by fluorescence polarization (FP) binding assays using a carboxyfluorescein (6FAM) -tagged dsDNA oligonucleotide containing the FKH consensus sequence (5′AAACAAACAAACAATC)
Summary
The Forkhead (FKH) transcription factor FOXM1 is a key regulator of the cell cycle and is overexpressed in most types of cancer. A number of genome-wide studies have mapped FOXM1 binding to the FKH target motif, while others have mapped the indirect binding of FOXM1 through its interaction with B-Myb or LIN9, a component of the MuvB complex [19]. These studies present conflicting models of FOXM1 recruitment to chromatin binding sites. Sadasivam et al [19] using HeLa cells, found that the FKH motif was enriched in genomic sites bound by LIN9 and B-Myb, which were predominantly located within cell cycle promoters.
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
