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Series GSE60032 Query DataSets for GSE60032
Status Public on Jun 11, 2015
Title FOXM1 binds directly to non-consensus sequences in the human genome
Organism Homo sapiens
Experiment type Genome binding/occupancy profiling by high throughput sequencing
Summary The Forkhead transcription factor, FOXM1, is a key regulator of the cell cycle and is over-expressed in most types of cancer. FOXM1, similar to other Forkhead (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 at FOXM1 binding sites 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 WT and DNA binding deficient FOXM1. An in vitro fluorescence polarization (FP) assay was used to identify point mutations in the DNA binding domain (DBD) of FOXM1 that inhibit binding to a FKH consensus sequence. Stable cell lines expressing either WT or DBD mutant green fluorescence protein (GFP)-tagged FOXM1 were utilized for genome-wide mapping studies comparing the distribution of the DBD mutant protein to the WT. This showed that interaction of the DBD of FOXM1 with target DNA is essential for recruitment. Moreover, analysis of protein interactome of the WT versus DBD mutant FOXM1 showed that the reduced recruitment is not due to the mutation inhibiting protein-protein interactions. This study showed that a functional DBD domain is essential for FOXM1 chromatin recruitment. Even in FOXM1 mutants that show almost complete loss of binding the protein-protein interactome 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 both canonical and non-canonical DNA sequences.
 
Overall design 15 samples, 40 bp single-ended ChIP-Seq libraries from cell lines with antibody for FOXM1 or expressing GFP-tagged FOXM1 (GFP-FOXM1), either wild type (GFP-FOXM1 WT) and with 2 different point mutations (GFP-FOXM1-HA and GFP-FOXM1-RA respectively). FOXM1: 2 replicates, samples labelled FOXM1_GTX; FOXM1-GFP WT: 5 replicates, samples labelled GFP and WT_GFP_FOXM1; FOXM1-GFP HA: 3 replicates, samples labelled GFP_FOXM1_HA; FOXM1-GFP RA: 2 replicates, samples labelled GFP_FOXM1_RA; input libraries: 3 different inputs for the respective experiments.
 
Contributor(s) Sanders DA, Gormally MV, Marsico G, Beraldi D, Tannahill D, Taylor C, Balasubramanian S
Citation(s) 26100407
Submission date Aug 01, 2014
Last update date May 15, 2019
Contact name Giovanni Marsico
E-mail(s) persego@gmail.com
Organization name CRUK Cambridge Institute
Street address Robinson Way
City Cambridge
ZIP/Postal code CB2 0RE
Country United Kingdom
 
Platforms (1)
GPL11154 Illumina HiSeq 2000 (Homo sapiens)
Samples (15)
GSM1464010 ds065_293T_Input
GSM1464011 ds066_293T_FOXM1_GTX_1
GSM1464012 ds067_293T_FOXM1_GTX_2
Relations
BioProject PRJNA257307
SRA SRP045190

Download family Format
SOFT formatted family file(s) SOFTHelp
MINiML formatted family file(s) MINiMLHelp
Series Matrix File(s) TXTHelp

Supplementary file Size Download File type/resource
GSE60032_RAW.tar 1.4 Mb (http)(custom) TAR (of BED)
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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