A misplaced lncRNA causes brachydactyly in humans
Philipp G. Maass, … , Friedrich C. Luft, Sylvia Bähring
Philipp G. Maass, … , Friedrich C. Luft, Sylvia Bähring
Published October 24, 2012
Citation Information: J Clin Invest. 2012;122(11):3990-4002. https://doi.org/10.1172/JCI65508.
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Research Article

A misplaced lncRNA causes brachydactyly in humans

Abstract

Translocations are chromosomal rearrangements that are frequently associated with a variety of disease states and developmental disorders. We identified 2 families with brachydactyly type E (BDE) resulting from different translocations affecting chromosome 12p. Both translocations caused downregulation of the parathyroid hormone-like hormone (PTHLH) gene by disrupting the cis-regulatory landscape. Using chromosome conformation capturing, we identified a regulator on chromosome 12q that interacts in cis with PTHLH over a 24.4-megabase distance and in trans with the sex-determining region Y-box 9 (SOX9) gene on chromosome 17q. The element also harbored a long noncoding RNA (lncRNA). Silencing of the lncRNA, PTHLH, or SOX9 revealed a feedback mechanism involving an expression-dependent network in humans. In the BDE patients, the human lncRNA was upregulated by the disrupted chromosomal association. Moreover, the lncRNA occupancy at the PTHLH locus was reduced. Our results document what we believe to be a novel in cis– and in trans–acting DNA and lncRNA regulatory feedback element that is reciprocally regulated by coding genes. Furthermore, our findings provide a systematic and combinatorial view of how enhancers encoding lncRNAs may affect gene expression in normal development.

Authors

Philipp G. Maass, Andreas Rump, Herbert Schulz, Sigmar Stricker, Lisanne Schulze, Konrad Platzer, Atakan Aydin, Sigrid Tinschert, Mary B. Goldring, Friedrich C. Luft, Sylvia Bähring

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Figure 4

DA125942 transcription and dependent regulation of SOX9, DA125942, and PTHLH.

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DA125942 transcription and dependent regulation of SOX9, DA125942, and ...
(A) CISTR-ACTS and CISTR-ACTF, with or without re18527, were cloned in a promoter and regulator lacking plasmid (pGl2-luci). In CISTR-ACTF–transfected C28/I2 cells, DA125942 was highly expressed. re18527 blocked the transcription; CISTR-ACTS produced no transcription. (B) SOX9 expression was knocked down relative to the scrambled siRNA control. Depleted PTHLH or DA125942 downregulated SOX9. siRNA-mediated depletion of PTHLH and SOX9 upregulated DA125942, and PTHLH expression was significantly reduced by PTHLH, DA125942, and SOX9 siRNA (n = 6). (C) Overexpression of PTHLH, SOX9, and DA125942 confirmed the expression-dependent network (n = 4). (D) STRING network. Expression array analysis revealed 22 organ morphogenesis genes (GO:0009887) showing interactions. Line thickness is indicative of physical or functional interaction confidence. Mesenchymal and prechondrogenic genes were differentially regulated. (E and F) Fibroblasts of affected BDE patients (AFF) and 3 nonaffected subjects (NON) were chondrogenically induced. DA125942 was upregulated in patients of both BDE families (n = 3). (G) RNA-ChIRP on C28/I2, nonaffected subject, and BDE patient chromatin. DA125942 was retrieved in contrast to GAPDH. (H and I) ChIRP on eluted C28/I2 DNA detected DA125942 binding at the PTHLH and SOX9 loci. Each amplicon was mapped by the UCSC custom tracks (numbered black boxes). (J and K) In t(4;12) and t(8;12), reduced lncRNA occupancy at the PTHLH locus was observed; binding at SOX9 was not affected (n = 2). ***P ≤ 0.001; **P ≤ 0.01; *P ≤ 0.05.