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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

GFP-tagged FOXM1 shows a similar genomic distribution to endogenous FOXM1. a Venn diagram showing the overlap between endogenous FOXM1 and GFP-FOXM1 binding sites in TetR HEK293 cells, using peaks in common in two replicates for each condition. b Motifs enriched in FOXM1 and GFP-FOXM1. Selected motifs identified in FOXM1 are also found in the GFP-FOXM1 peaks (full list in Additional file 1: Table S2). 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 FOXM1 peaks. c Examples of genomic regions showing three representative sites in common for endogenous FOXM1 and GFP-FOXM1 binding. d CEAS analysis comparing genomic distribution of endogenous FOXM1 binding events to GFP-FOXM1 in HEK293 cells
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Fig3: GFP-tagged FOXM1 shows a similar genomic distribution to endogenous FOXM1. a Venn diagram showing the overlap between endogenous FOXM1 and GFP-FOXM1 binding sites in TetR HEK293 cells, using peaks in common in two replicates for each condition. b Motifs enriched in FOXM1 and GFP-FOXM1. Selected motifs identified in FOXM1 are also found in the GFP-FOXM1 peaks (full list in Additional file 1: Table S2). 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 FOXM1 peaks. c Examples of genomic regions showing three representative sites in common for endogenous FOXM1 and GFP-FOXM1 binding. d CEAS analysis comparing genomic distribution of endogenous FOXM1 binding events to GFP-FOXM1 in HEK293 cells

Mentions: Two biological replicates were performed for both cell lines and 25–30 million reads were obtained for each sample (Additional file 1: Table S1) and binding peaks were called using Model-based Analysis for Chip-Seq (MACS) [34]. For endogenous FOXM1, 1,390 peaks were present in both replicates and for GFP-FOXM1 12,418 peaks were identified (Additional file 2), with a high level of concordance between replicates (approximately 78 % for FOXM1 and approximately 81 % for GFP-FOXM1). Comparison of the endogenous FOXM1 and GFP-FOXM1 peak locations (Fig. 3a) showed a high degree of overlap (approximately 88 %) indicating that the GFP-FOXM1 protein captures the majority of the endogenous FOXM1 binding sites. In the endogenous FOXM1 replicates approximately 50 % more peaks were called in one sample (3,970 compared to 1,722). When less stringent parameters were used for peak calling, using each replicate separately, the number of binding sites shared between endogenous FOXM1 and the GFP-FOXM1 increased to 3,173 representing approximately 26 % of the total GFP-FOXM1 binding sites. The approximately 10-fold increase in high confidence peaks called in the GFP-FOXM1 samples compared to endogenous FOXM1 is possibly due to the higher affinity of the GFP antibody and the relatively poor performance of commercial FOXM1 antibodies in ChIP-seq or the increased level of GFP-FOXM1 compared to endogenous FOXM1 protein in these cell lines.Fig. 3


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)

GFP-tagged FOXM1 shows a similar genomic distribution to endogenous FOXM1. a Venn diagram showing the overlap between endogenous FOXM1 and GFP-FOXM1 binding sites in TetR HEK293 cells, using peaks in common in two replicates for each condition. b Motifs enriched in FOXM1 and GFP-FOXM1. Selected motifs identified in FOXM1 are also found in the GFP-FOXM1 peaks (full list in Additional file 1: Table S2). 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 FOXM1 peaks. c Examples of genomic regions showing three representative sites in common for endogenous FOXM1 and GFP-FOXM1 binding. d CEAS analysis comparing genomic distribution of endogenous FOXM1 binding events to GFP-FOXM1 in HEK293 cells
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4492089&req=5

Fig3: GFP-tagged FOXM1 shows a similar genomic distribution to endogenous FOXM1. a Venn diagram showing the overlap between endogenous FOXM1 and GFP-FOXM1 binding sites in TetR HEK293 cells, using peaks in common in two replicates for each condition. b Motifs enriched in FOXM1 and GFP-FOXM1. Selected motifs identified in FOXM1 are also found in the GFP-FOXM1 peaks (full list in Additional file 1: Table S2). 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 FOXM1 peaks. c Examples of genomic regions showing three representative sites in common for endogenous FOXM1 and GFP-FOXM1 binding. d CEAS analysis comparing genomic distribution of endogenous FOXM1 binding events to GFP-FOXM1 in HEK293 cells
Mentions: Two biological replicates were performed for both cell lines and 25–30 million reads were obtained for each sample (Additional file 1: Table S1) and binding peaks were called using Model-based Analysis for Chip-Seq (MACS) [34]. For endogenous FOXM1, 1,390 peaks were present in both replicates and for GFP-FOXM1 12,418 peaks were identified (Additional file 2), with a high level of concordance between replicates (approximately 78 % for FOXM1 and approximately 81 % for GFP-FOXM1). Comparison of the endogenous FOXM1 and GFP-FOXM1 peak locations (Fig. 3a) showed a high degree of overlap (approximately 88 %) indicating that the GFP-FOXM1 protein captures the majority of the endogenous FOXM1 binding sites. In the endogenous FOXM1 replicates approximately 50 % more peaks were called in one sample (3,970 compared to 1,722). When less stringent parameters were used for peak calling, using each replicate separately, the number of binding sites shared between endogenous FOXM1 and the GFP-FOXM1 increased to 3,173 representing approximately 26 % of the total GFP-FOXM1 binding sites. The approximately 10-fold increase in high confidence peaks called in the GFP-FOXM1 samples compared to endogenous FOXM1 is possibly due to the higher affinity of the GFP antibody and the relatively poor performance of commercial FOXM1 antibodies in ChIP-seq or the increased level of GFP-FOXM1 compared to endogenous FOXM1 protein in these cell lines.Fig. 3

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