Human embryonic lung epithelial tips are multipotent progenitors that can be expanded in vitro as long-term self-renewing organoids.

The embryonic mouse lung is a widely used substitute for human lung development. For example, attempts to differentiate human pluripotent stem cells to lung epithelium rely on passing through progenitor states that have only been described in mouse. The tip epithelium of the branching mouse lung is a multipotent progenitor pool that self-renews and produces differentiating descendants. We hypothesized that the human distal tip epithelium is an analogous progenitor population and tested this by examining morphology, gene expression and in vitro self-renewal and differentiation capacity of human tips. These experiments confirm that human and mouse tips are analogous and identify signalling pathways that are sufficient for long-term self-renewal of human tips as differentiation-competent organoids. Moreover, we identify mouse-human differences, including markers that define progenitor states and signalling requirements for long-term self-renewal. Our organoid system provides a genetically-tractable tool that will allow these human-specific features of lung development to be investigated.

Lung epithelial tip progenitors integrate glucocorticoid- and STAT3-mediated signals to control progeny fate.

Insufficient alveolar gas exchange capacity is a major contributor to lung disease. During lung development, a population of distal epithelial progenitors first produce bronchiolar-fated and subsequently alveolar-fated progeny. The mechanisms controlling this bronchiolar-to-alveolar developmental transition remain largely unknown. We developed a novel grafting assay to test if lung epithelial progenitors are intrinsically programmed or if alveolar cell identity is determined by environmental factors. These experiments revealed that embryonic lung epithelial identity is extrinsically determined. We show that both glucocorticoid and STAT3 signalling can control the timing of alveolar initiation, but that neither pathway is absolutely required for alveolar fate specification; rather, glucocorticoid receptor and STAT3 work in parallel to promote alveolar differentiation. Thus, developmental acquisition of lung alveolar fate is a robust process controlled by at least two independent extrinsic signalling inputs. Further elucidation of these pathways might provide therapeutic opportunities for restoring alveolar capacity.

E2F2 and CREB cooperatively regulate transcriptional activity of cell cycle genes.

E2F2 is essential for the maintenance of T lymphocyte quiescence. To identify the full set of E2F2 target genes, and to gain further understanding of the role of E2F2 in transcriptional regulation, we have performed ChIP-chip analyses across the genome of lymph node-derived T lymphocytes. Here we show that during quiescence, E2F2 binds the promoters of a large number of genes involved in DNA metabolism and cell cycle regulation, concomitant with their transcriptional silencing. A comparison of ChIP-chip data with expression profiling data on resting E2f2(-)(/)(-) T lymphocytes identified a subset of 51 E2F2-specific target genes, most of which are upregulated on E2F2 loss. Luciferase reporter assays showed a retinoblastoma-independent role for E2F2 in the negative regulation of these target genes. Importantly, we show that the DNA binding activity of the transcription factor CREB contributes to E2F2-mediated repression of Mcm5 and Chk1 promoters. siRNA-mediated CREB knockdown, expression of a dominant negative KCREB mutant or disruption of CREB binding by mutating a CRE motif on Mcm5 promoter, relieved E2F2-mediated transcriptional repression. Taken together, our data uncover a new regulatory mechanism for E2F-mediated transcriptional control, whereby E2F2 and CREB cooperate in the transcriptional repression of a subset of E2F2 target genes.

Differential proteomics analysis reveals a role for E2F2 in the regulation of the Ahr pathway in T lymphocytes.

E2F transcription factors (E2F1-8) are best known for their role in cell proliferation, although it is clear that they regulate many other biological processes through the transcriptional modulation of distinct target genes. However, the specific set of genes regulated by each E2F remains to be characterized. To gain insight into the molecular pathways regulated by E2F2, we have analyzed the proteome of antigen receptor-activated T cells lacking E2F2. We report that loss of E2F2 results in a deregulated Aryl-hydrocarbon-receptor pathway. Proliferating E2F2(-/-) T lymphocytes expressed significantly higher levels of Aip, Ahr, and Arnt relative to wild-type (WT)(1) controls. The mechanism for increased levels of Aip appears straightforward, involving direct regulation of the Aip gene promoter by E2F2. Although the Ahr and Arnt promoters also bind E2F2, their regulation appears to be more complex. Nevertheless, exposure to the environmental xenobiotic 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a well-known exogenous ligand of the Ahr pathway, led to overexpression of the Ahr target gene Cyp1a1, and to increased sensitivity to TCDD-triggered apoptosis in E2F2(-/-) T cells compared with WT controls. These results suggest that E2F2 modulates cellular sensitivity to xenobiotic signals through the negative regulation of the Ahr pathway.

Multiple E2F-induced microRNAs prevent replicative stress in response to mitogenic signaling.

Transcription of microRNAs (miRNAs) is thought to be regulated similarly to that of protein-coding genes. However, how miRNAs are regulated during the cell division cycle is not well understood. We have analyzed the transcription profiles of miRNAs in response to mitogenic stimulation in primary fibroblasts. About 33% of the miRNAs expressed in these cells are induced upon exit from quiescence. Many of these miRNAs are specifically induced by E2F1 or E2F3 during the G(1)/S transition and are repressed in E2F1/3-knockout cells. At least four miRNA clusters, let-7a-d, let-7i, mir-15b-16-2, and mir-106b-25, are direct targets of E2F1 and E2F3 during G(1)/S and are repressed in E2F1/3-null cells. Interestingly, these miRNAs do not contribute to E2F-dependent entry into S phase but rather inhibit the G(1)/S transition by targeting multiple cell cycle regulators and E2F targets. In fact, E2F1 expression results in a significant increase in S-phase entry and DNA damage in the absence of these microRNAs. Thus, E2F-induced miRNAs contribute to limiting the cellular responses to E2F activation, thus preventing replicative stress. Given the known function of E2F of inducing other oncogenic miRNAs, control of miRNAs by E2F is likely to play multiple roles in cell proliferation and in proliferative diseases such as cancer.

E2F2 represses cell cycle regulators to maintain quiescence.

E2F transcription factors control diverse biological processes through regulation of target gene expression. However, the mechanism by which this regulation is established, and the relative contribution of each E2F member are still poorly defined. We have investigated the role of E2F2 in regulating cellular proliferation. We show that E2F2 is required for the normal G(0)/G(1) phase because targeted disruption of the E2F2 gene causes T cells to enter S phase early and to undergo accelerated cell division. A large set of E2F target genes involved in DNA replication and cell cycle progression (such as Mcm's, cyclins and Cdc2a) that are silent in G(0) and typically transcribed late in G(1) phase are already actively expressed in quiescent T cells and MEFs lacking E2F2. The classic E2F activators, E2F1 and E2F3, are largely dispensable for this process because compound loss of E2F1(-/-) and E2F2(-/-) produces a comparably shortened G(0)/G(1) phase, with early S phase entry. Likewise, shRNA knockdown of E2F3 does not alter significantly the E2F2(-/-) phenotype. Chromatin immunoprecipitation analysis indicates that in wild-type cells the promoters of the aberrantly early-transcribed genes are occupied by E2F2 in G(0), suggesting a direct role for E2F2 in transcriptional repression. We conclude that E2F2 functions to transcriptionally repress cell cycle genes to establish the G(0) state.

Diabetes and exocrine pancreatic insufficiency in E2F1/E2F2 double-mutant mice.

E2F transcription factors are thought to be key regulators of cell growth control. Here we use mutant mouse strains to investigate the function of E2F1 and E2F2 in vivo. E2F1/E2F2 compound-mutant mice develop nonautoimmune insulin-deficient diabetes and exocrine pancreatic dysfunction characterized by endocrine and exocrine cell dysplasia, a reduction in the number and size of acini and islets, and their replacement by ductal structures and adipose tissue. Mutant pancreatic cells exhibit increased rates of DNA replication but also of apoptosis, resulting in severe pancreatic atrophy. The expression of genes involved in DNA replication and cell cycle control was upregulated in the E2F1/E2F2 compound-mutant pancreas, suggesting that their expression is repressed by E2F1/E2F2 activities and that the inappropriate cell cycle found in the mutant pancreas is likely the result of the deregulated expression of these genes. Interestingly, the expression of ductal cell and adipocyte differentiation marker genes was also upregulated, whereas expression of pancreatic cell marker genes were downregulated. These results suggest that E2F1/E2F2 activity negatively controls growth of mature pancreatic cells and is necessary for the maintenance of differentiated pancreatic phenotypes in the adult.