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FOXM1 binds directly to non-consensus sequences in the human genome.

Sanders DA, Gormally MV, Marsico G, Beraldi D, Tannahill D, Balasubramanian S - Genome Biol. (2015)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Cancer Research UK, Cambridge Research Institute, Li Ka Shing Center, Robinson Way, Cambridge, CB2 0RE, UK. das1001@cam.ac.uk.

ABSTRACT

Background: The 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.

Results: An 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.

Conclusions: A 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.

No MeSH data available.


Related in: MedlinePlus

Proteomic analysis shows that the FOXM1 DBD mutants bind to same proteins as the WT. a Schematic diagram showing RIME analysis to identify FOXM1 co-binding proteins. b Coverage of GFP-FOXM1 and associated high-confidence interacting proteins. Yellow shading indicates regions of peptide coverage and the green shading shows post-translational modifications identified. (Prob = probability, # pep = number of peptides, %Cov = % protein coverage). c Table showing the average number of peptides identified for the six top proteins present in the WT GFP-FOXM1 pull-downs. d Co-immunoprecipitation showing pull-down of B-MYB, LIN9, and TF2B with a GFP antibody in extracts from HEK293 cells expressing WT, H287A, and R286A GFP-FOXM1 DBD mutants and GFP only. e Schematic diagram showing phosphorylation sites identified by Proteome viewer in a WT GFP-FOXM1 RIME sample. The color indicates the identification probability. f Diagram showing the position of previously identified phosphorylation sites in FOXM1b (red indicates serine and green threonine residues). The table indicates sites identified from the RIME analysis in WT and R286A DBD mutant GFP-FOXM1, with novel sites highlighted in green
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Fig6: Proteomic analysis shows that the FOXM1 DBD mutants bind to same proteins as the WT. a Schematic diagram showing RIME analysis to identify FOXM1 co-binding proteins. b Coverage of GFP-FOXM1 and associated high-confidence interacting proteins. Yellow shading indicates regions of peptide coverage and the green shading shows post-translational modifications identified. (Prob = probability, # pep = number of peptides, %Cov = % protein coverage). c Table showing the average number of peptides identified for the six top proteins present in the WT GFP-FOXM1 pull-downs. d Co-immunoprecipitation showing pull-down of B-MYB, LIN9, and TF2B with a GFP antibody in extracts from HEK293 cells expressing WT, H287A, and R286A GFP-FOXM1 DBD mutants and GFP only. e Schematic diagram showing phosphorylation sites identified by Proteome viewer in a WT GFP-FOXM1 RIME sample. The color indicates the identification probability. f Diagram showing the position of previously identified phosphorylation sites in FOXM1b (red indicates serine and green threonine residues). The table indicates sites identified from the RIME analysis in WT and R286A DBD mutant GFP-FOXM1, with novel sites highlighted in green

Mentions: To investigate whether the reduced DNA binding of the DBD mutant proteins is due to perturbation of protein-protein interactions involved in the recruitment, proteomic analysis of the FOXM1 interacting proteins was performed by RIME (Rapid immunoprecipitation mass spectrometry of endogenous proteins) [45] for both the WT GFP-FOXM1 and the R286A DBD mutant. Formaldehyde-fixed chromatin was isolated from HEK293 cells expressing either GFP only, GFP-FOXM1 WT, or GFP-FOXM1 R286A and immunoprecipitation was performed with an anti-GFP antibody followed by LC/MS-MS (Fig. 6), to identify associated proteins. To eliminate any non-specific interactions with GFP, which can occur through cross-linking of the GFP to highly abundant proteins such as keratins and ribosomal sub-units, proteins identified in the GFP only expressing cell line were not considered in the WT or R286A samples. High confidence interacting proteins were then identified using the Scaffold proteomic analysis tool [46], details of the proteins identified are shown in Additional file 4. In the WT, several proteins previously shown to interact with FOXM1 were identified, including components of the MuvB complex [19] (LIN9, LIN54), B-MYB [47] and PLK1 [30]. Figure 6b shows the sequence coverage from Scaffold for the top five high confidence proteins. These results show that the presence of the GFP epitope tag does not significantly affect the interaction of the GFP-FOXM1 fusion protein with known partners of endogenous FOXM1. Overlap of proteins identified in the WT and R286A replicates (identified by unique peptides present in at least three out of the four samples) revealed 21 common to both, while eight were present in the WT only and 15 in the R286A mutant (Additional file 1: Table S6 and Figure S10).Fig. 6


FOXM1 binds directly to non-consensus sequences in the human genome.

Sanders DA, Gormally MV, Marsico G, Beraldi D, Tannahill D, Balasubramanian S - Genome Biol. (2015)

Proteomic analysis shows that the FOXM1 DBD mutants bind to same proteins as the WT. a Schematic diagram showing RIME analysis to identify FOXM1 co-binding proteins. b Coverage of GFP-FOXM1 and associated high-confidence interacting proteins. Yellow shading indicates regions of peptide coverage and the green shading shows post-translational modifications identified. (Prob = probability, # pep = number of peptides, %Cov = % protein coverage). c Table showing the average number of peptides identified for the six top proteins present in the WT GFP-FOXM1 pull-downs. d Co-immunoprecipitation showing pull-down of B-MYB, LIN9, and TF2B with a GFP antibody in extracts from HEK293 cells expressing WT, H287A, and R286A GFP-FOXM1 DBD mutants and GFP only. e Schematic diagram showing phosphorylation sites identified by Proteome viewer in a WT GFP-FOXM1 RIME sample. The color indicates the identification probability. f Diagram showing the position of previously identified phosphorylation sites in FOXM1b (red indicates serine and green threonine residues). The table indicates sites identified from the RIME analysis in WT and R286A DBD mutant GFP-FOXM1, with novel sites highlighted in green
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Related In: Results  -  Collection

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Fig6: Proteomic analysis shows that the FOXM1 DBD mutants bind to same proteins as the WT. a Schematic diagram showing RIME analysis to identify FOXM1 co-binding proteins. b Coverage of GFP-FOXM1 and associated high-confidence interacting proteins. Yellow shading indicates regions of peptide coverage and the green shading shows post-translational modifications identified. (Prob = probability, # pep = number of peptides, %Cov = % protein coverage). c Table showing the average number of peptides identified for the six top proteins present in the WT GFP-FOXM1 pull-downs. d Co-immunoprecipitation showing pull-down of B-MYB, LIN9, and TF2B with a GFP antibody in extracts from HEK293 cells expressing WT, H287A, and R286A GFP-FOXM1 DBD mutants and GFP only. e Schematic diagram showing phosphorylation sites identified by Proteome viewer in a WT GFP-FOXM1 RIME sample. The color indicates the identification probability. f Diagram showing the position of previously identified phosphorylation sites in FOXM1b (red indicates serine and green threonine residues). The table indicates sites identified from the RIME analysis in WT and R286A DBD mutant GFP-FOXM1, with novel sites highlighted in green
Mentions: To investigate whether the reduced DNA binding of the DBD mutant proteins is due to perturbation of protein-protein interactions involved in the recruitment, proteomic analysis of the FOXM1 interacting proteins was performed by RIME (Rapid immunoprecipitation mass spectrometry of endogenous proteins) [45] for both the WT GFP-FOXM1 and the R286A DBD mutant. Formaldehyde-fixed chromatin was isolated from HEK293 cells expressing either GFP only, GFP-FOXM1 WT, or GFP-FOXM1 R286A and immunoprecipitation was performed with an anti-GFP antibody followed by LC/MS-MS (Fig. 6), to identify associated proteins. To eliminate any non-specific interactions with GFP, which can occur through cross-linking of the GFP to highly abundant proteins such as keratins and ribosomal sub-units, proteins identified in the GFP only expressing cell line were not considered in the WT or R286A samples. High confidence interacting proteins were then identified using the Scaffold proteomic analysis tool [46], details of the proteins identified are shown in Additional file 4. In the WT, several proteins previously shown to interact with FOXM1 were identified, including components of the MuvB complex [19] (LIN9, LIN54), B-MYB [47] and PLK1 [30]. Figure 6b shows the sequence coverage from Scaffold for the top five high confidence proteins. These results show that the presence of the GFP epitope tag does not significantly affect the interaction of the GFP-FOXM1 fusion protein with known partners of endogenous FOXM1. Overlap of proteins identified in the WT and R286A replicates (identified by unique peptides present in at least three out of the four samples) revealed 21 common to both, while eight were present in the WT only and 15 in the R286A mutant (Additional file 1: Table S6 and Figure S10).Fig. 6

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Cancer Research UK, Cambridge Research Institute, Li Ka Shing Center, Robinson Way, Cambridge, CB2 0RE, UK. das1001@cam.ac.uk.

ABSTRACT

Background: The 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.

Results: An 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.

Conclusions: A 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.

No MeSH data available.


Related in: MedlinePlus