Testis-specific Arf promoter expression in a transposase-aided BAC transgenic mouse model.

CDKN2A is an evolutionarily conserved gene encoding proteins implicated in tumor suppression, ocular development, aging, and metabolic diseases. Like the human form, mouse Cdkn2a encodes two distinct proteins-p16Ink4a, which blocks cyclin-dependent kinase activity, and p19Arf, which is best known as a positive regulator of the p53 tumor suppressor-and their functions have been well-studied in genetically engineered mouse models. Relatively little is known about how expression of the two transcripts is controlled in normal development and in certain disease states. To better understand their coordinate and transcript-specific expression in situ, we used a transposase-aided approach to generate a new BAC transgenic mouse model in which the first exons encoding Arf and Ink4a are replaced by fluorescent reporters. We show that mouse embryo fibroblasts generated from the transgenic lines faithfully display induction of each transgenic reporter in cell culture models, and we demonstrate the expected expression of the Arf reporter in the normal testis, one of the few places where that promoter is normally expressed. Interestingly, the TGFß-2-dependent induction of the Arf reporter in the eye-a process essential for normal eye development-does not occur. Our findings illustrate the value of BAC transgenesis in mapping key regulatory elements in the mouse by revealing the genomic DNA required for Cdkn2a induction in cultured cells and the developing testis, and the apparent lack of elements driving expression in the developing eye.

p19(Arf) limits primary vitreous cell proliferation driven by PDGF-B.

Arf encodes an important tumor suppressor, p19(Arf), which also plays a critical role to control hyperplasia in the primary vitreous during mouse eye development. In the absence of Arf, mice are born blind and display a phenotype closely mimicking severe forms of the human eye disease, persistent hyperplastic primary vitreous (PHPV). In this report, we characterize p19(Arf) expression in perivascular cells that normally populate the primary vitreous and express the Arf promoter. Using a new ex vivo model, we show that these cells respond to exogenous Tgfß, despite being isolated at a time when Tgfß has already turned on the Arf promoter. Treatment of the cells with PDGF-B ligand doubles the population of cells in S-phase and ectopic expression of Arf blunts that effect. We show this effect is mediated through Pdgfrß as expression of Arf represses expression of Pdgfrß mRNA and protein to approximately 60%. p53 is not required for Arf-dependent blockade of PDGF-B driven proliferation and repression of Pdgfrß protein as ectopic expression of Arf is still able to inhibit the 2-fold increase in the S-phase fraction of cells upon treatment with PDGF-B. Finally, induction of mature miR-34a, a microRNA previously identified to be regulated by p19(Arf) does not depend on p53 while the expression of the primary transcript does require p53. These data corroborate that, as in vivo, p19(Arf) functions to inhibit PDGF-B driven proliferation ex vivo.

Varied manifestations of persistent hyperplastic primary vitreous with graded somatic mosaic deletion of a single gene.

PURPOSE: Persistent hyperplastic primary vitreous (PHPV) represents a developmental eye disease known to have diverse manifestations ranging from a trivial remnant of hyaloid vessels to a dense fibrovascular mass causing lens opacity and retinal detachment. PHPV can be modeled in mice lacking individual genes, but certain features of such models differ from the clinical realm. For example, mice lacking the Arf gene have uniformly severe disease with consistent autosomal recessive disease penetrance. We tested whether the graded somatic loss of Arf in a subset of cells in chimeric mice mimics the range of disease in a non-heritable manner. METHODS: Wild type ↔ Arf(-/-) mouse chimeras were generated by morulae fusion, and when the mice were 10 weeks old, fundoscopic, slit-lamp, and histological evaluations were performed. The relative fraction of cells of the Arf(-/-) lineage was assessed with visual, molecular genetic, and histological analysis. Objective quantification of various aspects of the phenotype was correlated with the genotype. RESULTS: Sixteen chimeras were generated and shown to have low, medium, and high contributions of Arf(-/-) cells to tail DNA, the cornea, and the retinal pigment epithelium (RPE), with excellent correlation between chimerism in the tail DNA and the RPE. Phenotypic differences (coat color and severity of eye disease) were evident, objectively quantified, and found to correlate with the contribution of Arf(-/-) cells to the RPE and tail-derived DNA, but not the cornea. CONCLUSIONS: Generating animals composed of different numbers of Arf(-/-) cells mimicked the range of disease severity observed in patients with PHPV. This establishes the potential for full manifestations of PHPV to be caused by somatic mutations of a single gene during development.

p19Arf represses platelet-derived growth factor receptor ß by transcriptional and posttranscriptional mechanisms.

In addition to cancer surveillance, p19(Arf) plays an essential role in blocking signals stemming from platelet-derived growth factor receptor ß (Pdgfrß) during eye development, but the underlying mechanisms have not been clear. We now show that without Arf, pericyte hyperplasia in the eye results from enhanced Pdgfrß-dependent proliferation from embryonic day 13.5 (E13.5) of mouse development. Loss of Arf in the eye increases Pdgfrß expression. In cultured fibroblasts and pericyte-like cells, ectopic p19(Arf) represses and Arf knockdown enhances the expression of Pdgfrß mRNA and protein. Ectopic Arf also represses primary Pdgfrß transcripts and a plasmid driven by a minimal promoter, including one missing the CCAAT element required for high-level expression. p19(Arf) uses both p53-dependent and -independent mechanisms to control Pdgfrß. In vivo, without p53, Pdgfrß mRNA is elevated and eye development abnormalities resemble the Arf (-/-) phenotype. However, effects of p53 on Pdgfrß mRNA do not appear to be due to direct p53 or RNA polymerase II recruitment to the promoter. Although p19(Arf) controls Pdgfrß mRNA in a p53-dependent manner, it also blunts Pdgfrß protein expression by blocking new protein synthesis in the absence of p53. Thus, our findings demonstrate a novel capacity for p19(Arf) to control Pdgfrß expression by p53-dependent and -independent mechanisms involving RNA transcription and protein synthesis, respectively, to promote the vascular remodeling needed for normal vision.

The bHLH transcription factor Tcf21 is required for lineage-specific EMT of cardiac fibroblast progenitors.

The basic helix-loop-helix (bHLH) family of transcription factors orchestrates cell-fate specification, commitment and differentiation in multiple cell lineages during development. Here, we describe the role of a bHLH transcription factor, Tcf21 (epicardin/Pod1/capsulin), in specification of the cardiac fibroblast lineage. In the developing heart, the epicardium constitutes the primary source of progenitor cells that form two cell lineages: coronary vascular smooth muscle cells (cVSMCs) and cardiac fibroblasts. Currently, there is a debate regarding whether the specification of these lineages occurs early in the formation of the epicardium or later after the cells have entered the myocardium. Lineage tracing using a tamoxifen-inducible Cre expressed from the Tcf21 locus demonstrated that the majority of Tcf21-expressing epicardial cells are committed to the cardiac fibroblast lineage prior to initiation of epicardial epithelial-to-mesenchymal transition (EMT). Furthermore, Tcf21 null hearts fail to form cardiac fibroblasts, and lineage tracing of the null cells showed their inability to undergo EMT. This is the first report of a transcription factor essential for the development of cardiac fibroblasts. We demonstrate a unique role for Tcf21 in multipotent epicardial progenitors, prior to the process of EMT that is essential for cardiac fibroblast development.

Epicardial-derived cell epithelial-to-mesenchymal transition and fate specification require PDGF receptor signaling.

RATIONALE: In early heart development, platelet-derived growth factor (PDGF) receptor expression in the heart ventricles is restricted to the epicardium. Previously, we showed that PDGFRß is required for coronary vascular smooth muscle cell (cVSMC) development, but a role for PDGFRα has not been identified. Therefore, we investigated the combined and independent roles of these receptors in epicardial development. OBJECTIVE: To understand the contribution of PDGF receptors in epicardial development and epicardial-derived cell fate determination. METHODS AND RESULTS: By generating mice with epicardial-specific deletion of the PDGF receptors, we found that epicardial epithelial-to-mesenchymal transition (EMT) was defective. Sox9, an SRY-related transcription factor, was reduced in PDGF receptor-deficient epicardial cells, and overexpression of Sox9 restored epicardial migration, actin reorganization, and EMT gene expression profiles. The failure of epicardial EMT resulted in hearts that lacked epicardial-derived cardiac fibroblasts and cVSMC. Loss of PDGFRα resulted in a specific disruption of cardiac fibroblast development, whereas cVSMC development was unperturbed. CONCLUSIONS: Signaling through both PDGF receptors is necessary for epicardial EMT and formation of epicardial-mesenchymal derivatives. PDGF receptors also have independent functions in the development of specific epicardial-derived cell fates.

Depression, social support, and beta-adrenergic transcription control in human ovarian cancer.

Motivated by previous indications that beta-adrenergic signaling can regulate tumor cell gene expression in model systems, we sought to determine whether similar dynamics occur in primary human ovarian cancer. DNA microarray analyses of 10 ovarian carcinomas identified 266 human transcripts that were differentially expressed in tumors from patients with elevated biobehavioral risk factors (high depressive symptoms and low social support) relative to grade- and stage-matched tumors from low-risk patients. Promoter-based bioinformatic analyses indicated increased activity of several beta-adrenergically-linked transcription control pathways, including CREB/ATF, NF-kappaB/Rel, STAT, and Ets family transcription factors. Consistent with increased beta-adrenergic signaling, high biobehavioral risk patients also showed increased intra-tumor concentrations of norepinephrine (but no difference in plasma norepinephrine). These data show that genome-wide transcriptional profiles are significantly altered in tumors from patients with high behavioral risk profiles, and they identify beta-adrenergic signal transduction as a likely mediator of those effects.

Social regulation of gene expression in human leukocytes.

BACKGROUND: Social environmental influences on human health are well established in the epidemiology literature, but their functional genomic mechanisms are unclear. The present study analyzed genome-wide transcriptional activity in people who chronically experienced high versus low levels of subjective social isolation (loneliness) to assess alterations in the activity of transcription control pathways that might contribute to increased adverse health outcomes in social isolates. RESULTS: DNA microarray analysis identified 209 genes that were differentially expressed in circulating leukocytes from 14 high- versus low-lonely individuals, including up-regulation of genes involved in immune activation, transcription control, and cell proliferation, and down-regulation of genes supporting mature B lymphocyte function and type I interferon response. Promoter-based bioinformatic analyses showed under-expression of genes bearing anti-inflammatory glucocorticoid response elements (GREs; p = 0.032) and over-expression of genes bearing response elements for pro-inflammatory NF-kappaB/Rel transcription factors (p = 0.011). This reciprocal shift in pro- and anti-inflammatory signaling was not attributable to differences in circulating cortisol levels, or to other demographic, psychological, or medical characteristics. Additional transcription control pathways showing differential activity in bioinformatic analyses included the CREB/ATF, JAK/STAT, IRF1, C/EBP, Oct, and GATA pathways. CONCLUSION: These data provide the first indication that human genome-wide transcriptional activity is altered in association with a social epidemiological risk factor. Impaired transcription of glucocorticoid response genes and increased activity of pro-inflammatory transcription control pathways provide a functional genomic explanation for elevated risk of inflammatory disease in individuals who experience chronically high levels of subjective social isolation.