Limits...
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

FOXM1 transcriptional activity requires direct chromatin interaction involving recruitment to non-consensus sequences. a qPCR analysis of the mRNA transcript levels in GFP-FOXM1 WT or mutant (H287A or R286A) cell lines treated ± doxycycline (1 μg/mL) for 24 h showing the relative change in the levels of FOXM1B, AURKB, CCNB1, CDC25B, CENPF, and PLK1. In each case the data are normalized to the minus doxycycline control. b Binding curves measured by fluorescence polarization analysis (assay details in the Materials and methods section), showing binding affinity of GST-FOXM1B DBD for 16-mer [FAM]dsDNA sequences present in FOXM1 binding peaks from the ChIP-seq dataset compared to the FKH consensus. The plot shows the fraction bound with increasing protein concentration. The table shows the Kd values ± SD determined for each sequence. c Illustration of alternative models proposed the recruitment of FOXM1 to chromatin. (1) Direct DNA binding of FOXM1 at promoter sites containing a FKH consensus motif and interaction with MuvB and B-Myb. (2) FOXM1 is recruited by MuvB complex and does not directly bind to the DNA. (3) FOXM1 binds directly at non-consensus sequences facilitated by interaction with MuvB and B-Myb. Arrow indicates transcription start site of target gene. Data representative of triplicate experiments ± SD. (*) P <0.05, (**) P <0.01, (***) P <0.001
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
getmorefigures.php?uid=PMC4492089&req=5

Fig7: FOXM1 transcriptional activity requires direct chromatin interaction involving recruitment to non-consensus sequences. a qPCR analysis of the mRNA transcript levels in GFP-FOXM1 WT or mutant (H287A or R286A) cell lines treated ± doxycycline (1 μg/mL) for 24 h showing the relative change in the levels of FOXM1B, AURKB, CCNB1, CDC25B, CENPF, and PLK1. In each case the data are normalized to the minus doxycycline control. b Binding curves measured by fluorescence polarization analysis (assay details in the Materials and methods section), showing binding affinity of GST-FOXM1B DBD for 16-mer [FAM]dsDNA sequences present in FOXM1 binding peaks from the ChIP-seq dataset compared to the FKH consensus. The plot shows the fraction bound with increasing protein concentration. The table shows the Kd values ± SD determined for each sequence. c Illustration of alternative models proposed the recruitment of FOXM1 to chromatin. (1) Direct DNA binding of FOXM1 at promoter sites containing a FKH consensus motif and interaction with MuvB and B-Myb. (2) FOXM1 is recruited by MuvB complex and does not directly bind to the DNA. (3) FOXM1 binds directly at non-consensus sequences facilitated by interaction with MuvB and B-Myb. Arrow indicates transcription start site of target gene. Data representative of triplicate experiments ± SD. (*) P <0.05, (**) P <0.01, (***) P <0.001

Mentions: To investigate the effect of the DBD mutations on FOXM1-regulated gene expression, qPCR was used to measure the expression levels of several known target genes (Fig. 7a). Transcript levels were compared in the WT and DBD mutant cells (H287A and R286A) between ± doxycycline. The level of GFP-FOXM1B induction was first measured to ensure comparable expression of the WT and DBD mutants. Indeed by qPCR, the addition of doxycycline addition led to an approximately 13-fold increase in GFP-FOXM1 expression in both the WT and DBD mutant cell lines compared to the non-induced WT GFP-FOXM1 cells (Fig. 7a). Next, the expression of seven known FOXM1 target genes was measured; AURKB, CENPF, KNSTRN, CCNB1, CDC25B, NEK2, and PLK1. In each case, a significant increase in expression was observed in the GFP WT-FOXM1 cell line following induction by doxycycline (P <0.05). Although the relative increase in expression was small (in the range of 1.3-fold for AURKB to 1.8-fold for NEK2), this is in the context of endogenous FOXM1 and comparable to previous studies [3, 31]. In contrast, for the R286A DBD mutant there was no significant difference in transcript levels following induction, whereas for the H287A DBD mutant, three transcripts (CENPF, CCNB1, and PLK1) showed no significant change on induction This latter result may reflect a higher residual level of DNA binding activity that is observed in the FP assay and by ChIP-seq when compared to R286A. This is supported by inspection of the ChIP-seq data for three transcripts (KNSTRN, CDC25B, and NEK2) that show different responses in the H287A compared to the R286A mutant (Additional file 1: Figure S13). In each case the peak in the promoter region of the gene is significantly smaller in the R286A sample.Fig. 7


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)

FOXM1 transcriptional activity requires direct chromatin interaction involving recruitment to non-consensus sequences. a qPCR analysis of the mRNA transcript levels in GFP-FOXM1 WT or mutant (H287A or R286A) cell lines treated ± doxycycline (1 μg/mL) for 24 h showing the relative change in the levels of FOXM1B, AURKB, CCNB1, CDC25B, CENPF, and PLK1. In each case the data are normalized to the minus doxycycline control. b Binding curves measured by fluorescence polarization analysis (assay details in the Materials and methods section), showing binding affinity of GST-FOXM1B DBD for 16-mer [FAM]dsDNA sequences present in FOXM1 binding peaks from the ChIP-seq dataset compared to the FKH consensus. The plot shows the fraction bound with increasing protein concentration. The table shows the Kd values ± SD determined for each sequence. c Illustration of alternative models proposed the recruitment of FOXM1 to chromatin. (1) Direct DNA binding of FOXM1 at promoter sites containing a FKH consensus motif and interaction with MuvB and B-Myb. (2) FOXM1 is recruited by MuvB complex and does not directly bind to the DNA. (3) FOXM1 binds directly at non-consensus sequences facilitated by interaction with MuvB and B-Myb. Arrow indicates transcription start site of target gene. Data representative of triplicate experiments ± SD. (*) P <0.05, (**) P <0.01, (***) P <0.001
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4492089&req=5

Fig7: FOXM1 transcriptional activity requires direct chromatin interaction involving recruitment to non-consensus sequences. a qPCR analysis of the mRNA transcript levels in GFP-FOXM1 WT or mutant (H287A or R286A) cell lines treated ± doxycycline (1 μg/mL) for 24 h showing the relative change in the levels of FOXM1B, AURKB, CCNB1, CDC25B, CENPF, and PLK1. In each case the data are normalized to the minus doxycycline control. b Binding curves measured by fluorescence polarization analysis (assay details in the Materials and methods section), showing binding affinity of GST-FOXM1B DBD for 16-mer [FAM]dsDNA sequences present in FOXM1 binding peaks from the ChIP-seq dataset compared to the FKH consensus. The plot shows the fraction bound with increasing protein concentration. The table shows the Kd values ± SD determined for each sequence. c Illustration of alternative models proposed the recruitment of FOXM1 to chromatin. (1) Direct DNA binding of FOXM1 at promoter sites containing a FKH consensus motif and interaction with MuvB and B-Myb. (2) FOXM1 is recruited by MuvB complex and does not directly bind to the DNA. (3) FOXM1 binds directly at non-consensus sequences facilitated by interaction with MuvB and B-Myb. Arrow indicates transcription start site of target gene. Data representative of triplicate experiments ± SD. (*) P <0.05, (**) P <0.01, (***) P <0.001
Mentions: To investigate the effect of the DBD mutations on FOXM1-regulated gene expression, qPCR was used to measure the expression levels of several known target genes (Fig. 7a). Transcript levels were compared in the WT and DBD mutant cells (H287A and R286A) between ± doxycycline. The level of GFP-FOXM1B induction was first measured to ensure comparable expression of the WT and DBD mutants. Indeed by qPCR, the addition of doxycycline addition led to an approximately 13-fold increase in GFP-FOXM1 expression in both the WT and DBD mutant cell lines compared to the non-induced WT GFP-FOXM1 cells (Fig. 7a). Next, the expression of seven known FOXM1 target genes was measured; AURKB, CENPF, KNSTRN, CCNB1, CDC25B, NEK2, and PLK1. In each case, a significant increase in expression was observed in the GFP WT-FOXM1 cell line following induction by doxycycline (P <0.05). Although the relative increase in expression was small (in the range of 1.3-fold for AURKB to 1.8-fold for NEK2), this is in the context of endogenous FOXM1 and comparable to previous studies [3, 31]. In contrast, for the R286A DBD mutant there was no significant difference in transcript levels following induction, whereas for the H287A DBD mutant, three transcripts (CENPF, CCNB1, and PLK1) showed no significant change on induction This latter result may reflect a higher residual level of DNA binding activity that is observed in the FP assay and by ChIP-seq when compared to R286A. This is supported by inspection of the ChIP-seq data for three transcripts (KNSTRN, CDC25B, and NEK2) that show different responses in the H287A compared to the R286A mutant (Additional file 1: Figure S13). In each case the peak in the promoter region of the gene is significantly smaller in the R286A sample.Fig. 7

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