To identify direct transcriptional targets of Nkx2-1 that could be effectors of its developmental functions we performed ChIP-chip analyses in early and late developing mouse lung. The differential expression pattern of Nkx2-1 at these developmental stages allowed us to evaluate Nkx2-1 targets in cell populations undergoing proliferation at E11.5 and differentiation at E19.5. In this work, we identified a preferential role for Nkx2-1 in direct transcriptional regulation of proliferation-related genes in early development and of ion transport genes in late development.
Nkx2-1 regulation of lung cell proliferation and survival was previously shown in development and tumor cell lines 
, but the effector genes directly regulated by Nkx2-1 were largely unknown. Amongst several proliferation-related genes targeted by Nkx2-1, we identified E2f3, cyclins Ccnb1 and Ccnb2, and c-Met. E2f3 controls the rate of cell proliferation by controlling the G1/S transition and the initiation of DNA synthesis 
and is expressed in the lung epithelium in early development 
. Cyclins Ccnb1 and Ccnb2 regulate the G2/M phase transition and are ubiquitously expressed in the lung during development. Ccnb1, E2f3 and other proliferation genes are mostly bound by Nkx2-1 at E11.5 but not at E19.5. Binding of Nkx2-1 to the promoters of these genes correlates with increased expression (Figure S3
), and with proliferative state of the epithelial cells in early lung development. c-Met is a proto-oncogene and the HGF receptor tyrosine kinase expressed in E13 mouse lung epithelium and thereafter, where it is involved in mitosis, migration and morphogenesis 
. Reduction of Nkx2-1 expression in cell lines alters expression of these genes, and slows down cell cycle progression. In vivo, the absence of Nkx2-1 results in impaired lung epithelial lineage expansion and branching morphogenesis. These findings make us speculate that reduced expression of genes involved in cell proliferation and progression of the cell cycle may contribute to the hypomorphic lung phenotype observed in Nkx2-1 null embryos 
. It will be interesting in the future to determine if altered expression of the genes identified precludes distal lung epithelial progenitor cells to proliferate and engage in the process of branching morphogenesis.
A different context is observed at E19.5, when Nkx2-1 expressing cells are differentiating and preparing for the rapid absorption of luminal fluid and for the first breath 
. Nkx2-1 binding to ion transport genes in distal lung epithelial cells at E19.5 suggests that Nkx2-1 participates in differentiation of the distal lung epithelium to perform these functions at birth. Gene expression analyses of E18 lungs harboring a Nkx2-1 phosphorylation-deficient mutant also show reduced expression of genes that regulate fluid and electrolyte transport 
supporting a direct link between Nkx2-1 and these functions.
Our results may also have important implications for understanding NKX2-1 functions in lung cancer. A link between development and tumorigenesis has been suggested in different cancers and their corresponding organ of origin 
. Genomic associations between human lung cancer subtypes and developing mouse lung indicated that tumors with genomic profiles similar to early lung development correlate to poorer patient's prognosis 
while tumors with gene expression profiles similar to more differentiated lung cell phenotypes correlate to better patient's prognosis. Developmental genes expressed in tumors, such as NKX2-1 may underlie these associations. Multiple evidences support a dual role for NKX2-1 as a proto-oncogene and tumor suppressor gene in lung cancer. NKX2-1 is considered a lineage specific oncogene since its expression is increased or amplified in some lung tumors 
. In other analyses NKX2-1 is considered a good prognostic factor, since patients with NSCLC showing high levels of NKX2-1 or amplification of the locus have a better prognosis than those that have lost NKX2-1 expression 
. NKX2-1 was also proposed as a suppressor of lung adenocarcinoma progression in a mouse model of lung cancer 
. NKX2-1 target genes, effectors of these functions in lung tumors are also unknown. NKX2-1 and some human homologues of the targets identified in development, including E2F3, CCNB1, CCNB2 and c-MET have been proposed as independent lung tumor markers and prognostic genes. E2F3 is overexpressed in 55–70% squamous cell carcinomas and 79% of adenocarcinomas of the lung. 
, and is associated with high Ki67 in invasive cancers 
. Increased expression of CCNB1 in NSCLC was suggested as a poor prognostic parameter 
. CCNB2 and c-MET are also over expressed in adenocarcinomas 
. Our findings point to NKX2-1 as a direct transcriptional regulator of these independent markers of lung tumorigenesis modulating their level of expression at different stages of tumor progression.
Comparison of mouse lung development and human lung cancer data sets identified cell cycle and proliferation as the largest gene categories involved in both processes. Since early development in most organs involves significant cell proliferation, it is not surprising that most similarities between NKX2-1 targets in early lung development and tumors are related to cell cycle and proliferation genes 
. It is possible that tumor cells maintaining lung-lineage characteristics use tissue/cell specific factors including NKX2-1 to control proliferation and other functions. In addition to the genes identified in these studies, there may be other genes uniquely regulated by NKX2-1 in tumors and not in development; to identify those genes it will be necessary, in the future, to analyze direct NKX2-1 binding in primary tumors or alternatively in tumor cell lines.
It is intriguing that many cell proliferation genes inversely correlate to the levels of NKX2-1 in NSCLC. This inverse correlation may explain the poorer prognosis of patients with NSCLC with low levels of NKX2-1. To determine if the reverse correlation is due to repression by direct NKX2-1 binding, ChIP analyses may be performed in human tumor tissues or tumor cell lines. Alternatively molecular analyses of the promoters of these genes in cell lines may provide information about the repression of these genes by Nkx2-1 binding. These experiments will be the focus of our future studies. Adenocarcinomas sub-classification based on gene expression profiling was proposed to improve prediction of malignant potential and prognosis 
. The associations identified in our studies may contribute to the molecular classification of these tumors and clarify NSCLC heterogeneity, holding great potential to increase the understanding of this disease.
Our findings point to potential molecular mechanisms by which Nkx2-1 may differentially regulate transcriptional activity. First, inverse correlation in expression level of NKX2-1 and targets in tumors, and of Nkx2-1 and c-Met in MLE15 cells suggests a more widespread role of Nkx2-1 in transcriptional repression. This effect could be by direct binding or, alternatively, by recruitment or activation of transcriptional repressors by Nkx2-1 to down-regulate particular genes. Nkx2-1 has been mostly linked to transcriptional activation in lung and other organs 
, although neuropilin-2 
and RAGE 
have been reported to be down-regulated by direct binding of Nkx2-1 to a cis-element in their promoters strongly supporting Nkx2-1 repressor activity.
Second, there are target genes bound by Nkx2-1 at both developmental time points, whereas others are bound only at E11.5 or E19.5. Interactions with alternative co-factors differentially expressed at each time point might result in differential affinity and binding to alternative targets 
. Different isoforms and/or modifications of Nkx2-1 proteins by phosphorylation, acetylation or oxidation may affect affinity for particular cis
-elements or interactions to different co-factors at each time point 
. Identification of different forms of Nkx2-1 protein at E11.5 and E19.5 will be necessary to fully understand the different targets in alternative cell contexts.
The specificity of Nkx2-1 binding has also been linked to promoter structure 
. Differences in chromatin modifications surrounding these cis
-elements in different cell contexts could affect affinity of Nkx2-1 proteins. For example binding of Nkx2-1 to the Sftpb promoter is prevented by DNA methylation of the Sftpb promoter in non-expressing tissues, such as thyroid 
. To fully understand the differences in binding patterns in different cell contexts we will need to identify the consensus sequences and the chromatin modifications of the binding sites in genomic regions only bound at E11.5 or only bound at E19.5. Future analyses will be focused in discerning these alternatives and characterizing Nkx2-1 binding sites in different contexts.
Finally, we observed strong binding to brain and thyroid genes not expressed at detectable levels in developing lung, suggesting that binding of Nkx2-1 does not imply activation of transcription. In certain cases, binding may precede activation such as in the case of Sftpa and may prime the gene for activation upon recruitment of other transcription complexes and/or co-factors to the promoter. The identification of unique Nkx2-1 targets at E11.5 and E19.5 will facilitate the evaluation of possible mechanisms that control specificity.
Overall, we provide novel insights into biological processes regulated by Nkx2-1 in different cell contexts in development, and cancer. We identified Nkx2-1 direct target genes in mouse lung epithelium that are primary effectors of Nkx2-1 functions, in particular cell proliferation genes. We showed that expression levels of the target genes depend on NKX2-1 levels in NSCLC. NKX2-1 has been associated to longer, similar or shorter patient survival in NSCLC, depending on expression levels 
. Therefore, evaluation of NKX2-1 expression levels relative to its downstream targets will provide a way to sub-classify NSCLCs, and understand the mechanisms underlying associations to patient survival.