The transcription factor Nkx2-1 is the earliest known marker of lung epithelial fate and a master regulator of lung development and homeostasis. Mutations in Nkx2-1 lead to severe developmental defects in the lung and lethal respiratory distress. Nkx2-1 is also a major regulator of proliferation and metastatic potential in lung cancer Despite the importance of Nkx2-1 in lung development and disease, little is known about what directly regulates its expression. Recently, our lab identified a long noncoding RNA (lncRNA), named NANCI (Nkx2-1 associated noncoding intergenic RNA), which promotes Nkx2-1 expression during lung development. During development, NANCI and Nkx2-1 have similar expression patterns in both mouse and human lungs. Knockdown of NANCI in mouse or human cell lines leads to decreased expression of Nkx2-1 and its target genes, suggesting a conserved role for NANCI in regulating Nkx2-1 expression. Furthermore knockdown of NANCI in the developing mouse lung results in phenotypes associated with Nkx2-1 haploinsufficiency. While these data suggest NANCI promotes Nkx2-1 expression and thus plays a major role in regulating lung development and homeostasis, the scope of NANCI's activity during development is unclear. Knockdown of NANCI in vitro results in disrupted expression of many genes involved in lung development that are not known targets of Nkx2-1, suggesting NANCI may regulate genes other than Nkx2-1. To determine NANCI's activity during lung development, a NANCI knockout line (NANCICreERT2:tdTR) will be characterized as part of this proposal (Aim1a). In this line the first splice donor site of NANCI has been replaced with a polyadenylated reporter construct encoding a tamoxifen inducible Cre recombinase and a red fluorescent protein (RFP). This will prevent transcription of most of the NANCI transcript and allow for marking and fate-mapping of NANCI/RFP expressing cells. Immunohistochemistry, RNA-Seq, and qPCR will be used to compare and contrast NANCICreERT2:tdTR/CreERT2:tdTR mice to Nkx2-1-/- and WT mice to determine the role NANCI during development and whether NANCI has Nkx2-1 independent functions (Aim 1b). The mechanism by which NANCI regulates gene expression is also unknown. Preliminary mass spectrometry data suggests that NANCI interacts with the Tip60 histone acetyltransferase (HAT) complex. This chromatin modifier complex generates histone acetyl marks that loosen chromatin to promote gene expression, suggesting NANCI may regulate gene expression by affecting localization of the Tip60 HAT complex. In this proposal, NANCI's interaction with the Tip60 HAT complex will first be verified using RNA pulldown followed by Western blots (Aim 2a). To determine if this interaction is important for NANCI's activity, I will first knockdown the Tip60 HAT complex and determine the effect on NANCI target genes. I will then see if Tip60 localization and histone acetylation marks are affected by NANCI knockdown (Aim 2b). Altogether this proposal will elucidate the scope and mechanism of NANCI's activity during lung development.
Nkx2-1 is a master regulator of lung development that has been implicated in developmental disorders as well as regulation of lung cancer progression. While little is known about what regulates Nkx2-1's expression, the long noncoding RNA NANCI was recently shown to promote Nkx2-1 expression during development. To better understand NANCI's role in lung development and disease this study will characterize the effects of knocking out NANCI and the mechanism by which NANCI regulates gene expression
| Herriges, Michael J; Tischfield, David J; Cui, Zheng et al. (2017) The NANCI-Nkx2.1 gene duplex buffers Nkx2.1 expression to maintain lung development and homeostasis. Genes Dev 31:889-903 |
