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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

Generation of inducible GFP-FOXM1 expressing HEK293 cell lines. a Western blot showing induction of GFP-FOXM1B expression following addition of doxycycline for 24 h while levels of endogenous FOXM1 are unchanged. The blot was probed using antibodies for FOXM1 and ACTB. b Quantitative PCR (qPCR) showing RNA expression levels of total FOXM1 (GFP-FOXM1 and endogenous) and endogenous FOXM1 only (FOXM1-UTR). c qPCR of FOXM1 target genes transcript levels in GFP-FOXM1 cells treated ± doxycycline for 24 h and expressed relative to the levels in the parent HEK293 cells (Negative) for each transcript. Data show triplicate experiments ± SEM. d Western blot showing protein levels of GFP-FOXM1 in WT and mutant cell lines treated ± doxycycline (Dox) at 1 μg/mL for 24 h
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Fig2: Generation of inducible GFP-FOXM1 expressing HEK293 cell lines. a Western blot showing induction of GFP-FOXM1B expression following addition of doxycycline for 24 h while levels of endogenous FOXM1 are unchanged. The blot was probed using antibodies for FOXM1 and ACTB. b Quantitative PCR (qPCR) showing RNA expression levels of total FOXM1 (GFP-FOXM1 and endogenous) and endogenous FOXM1 only (FOXM1-UTR). c qPCR of FOXM1 target genes transcript levels in GFP-FOXM1 cells treated ± doxycycline for 24 h and expressed relative to the levels in the parent HEK293 cells (Negative) for each transcript. Data show triplicate experiments ± SEM. d Western blot showing protein levels of GFP-FOXM1 in WT and mutant cell lines treated ± doxycycline (Dox) at 1 μg/mL for 24 h

Mentions: The Flp-In system (Invitrogen) was next used to generate stable cell lines expressing GFP-FOXM1 WT by the targeted insertion of an expression construct at a single transcriptionally active genomic site. This system was utilized to ensure that the WT and mutant FOXM1 proteins were expressed at equivalent levels following induction. HEK293Flp-In cells stably expressing the Tet repressor at high levels were first generated by transfection of a TetR plasmid under the control of a CMV promoter. These cells were transfected with WT or mutant FOXM1-GFP plasmids together with Flp recombinase plasmid to generate inducible cell lines. In the absence of doxycycline, no detectable expression of the GFP-FOXM1 protein or transcript was observed (Fig. 2a and b), while expression was induced after addition of doxycycline at concentrations above 1 ng/mL, giving approximately 50-fold higher levels of total FOXM1 protein (1000 ng/mL) compared to the uninduced cells. There was no significant change in the level of the endogenous FOXM1 protein or mRNA following overexpression of the GFP-FOXM1 as shown by western blotting and qPCR for the FOXM1 UTR [33]. This result contrasts the hypothesis, proposed by Halasi et al. [33], that FOXM1 expression is primarily regulated by a positive auto-regulatory loop. Isoform specific qPCR (Additional file 1: Figure S3) showed that only GFP-FOXM1B protein was significantly upregulated following doxycycline induction.Fig. 2


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)

Generation of inducible GFP-FOXM1 expressing HEK293 cell lines. a Western blot showing induction of GFP-FOXM1B expression following addition of doxycycline for 24 h while levels of endogenous FOXM1 are unchanged. The blot was probed using antibodies for FOXM1 and ACTB. b Quantitative PCR (qPCR) showing RNA expression levels of total FOXM1 (GFP-FOXM1 and endogenous) and endogenous FOXM1 only (FOXM1-UTR). c qPCR of FOXM1 target genes transcript levels in GFP-FOXM1 cells treated ± doxycycline for 24 h and expressed relative to the levels in the parent HEK293 cells (Negative) for each transcript. Data show triplicate experiments ± SEM. d Western blot showing protein levels of GFP-FOXM1 in WT and mutant cell lines treated ± doxycycline (Dox) at 1 μg/mL for 24 h
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig2: Generation of inducible GFP-FOXM1 expressing HEK293 cell lines. a Western blot showing induction of GFP-FOXM1B expression following addition of doxycycline for 24 h while levels of endogenous FOXM1 are unchanged. The blot was probed using antibodies for FOXM1 and ACTB. b Quantitative PCR (qPCR) showing RNA expression levels of total FOXM1 (GFP-FOXM1 and endogenous) and endogenous FOXM1 only (FOXM1-UTR). c qPCR of FOXM1 target genes transcript levels in GFP-FOXM1 cells treated ± doxycycline for 24 h and expressed relative to the levels in the parent HEK293 cells (Negative) for each transcript. Data show triplicate experiments ± SEM. d Western blot showing protein levels of GFP-FOXM1 in WT and mutant cell lines treated ± doxycycline (Dox) at 1 μg/mL for 24 h
Mentions: The Flp-In system (Invitrogen) was next used to generate stable cell lines expressing GFP-FOXM1 WT by the targeted insertion of an expression construct at a single transcriptionally active genomic site. This system was utilized to ensure that the WT and mutant FOXM1 proteins were expressed at equivalent levels following induction. HEK293Flp-In cells stably expressing the Tet repressor at high levels were first generated by transfection of a TetR plasmid under the control of a CMV promoter. These cells were transfected with WT or mutant FOXM1-GFP plasmids together with Flp recombinase plasmid to generate inducible cell lines. In the absence of doxycycline, no detectable expression of the GFP-FOXM1 protein or transcript was observed (Fig. 2a and b), while expression was induced after addition of doxycycline at concentrations above 1 ng/mL, giving approximately 50-fold higher levels of total FOXM1 protein (1000 ng/mL) compared to the uninduced cells. There was no significant change in the level of the endogenous FOXM1 protein or mRNA following overexpression of the GFP-FOXM1 as shown by western blotting and qPCR for the FOXM1 UTR [33]. This result contrasts the hypothesis, proposed by Halasi et al. [33], that FOXM1 expression is primarily regulated by a positive auto-regulatory loop. Isoform specific qPCR (Additional file 1: Figure S3) showed that only GFP-FOXM1B protein was significantly upregulated following doxycycline induction.Fig. 2

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