RT Journal
A1 Ibrahim, Daniel M.
A1 Hansen, Peter
A1 Rödelsperger, Christian
A1 Stiege, Asita C.
A1 Doelken, Sandra C.
A1 Horn, Denise
A1 Jäger, Marten
A1 Janetzki, Catrin
A1 Krawitz, Peter
A1 Leschik, Gundula
A1 Wagner, Florian
A1 Scheuer, Till
A1 Schmidt-von Kegler, Mareen
A1 Seemann, Petra
A1 Timmermann, Bernd
A1 Robinson, Peter N.
A1 Mundlos, Stefan
A1 Hecht, Jochen
T1 Distinct global shifts in genomic binding profiles of limb malformation-associated HOXD13 mutations
JF Genome Research 
JO Genome Research 
YR 2013 
FD December 01 
VO 23 
IS 12 
SP 2091 
OP 2102 
DO 10.1101/gr.157610.113 
UL http://genome.cshlp.org/content/23/12/2091.abstract 
AB 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.