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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

Mutation of the DBD of FOXM1 inhibits binding genome-wide. a Hierarchical clustering analysis of the WT and DBD mutant GFP-FOXM1 replicate ChIP-seq datasets showing that the samples separate well into distinct treatment groups. b Venn diagram showing the overlap between binding regions identified by ChIP-seq analysis in 293 cells expressing GFP-FOXM1 WT and the GFP-FOXM1 mutants H287A or R286A. c CEAS analysis comparing genomic distribution of GFP-FOXM1 binding events in WT versus DBD mutants. d Examples of genomic regions showing three representative promoter binding sites in which binding peaks are only identified for GFP-FOXM1 WT and are not present in the DBD mutant cell lines. e Motifs enriched in WT GFP-FOXM1 and the mutant R286A-H287A (full list in Additional file 1: Table S4). P values and percentages in brackets represent the statistical significance and the percentage of peaks for each motif, respectively. Motif sequences are sorted according to their proportional representation in WT GFP-FOXM1 peaks (NS = non-significant)
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Fig4: Mutation of the DBD of FOXM1 inhibits binding genome-wide. a Hierarchical clustering analysis of the WT and DBD mutant GFP-FOXM1 replicate ChIP-seq datasets showing that the samples separate well into distinct treatment groups. b Venn diagram showing the overlap between binding regions identified by ChIP-seq analysis in 293 cells expressing GFP-FOXM1 WT and the GFP-FOXM1 mutants H287A or R286A. c CEAS analysis comparing genomic distribution of GFP-FOXM1 binding events in WT versus DBD mutants. d Examples of genomic regions showing three representative promoter binding sites in which binding peaks are only identified for GFP-FOXM1 WT and are not present in the DBD mutant cell lines. e Motifs enriched in WT GFP-FOXM1 and the mutant R286A-H287A (full list in Additional file 1: Table S4). P values and percentages in brackets represent the statistical significance and the percentage of peaks for each motif, respectively. Motif sequences are sorted according to their proportional representation in WT GFP-FOXM1 peaks (NS = non-significant)

Mentions: Each replicate for each cell line resulted in 25–30 million reads (Additional file 1: Table S1). For each construct, peaks in common between each replicate showed a good level of concordance. WT, H287A and R286A replicates showed 7,473 (72 % overlap), 1,169 (60 % overlap) and 804 shared binding events (81 % overlap), respectively (Additional file 2). Comparing this GFP-FOXM1 dataset with that in the preceding section again showed a high level of overlap with 6,443 shared peaks (86 % concordance), further confirming the high degree of reproducibility for the GFP-FOXM1 ChIP-seq. Furthermore, hierarchical clustering (Fig. 4a) showed that the replicate samples grouped together indicating a reproducible dataset. Overlapping the peaks from the WT and DBD FOXM1 mutants (Fig. 4b) showed that the majority of H287A (86.5 %) and R286A (98.5 %) peaks are a subset of WT GFP-FOXM1 binding sites. For H287A, although 156 peaks did not overlap with the WT or R286A peaks, most overlapped with at least one WT replicate, suggesting that they are not novel binding sites. The majority of R286A peaks are also shared by H287A (approximately 57 %). Of particular note is the observation that both FOXM1 DBD mutations significantly reduce the overall level of genomic binding. The R286A mutation was more effective at reducing binding compared to H287A, which is consistent with the in vitro DBD FP analysis showing that R286A has significantly lower binding than H287A (Fig. 1c) for the FKH consensus.Fig. 4


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)

Mutation of the DBD of FOXM1 inhibits binding genome-wide. a Hierarchical clustering analysis of the WT and DBD mutant GFP-FOXM1 replicate ChIP-seq datasets showing that the samples separate well into distinct treatment groups. b Venn diagram showing the overlap between binding regions identified by ChIP-seq analysis in 293 cells expressing GFP-FOXM1 WT and the GFP-FOXM1 mutants H287A or R286A. c CEAS analysis comparing genomic distribution of GFP-FOXM1 binding events in WT versus DBD mutants. d Examples of genomic regions showing three representative promoter binding sites in which binding peaks are only identified for GFP-FOXM1 WT and are not present in the DBD mutant cell lines. e Motifs enriched in WT GFP-FOXM1 and the mutant R286A-H287A (full list in Additional file 1: Table S4). P values and percentages in brackets represent the statistical significance and the percentage of peaks for each motif, respectively. Motif sequences are sorted according to their proportional representation in WT GFP-FOXM1 peaks (NS = non-significant)
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4492089&req=5

Fig4: Mutation of the DBD of FOXM1 inhibits binding genome-wide. a Hierarchical clustering analysis of the WT and DBD mutant GFP-FOXM1 replicate ChIP-seq datasets showing that the samples separate well into distinct treatment groups. b Venn diagram showing the overlap between binding regions identified by ChIP-seq analysis in 293 cells expressing GFP-FOXM1 WT and the GFP-FOXM1 mutants H287A or R286A. c CEAS analysis comparing genomic distribution of GFP-FOXM1 binding events in WT versus DBD mutants. d Examples of genomic regions showing three representative promoter binding sites in which binding peaks are only identified for GFP-FOXM1 WT and are not present in the DBD mutant cell lines. e Motifs enriched in WT GFP-FOXM1 and the mutant R286A-H287A (full list in Additional file 1: Table S4). P values and percentages in brackets represent the statistical significance and the percentage of peaks for each motif, respectively. Motif sequences are sorted according to their proportional representation in WT GFP-FOXM1 peaks (NS = non-significant)
Mentions: Each replicate for each cell line resulted in 25–30 million reads (Additional file 1: Table S1). For each construct, peaks in common between each replicate showed a good level of concordance. WT, H287A and R286A replicates showed 7,473 (72 % overlap), 1,169 (60 % overlap) and 804 shared binding events (81 % overlap), respectively (Additional file 2). Comparing this GFP-FOXM1 dataset with that in the preceding section again showed a high level of overlap with 6,443 shared peaks (86 % concordance), further confirming the high degree of reproducibility for the GFP-FOXM1 ChIP-seq. Furthermore, hierarchical clustering (Fig. 4a) showed that the replicate samples grouped together indicating a reproducible dataset. Overlapping the peaks from the WT and DBD FOXM1 mutants (Fig. 4b) showed that the majority of H287A (86.5 %) and R286A (98.5 %) peaks are a subset of WT GFP-FOXM1 binding sites. For H287A, although 156 peaks did not overlap with the WT or R286A peaks, most overlapped with at least one WT replicate, suggesting that they are not novel binding sites. The majority of R286A peaks are also shared by H287A (approximately 57 %). Of particular note is the observation that both FOXM1 DBD mutations significantly reduce the overall level of genomic binding. The R286A mutation was more effective at reducing binding compared to H287A, which is consistent with the in vitro DBD FP analysis showing that R286A has significantly lower binding than H287A (Fig. 1c) for the FKH consensus.Fig. 4

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