title: Distinct global shifts in genomic binding profiles of limb malformation-associated HOXD13 mutations.
abstract: Gene regulation by transcription factors (TFs) determines developmental programs and cell identity. Consequently, mutations in TFs can lead to dramatic phenotypes in humans by disrupting gene regulation. To date, the molecular mechanisms that actually cause these phenotypes have been difficult to address experimentally. ChIP-seq, which couples chromatin immunoprecipitation with high-throughput sequencing, allows TF function to be investigated on a genome-wide scale, enabling new approaches for the investigation of gene regulation. Here, we present the application of ChIP-seq to explore the effect of missense mutations in TFs on their genome-wide binding profile. Using a retroviral expression system in chicken mesenchymal stem cells, we elucidated the mechanism underlying a novel missense mutation in HOXD13 (Q317K) associated with a complex hand and foot malformation phenotype. The mutated glutamine (Q) is conserved in most homeodomains, a notable exception being bicoid-type homeodomains that have lysine (K) at this position. Our results show that the mutation results in a shift in the binding profile of the mutant toward a bicoid/PITX1 motif. Gene expression analysis and functional assays using in vivo overexpression studies confirm that the mutation results in a partial conversion of HOXD13 into a TF with bicoid/PITX1 properties. A similar shift was not observed with another mutation, Q317R, which is associated with brachysyndactyly, suggesting that the bicoid/PITX1-shift observed for Q317K might be related to the severe clinical phenotype. The methodology described can be used to investigate a wide spectrum of TFs and mutations that have not previously been amenable to ChIP-seq experiments.
authors.affiliation: Berlin-Brandenburg Center for Regenerative Therapies (BCRT), Charité Universitätsmedizin Berlin, 13353 Berlin, Germany;
authors.forename: Daniel M
authors.initials: DM
authors.lastname: Ibrahim
authors.forename: Peter
authors.initials: P
authors.lastname: Hansen
authors.forename: Christian
authors.initials: C
authors.lastname: Rödelsperger
authors.forename: Asita C
authors.initials: AC
authors.lastname: Stiege
authors.forename: Sandra C
authors.initials: SC
authors.lastname: Doelken
authors.forename: Denise
authors.initials: D
authors.lastname: Horn
authors.forename: Marten
authors.initials: M
authors.lastname: Jäger
authors.forename: Catrin
authors.initials: C
authors.lastname: Janetzki
authors.forename: Peter
authors.initials: P
authors.lastname: Krawitz
authors.forename: Gundula
authors.initials: G
authors.lastname: Leschik
authors.forename: Florian
authors.initials: F
authors.lastname: Wagner
authors.forename: Till
authors.initials: T
authors.lastname: Scheuer
authors.forename: Mareen
authors.initials: M
authors.lastname: Schmidt-von Kegler
authors.forename: Petra
authors.initials: P
authors.lastname: Seemann
authors.forename: Bernd
authors.initials: B
authors.lastname: Timmermann
authors.forename: Peter N
authors.initials: PN
authors.lastname: Robinson
authors.forename: Stefan
authors.initials: S
authors.lastname: Mundlos
authors.forename: Jochen
authors.initials: J
authors.lastname: Hecht
mesh_terms: D000818:Animals; D001665:Binding Sites; D002642:Chick Embryo; D047369:Chromatin Immunoprecipitation; D005260:Female; D020869:Gene Expression Profiling; D015894:Genome, Human; D005973:Glutamine / Q000235:genetics; D018398:Homeodomain Proteins / Q000235:genetics* / Q000378:metabolism*; D006801:Humans; D017880:Limb Deformities, Congenital / Q000235:genetics*; D059630:Mesenchymal Stem Cells / Q000378:metabolism; D020125:Mutation, Missense; D020411:Oligonucleotide Array Sequence Analysis; D051761:Paired Box Transcription Factors / Q000235:genetics / Q000378:metabolism*; D010641:Phenotype; D057050:Receptor Tyrosine Kinase-like Orphan Receptors / Q000235:genetics; D014157:Transcription Factors / Q000235:genetics* / Q000378:metabolism*
chemical_list: C513184:HOXD13 protein, human; D018398:Homeodomain Proteins; D051761:Paired Box Transcription Factors; D014157:Transcription Factors; C101485:homeobox protein PITX1; D005973:Glutamine; C078717:ROR2 protein, human; D057050:Receptor Tyrosine Kinase-like Orphan Receptors