Gene 'dark matter' controls embryo lung growth
Only about two percent of our genome converts into proteins. A much higher percentage is transcribed into RNA — sometimes referred to as "genomic dark matter." Given the vast amount of RNA, scientists believe it must impact fetal development.
Large-scale genomic sequencing has allowed investigators to identify thousands of non-coding RNAs. Small non-coding RNAs - including microRNAs - are known to be important in turning on and off genes needed for tissue development. On the other hand, the function of long non-coding RNAs — lncRNAs — is less understood.
Edward Morrisey, PhD, professor of Medicine, Cell and Developmental Biology, Perelman School of Medicine at the University of Pennsylvania, and scientific director of the Penn Institute for Regenerative Medicine, has identified hundreds of lncRNAs in his research.
lncRNAs, "genomic dark matter," is expressed in fetal development as well as in our adult lung. Featured on the cover of Genes and Development, Morrisey's lab has found that lung lncRNAs appear to regulate the opening and closing of the DNA scaffold.
Morrisey's team identified 363 long non-coding RNAs in the lungs and foregut (also known as endoderm) layers of embryonic mice.
"We have defined a new association: long non-coding RNAs having transcription factor proteins bound to specific DNA sequences to control cell identity and function.
This association is important for lung development in mouse embryos, and we believe at least one of those long non-coding RNAs is important in human lung function."
Edward E. Morrisey, PhD, professor of Medicine, Cell and Developmental Biology at the Perelman School of Medicine, University of Pennsylvania; and, scientific director of the Penn Institute for Regenerative Medicine.
Morrisey's team found that a lncRNA called NANCI regulates the critical transcription factor Nkx2.1.
Nkx2.1 is the first lung molecular marker appearing in mouse and human development — and is essential for lungs to mature properly in either species.
NANCI appears to act prior to creation of Nkx2.1, but after Wnt signaling which is critical to later lung development. Turning off NANCI decreases Nkx2.1 which in turn decreases creation of surfactant proteins. Surfactant protects the lining of lung airways and marks invading bacteria for destruction by the immune system.
A single copy mutation of NKX2.1 leads to Brain-Lung-Thyroid Syndrome with the infant in respiratory distress immediately following birth. "There is also a report of a patient with a deletion in NANCI — but not NKX2.1 — who has Brain-Lung-Thyroid Syndrome. This suggests that mutations in NANCI (and perhaps other lncRNAs) may underlie human disease," explains Morrisey.
In addition to NANCI, the team identified another lncRNA, which they named LL34. LL34 regulates retinoic acid signaling, another important pathway for early lung development and highly expressed in the developing foregut layers.
The foregut of an embryo generates multiple tissue types including lung, thyroid and liver. Turning off LL34 leads to decreased retinoic acid signaling, suggesting lncRNA plays an early and important role in development of the lung and other critical organs.
Other lncRNAs expressed in the lung, such as MALAT1, play a role in cancer progression. However, data generated by the Morrisey lab provides a unique insight into which lncRNAs regulate development of the lung only.
"We are hopeful that this new data provides the foundation for a better understanding of how the non-coding transcriptome regulates tissue development and also maintenance of adult tissues," says Morrisey. Future work will be directed towards understanding how these lncRNAs may also contribute to adult lung regeneration.
Long noncoding RNAs (lncRNAs) are thought to play important roles in regulating gene transcription, but few have well-defined expression patterns or known biological functions during mammalian development. Using a conservative pipeline to identify lncRNAs that have important biological functions, we identified 363 lncRNAs in the lung and foregut endoderm. Importantly, we show that these lncRNAs are spatially correlated with transcription factors across the genome. In-depth expression analyses of lncRNAs with genomic loci adjacent to the critical transcription factors Nkx2.1, Gata6, Foxa2 (forkhead box a2), and Foxf1 mimic the expression patterns of their protein-coding neighbor. Loss-of-function analysis demonstrates that two lncRNAs, LL18/NANCI (Nkx2.1-associated noncoding intergenic RNA) and LL34, play distinct roles in endoderm development by controlling expression of critical developmental transcription factors and pathways, including retinoic acid signaling. In particular, we show that LL18/NANCI acts upstream of Nkx2.1 and downstream from Wnt signaling to regulate lung endoderm gene expression. These studies reveal that lncRNAs play an important role in foregut and lung endoderm development by regulating multiple aspects of gene transcription, often through regulation of transcription factor expression.
Additional coauthors are Michael J. Herriges, Michael P. Morley, Komal S. Rathi, Tien Peng, and Kathleen M. Stewart, all from Penn and Daniel T. Swarr, from The Children's Hospital of Philadelphia.
Studies in the Morrisey lab were supported by funding from the National Heart and Lung Institute (HL100405, HL110942).
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