Altekoester, Ann-Kristin
(2019).
Decoding the transcriptional landscape of Nkx2-5 in heart development and disease.
PhD thesis, Universität zu Köln.
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Abstract
A significant amount of the mammalian genome previously found to not code for proteins and considered ‘junk’, is actually pervasively transcribed and specifies a dynamic network of regulatory RNAs, the most abundant of which are long non-coding RNAs (lncRNAs). lncRNAs are critical players in many biological processes including epigenetic, transcriptional and post-transcriptional control as well as protein metabolism. Many exhibit cell-type or developmentally specific expression patterns and are associated with human disease. Interestingly, the majority of single nucleotide polymorphisms (SNPs) identified through genome wide association studies (GWAS) map to non-coding regions of the genome, which could potentially affect ncRNAs, given that ~80% of the human genome is transcribed. Here, we show that the key cardiac transcriptional regulator, Nkx2-5, is, in humans and mouse, surrounded by two previously uncharacterised, long cardiac transcripts, which we named NkxDS and NkxUS. In humans, NKX2-5 mutations are commonly associated with congenital heart disease and a variety of specific cardiac malformations. Several lncRNAs have also been identified to be crucial for cardiac function as well as cardiac lineage commitment. At least 100 lncRNAs have been found to be cardiac specific, however, for most, their functional mechanisms, especially in vivo, remain unknown. This work aimed to characterise the two novel transcripts in order to explain whether NkxUS and NkxDS regulate Nkx2-5 itself or other processes important for heart function, development and/or disease. This work shows that both mouse transcripts are nuclear and cardiac-enriched, expressed in the heart throughout development and are also expressed specifically in the human heart. NkxDS is a novel long isoform of Nkx2-5 as it contains the Nkx2-5 protein-coding sequence. Therefore, Nkx2-5 may have a protein-coding function as well as a non-coding role within the nucleus. In contrast, NkxUS is an independent transcript to Nkx2-5. Downregulation of mouse NkxUS expression in the HL-1 cardiac cell line displayed an upregulation of transcripts involved in cardiac contraction and conduction. Moreover, NkxUS interacting partners included proteins localising to the nucleus and mitochondria, which are involved in metabolism and in the regulation of transcription. Interestingly, a GWAS SNP associated with an increased resting heart rate lies within and disrupts a 600nt RNA structure conserved between mouse and human NkxUS. Analysis of mice harboring a partial deletion of this structure, generated by CRISPR/Cas9 technology, revealed no developmental defects, nor did the deleted structure have any effect on Nkx2-5 expression. However, remarkably, a similar phenotype as described for humans harboring the GWAS SNP was observed, with homozygous mice exhibiting an increased resting heart rate. The phenotype was shown to result from an elevated pacemaker activity, most likely due to enhanced signaling through the cAMP pathway caused possibly by an upregulation of ion channels. In conclusion, this work has discovered NkxDS as a new isoform of the master cardiac transcription factor Nkx2-5 and has shown that a conserved RNA structure within NkxUS plays an important role in the regulation of resting heart rate. These results provide new insights into the functional importance of lncRNA secondary structures and their involvement in GWAS SNPs associated traits and diseases.
| Item Type: | Thesis (PhD thesis) | ||||||||
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| URN: | urn:nbn:de:hbz:38-94751 | ||||||||
| Date: | 26 March 2019 | ||||||||
| Language: | English | ||||||||
| Faculty: | Faculty of Mathematics and Natural Sciences | ||||||||
| Divisions: | Faculty of Mathematics and Natural Sciences > Department of Biology > Institute for Genetics | ||||||||
| Subjects: | Life sciences | ||||||||
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| Date of oral exam: | 25 March 2019 | ||||||||
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| Refereed: | Yes | ||||||||
| URI: | http://kups.ub.uni-koeln.de/id/eprint/9475 |
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