Integrated proteomic and transcriptomic profiling of mouse lung development and Nmyc target genes

Abstract

Although microarray analysis has provided information regarding the dynamics of gene expression during development of the mouse lung, no extensive correlations have been made to the levels of corresponding protein products. Here, we present a global survey of protein expression during mouse lung organogenesis from embryonic day E13.5 until adulthood using gel‐free two‐dimensional liquid chromatography coupled to shotgun tandem mass spectrometry (MudPIT). Mathematical modeling of the proteomic profiles with parallel DNA microarray data identified large groups of gene products with statistically significant correlation or divergence in coregulation of protein and transcript levels during lung development. We also present an integrative analysis of mRNA and protein expression in Nmyc loss‐ and gain‐of‐function mutants. This revealed a set of 90 positively and negatively regulated putative target genes. These targets are evidence that Nmyc is a regulator of genes involved in mRNA processing and a repressor of the imprinted gene Igf2r in the developing lung. ### Synopsis The lung is a complex and highly organized tissue consisting of an epithelium in contact with the air, a mesenchyme layer allowing for the expansion and contraction of the lung during breathing and a complex vasculature to bring blood close to the site of gas exchange. The development of the lung is well defined morphologically and many genes have been shown to the critical for correct development using mouse genetic models such as gene knockout and misexpression ([Kimura et al , 1996][1]; [Sekine et al , 1999][2]). Several global microarray expression studies have investigated the profile of gene expression during normal lung development ([Mariani et al , 2002][3]; [Bonner et al , 2003][4]; [Lu et al , 2004a][5]). However, the expression levels and subcellular localization of the cognate proteins are largely unknown. Here, we report the profiling of proteins in the lung by gel‐free two‐dimensional liquid chromatography coupled to shotgun tandem mass spectrometry (MudPIT) over six developmental time points covering most of the significant stages of lung development. A prefractionation into organellar compartments (cytosol, nucleus and mitochondria) was performed to assay both tissue and subcellular specificity from the same sample preparation ([Kislinger et al , 2003][6], [2006][7]). Comparison of the proteomic data with mRNA expression profiles revealed a large number of gene products (protein and mRNA) that are coordinately regulated during development ([Figure 5A][8]). We were also able to identify a smaller group of ∼30 genes whose levels of mRNA and protein expression are uncorrelated ([Figure 5D][8]), suggesting regulation via post‐transcriptional or post‐translational control mechanisms. Having established a baseline of normal lung development, we next characterized the molecular changes that take place in mutant genetic backgrounds. One of the most powerful tools in understanding gene function is the combination of loss‐of‐function (nulls or hypomorophs) and gain‐of‐function (misexpression or overexpression) mutants. We used loss‐ and gain‐of‐function mutants of the gene Nmyc , a transcription factor that has previously been shown to be critical for lung development ([Moens et al , 1992][9], [1993][10]; [Okubo et al , 2005][11]), to molecularly characterize its function in lung development by proteomics and microarray profiling. By combining these data sets, we identified several potential direct targets of Nmyc regulation ([Figure 6][12]). Furthermore, as Nmyc can function as both a transcriptional activator and repressor, we were able to classify these target genes as being activated or repressed by Nmyc. Along with several known targets of Nmyc, we also identified many genes involved in mRNA processing, splicing and export that appeared positively regulated by Nmyc. We identified four genes that appeared to be repressed by Nmyc including Igf2r an imprinted gene. In summary, we have shown that the technique of gel‐free two‐dimensional liquid chromatography coupled to shotgun tandem mass spectrometry (MudPIT) can be used to profile embryonic tissues during development. Mining of protein profiles and protein–protein interaction networks was used to identify proteins with potential developmental importance. Finally, integrative analysis of protein and mRNA levels in Nmyc hypomorph and overexpressing mutant mice identified a list of possible direct Nmyc target genes. Mol Syst Biol. 3: 109 [1]: #ref-34 [2]: #ref-58 [3]: #ref-44 [4]: #ref-7 [5]: #ref-42 [6]: #ref-37 [7]: #ref-36 [8]: #F5 [9]: #ref-45 [10]: #ref-46 [11]: #ref-52 [12]: #F6