Distinct global shifts in genomic binding profiles of limb malformation-associated HOXD13 mutations.
Distinct global shifts in genomic binding profiles of limb malformation-associated HOXD13 mutations.
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DOI：
10.1101/gr.157610.113
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发表时间：
2013-12
期刊：
影响因子：
7
通讯作者：
Hecht J
中科院分区：
文献类型：
--
作者：
Ibrahim DM;Hansen P;Rödelsperger C;Stiege AC;Doelken SC;Horn D;Jäger M;Janetzki C;Krawitz P;Leschik G;Wagner F;Scheuer T;Schmidt-von Kegler M;Seemann P;Timmermann B;Robinson PN;Mundlos S;Hecht J
转录因子（TF）的基因调控决定了发育程序和细胞特性。因此，转录因子的突变可以通过破坏基因调控而导致人类的显著表型。迄今为止，实际上导致这些表型的分子机制一直难以通过实验解决。ChIP-seq将染色质免疫沉淀与高通量测序相结合，允许在全基因组范围内研究TF功能，从而为研究基因调控提供了新的方法。在这里，我们提出了ChIP-seq的应用，以探索TF中的错义突变对其全基因组结合谱的影响。利用鸡间充质干细胞中的逆转录病毒表达系统，我们阐明了HOXD 13（Q317 K）中与复杂手足畸形表型相关的新型错义突变的机制。突变的谷氨酰胺（Q）在大多数同源结构域中是保守的，一个值得注意的例外是在该位置具有赖氨酸（K）的bicoid型同源结构域。我们的研究结果表明，突变的结果在一个转变的结合模式的突变体向bicoid/PITX 1基序。使用体内过表达研究的基因表达分析和功能测定证实，突变导致HOXD 13部分转化为具有bicoid/PITX 1特性的TF。与短并指相关的另一个突变Q317 R没有观察到类似的变化，这表明Q317 K观察到的bicoid/PITX 1变化可能与严重的临床表型有关。所描述的方法可用于研究以前不适合ChIP-seq实验的广泛的TF和突变。
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.
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影响因子：
8
作者：
Dorman, CM;Johnson, SE
通讯作者：
Johnson, SE
影响因子：
64.5
作者：
Junion, Guillaume;Spivakov, Mikhail;Furlong, Eileen E. M.
通讯作者：
Furlong, Eileen E. M.
影响因子：
4
作者：
Debeer, P;Bacchelli, C;Goodman, FR
通讯作者：
Goodman, FR
DOI：
10.1002/ajmg.10776
发表时间：
2002-10-15
期刊：
AMERICAN JOURNAL OF MEDICAL GENETICS
影响因子：
--
作者：
Goodman, FR
通讯作者：
Goodman, FR
影响因子：
64.5
作者：
Berger, Michael F.;Badis, Gwenael;Hughes, Timothy R.
通讯作者：
Hughes, Timothy R.