The importance of detailed epigenomic profiling of different cell types within organs.

The human body consists of hundreds of kinds of cells specified from a single genome overlaid with cell type-specific epigenetic information. Comprehensively profiling the body's distinct epigenetic landscapes will allow researchers to verify cell types used in regenerative medicine and to determine the epigenetic effects of disease, environmental exposures and genetic variation. Key marks/factors that should be investigated include regions of nucleosome-free DNA accessible to regulatory factors, histone marks defining active enhancers and promoters, DNA methylation levels, regulatory RNAs, and factors controlling the three-dimensional conformation of the genome. Here we use the lung to illustrate the importance of investigating an organ's purified cell epigenomes, and outline the challenges and promise of realizing a comprehensive catalog of primary cell epigenomes

Transcriptomic profiling of primary alveolar epithelial cell differentiation in human and rat

AbstractCell-type specific gene regulation is a key to gaining a full understanding of how the distinct phenotypes of differentiated cells are achieved and maintained. Here we examined how changes in transcriptional activation during alveolar epithelial cell (AEC) differentiation determine phenotype. We performed transcriptomic profiling using in vitro differentiation of human and rat primary AEC. This model recapitulates in vitro an in vivo process in which AEC transition from alveolar type 2 (AT2) cells to alveolar type 1 (AT1) cells during normal maintenance and regeneration following lung injury. Here we describe in detail the quality control, preprocessing, and normalization of microarray data presented within the associated study (Marconett et al., 2013). We also include R code for reproducibility of the referenced data and easily accessible processed data tables

Transcriptomic Profiling of Primary Alveolar Epithelial Cell Differentiation in Human and Rat.

Cell-type specific gene regulation is key to gaining a full understanding of how the distinct phenotypes of differentiated cells are achieved and maintained. Here we examined how changes in transcriptional activation during alveolar epithelial cell (AEC) differentiation determine phenotype. We performed transcriptomic profiling using in vitro differentiation of human and rat primary AEC. This model recapitulates in vitro an in vivo process in which AEC transition from alveolar type 2 (AT2) cells to alveolar type 1 (AT1) cells during normal maintenance and regeneration following lung injury. Here we describe in detail the quality control, preprocessing, and normalization of microarray data presented within the associated study [1]. We also include R code for reproducibility of the referenced data and easily accessible processed data tables

The importance of detailed epigenomic profiling of different cell types within organs.

The human body consists of hundreds of kinds of cells specified from a single genome overlaid with cell type-specific epigenetic information. Comprehensively profiling the body's distinct epigenetic landscapes will allow researchers to verify cell types used in regenerative medicine and to determine the epigenetic effects of disease, environmental exposures and genetic variation. Key marks/factors that should be investigated include regions of nucleosome-free DNA accessible to regulatory factors, histone marks defining active enhancers and promoters, DNA methylation levels, regulatory RNAs, and factors controlling the three-dimensional conformation of the genome. Here we use the lung to illustrate the importance of investigating an organ's purified cell epigenomes, and outline the challenges and promise of realizing a comprehensive catalog of primary cell epigenomes

Subunit-specific coordinate upregulation of sodium pump activity in alveolar epithelial cells by lentivirus-mediated gene transfer

Resolution of alveolar edema depends on active ion transport by sodium pumps located on the basolateral surface of alveolar epithelial cells (AECs), suggesting that upregulation of sodium pump activity may facilitate clearance of edema fluid. We have investigated the use of lentiviral vectors to augment sodium pump activity via gene transfer of sodium pump subunits to AECs. Full-length cDNA for the alpha(1) or beta(1) subunit of rat Na(+),K(+)-ATPase was cloned into the lentiviral vector pRRLsin.hCMV.IRES.EGFP. Rat AECs in primary culture were transduced on day 4 with lentiviral vectors pseudotyped with vesicular stomatitis virus glycoprotein G. Transduction with lentiviral vectors encoding either alpha(1) subunit (Lenti-alpha(1)-EGFP) or beta(1) subunit (Lenti-beta(1)-EGFP) led to dose-dependent increases in mRNA and protein for the corresponding subunit. Transduction with Lenti-beta(1)-EGFP was accompanied by coordinate upregulation of endogenous alpha(1) expression, whereas endogenous beta(1) expression was unchanged after transduction with Lenti-alpha(1)-EGFP. Consistent with these findings, transduction with Lenti-beta(1)-EGFP, but not Lenti-alpha(1)-EGFP, led to augmentation of sodium pump activity as a result of increases in Na(+),K(+)-ATPase holoenzyme. Sodium pump alpha(2) subunit and sodium channel protein did not change after Lenti-beta(1)-EGFP transduction. These results demonstrate that overall sodium pump activity can be efficiently upregulated in AECs specifically via gene transfer of the sodium pump beta(1) subunit and support the feasibility of lentivirus-mediated gene transfer to augment alveolar fluid clearance

FOXO1 Couples KGF and PI-3K/AKT Signaling to NKX2.1-Regulated Differentiation of Alveolar Epithelial Cells

NKX2.1 is a master regulator of lung morphogenesis and cell specification; however, interactions of NKX2.1 with various transcription factors to regulate cell-specific gene expression and cell fate in the distal lung remain incompletely understood. FOXO1 is a key regulator of stem/progenitor cell maintenance/differentiation in several tissues but its role in the regulation of lung alveolar epithelial progenitor homeostasis has not been evaluated. We identified a novel role for FOXO1 in alveolar epithelial cell (AEC) differentiation that results in the removal of NKX2.1 from surfactant gene promoters and the subsequent loss of surfactant expression in alveolar epithelial type I-like (AT1-like) cells. We found that the FOXO1 forkhead domain potentiates a loss of surfactant gene expression through an interaction with the NKX2.1 homeodomain, disrupting NKX2.1 binding to the SFTPC promoter. In addition, blocking PI-3K/AKT signaling reduces phosphorylated FOXO-1 (p-FOXO1), allowing accumulated nuclear FOXO1 to interact with NKX2.1 in differentiating AEC. Inhibiting AEC differentiation in vitro with keratinocyte growth factor (KGF) maintained an AT2 cell phenotype through increased PI3K/AKT-mediated FOXO1 phosphorylation, resulting in higher levels of surfactant expression. Together these results indicate that FOXO1 plays a central role in AEC differentiation by directly binding NKX2.1 and suggests an essential role for FOXO1 in mediating AEC homeostasis