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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

Binding of the GFP-FOXM1 DBD mutants is significantly reduced genome-wide. a Heat map comparing binding events in WT FOXM1 and both FOXM1 DBD mutants. The window represents ± 5 kb regions centered on WT GFP-FOXM1 binding events with a second plot (on right) showing the average signal intensity of differential bound peaks. b Three examples of genomic regions showing binding peaks where binding is reduced in the GFP-FOXM1 DBD mutants compared to the WT. (c and d) Differential binding analysis (DBA) was used to identify significantly (FDR <0.05) differentially bound peaks in the WT GFP-FOXM1 compared to H287A DBD mutant or the R286A DBD mutant GFP-FOXM1 cell lines. The red dots represent peaks where FOXM1 binding is significantly increased/decreased compared to the WT
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Fig5: Binding of the GFP-FOXM1 DBD mutants is significantly reduced genome-wide. a Heat map comparing binding events in WT FOXM1 and both FOXM1 DBD mutants. The window represents ± 5 kb regions centered on WT GFP-FOXM1 binding events with a second plot (on right) showing the average signal intensity of differential bound peaks. b Three examples of genomic regions showing binding peaks where binding is reduced in the GFP-FOXM1 DBD mutants compared to the WT. (c and d) Differential binding analysis (DBA) was used to identify significantly (FDR <0.05) differentially bound peaks in the WT GFP-FOXM1 compared to H287A DBD mutant or the R286A DBD mutant GFP-FOXM1 cell lines. The red dots represent peaks where FOXM1 binding is significantly increased/decreased compared to the WT

Mentions: It is striking that genomic sites where DBD mutant binding is still retained are predominantly those with the most significantly enriched binding in the WT GFP-FOXM1 set. For example, 77 % of the joint H287A/R286A peaks are within the top 1000 FOXM1 WT peaks (13 % of all FOXM1 WT peaks) ranked by FDR. This suggests that mutation of key DBD residues ablates binding to lower affinity sites and that only higher affinity sites are retained with the DBD mutant proteins. This phenomenon is further illustrated in the heat map in Fig. 5a, where the average signal intensity for all the binding events for each protein is shown aligned to the WT GFP-FOXM1. It can be seen that the highest intensity WT GFP-FOXM1 binding events are preserved in both DBD mutant ChIP-seq datasets, although with an overall loss in the average signal density. We also confirmed this observation by ChIP-PCR for three known promoter binding sites of FOXM1 (Additional file 1: Figure S9). Figure 5b illustrates three examples of differential binding of the DBD mutant proteins compared to the GFP-WT FOXM1 at high-affinity genomic binding sites.Fig. 5


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)

Binding of the GFP-FOXM1 DBD mutants is significantly reduced genome-wide. a Heat map comparing binding events in WT FOXM1 and both FOXM1 DBD mutants. The window represents ± 5 kb regions centered on WT GFP-FOXM1 binding events with a second plot (on right) showing the average signal intensity of differential bound peaks. b Three examples of genomic regions showing binding peaks where binding is reduced in the GFP-FOXM1 DBD mutants compared to the WT. (c and d) Differential binding analysis (DBA) was used to identify significantly (FDR <0.05) differentially bound peaks in the WT GFP-FOXM1 compared to H287A DBD mutant or the R286A DBD mutant GFP-FOXM1 cell lines. The red dots represent peaks where FOXM1 binding is significantly increased/decreased compared to the WT
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4492089&req=5

Fig5: Binding of the GFP-FOXM1 DBD mutants is significantly reduced genome-wide. a Heat map comparing binding events in WT FOXM1 and both FOXM1 DBD mutants. The window represents ± 5 kb regions centered on WT GFP-FOXM1 binding events with a second plot (on right) showing the average signal intensity of differential bound peaks. b Three examples of genomic regions showing binding peaks where binding is reduced in the GFP-FOXM1 DBD mutants compared to the WT. (c and d) Differential binding analysis (DBA) was used to identify significantly (FDR <0.05) differentially bound peaks in the WT GFP-FOXM1 compared to H287A DBD mutant or the R286A DBD mutant GFP-FOXM1 cell lines. The red dots represent peaks where FOXM1 binding is significantly increased/decreased compared to the WT
Mentions: It is striking that genomic sites where DBD mutant binding is still retained are predominantly those with the most significantly enriched binding in the WT GFP-FOXM1 set. For example, 77 % of the joint H287A/R286A peaks are within the top 1000 FOXM1 WT peaks (13 % of all FOXM1 WT peaks) ranked by FDR. This suggests that mutation of key DBD residues ablates binding to lower affinity sites and that only higher affinity sites are retained with the DBD mutant proteins. This phenomenon is further illustrated in the heat map in Fig. 5a, where the average signal intensity for all the binding events for each protein is shown aligned to the WT GFP-FOXM1. It can be seen that the highest intensity WT GFP-FOXM1 binding events are preserved in both DBD mutant ChIP-seq datasets, although with an overall loss in the average signal density. We also confirmed this observation by ChIP-PCR for three known promoter binding sites of FOXM1 (Additional file 1: Figure S9). Figure 5b illustrates three examples of differential binding of the DBD mutant proteins compared to the GFP-WT FOXM1 at high-affinity genomic binding sites.Fig. 5

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