Frances A. High

Hlx is induced by and genetically interacts with T-bet to promote heritable T H 1 gene induction

Type 1 helper T (TH1) cells are essential for cellular immunity, but their ontogeny, maturation and durability remain poorly understood. By constructing a dominant-negative form of T-bet, we were able to determine the role played by this lineage-inducing trans-activator in the establishment and maintenance of heritable TH1 gene expression. Optimal induction of interferon-γ (IFN-γ) expression required genetic interaction between T-bet and its target, the homeoprotein Hlx. In fully mature TH1 cells, reiteration of IFN-γ expression and stable chromatin remodeling became relatively independent of T-bet activity and coincided with demethylation of DNA. In contrast, some lineage attributes, such as expression of IL-12Rβ2 (interleukin 12 receptor β2), required ongoing T-bet activity in mature TH1 cells and their progeny. These findings suggest that heritable states of gene expression might be maintained by continued expression of the inducing factor or by a mechanism that confers a stable imprint of the induced state.

The multifaceted role of Notch in cardiac development and disease.

Notch receptors and their cognate ligands transduce crucial signals between cells in various tissues, and have been conserved across millions of years of evolution. Mutations in Notch signalling components result in congenital heart defects in humans and mice, demonstrating an essential role for Notch in cardiovascular development. The results of recent experiments implicate this signalling pathway in many stages of heart development, and provide mechanistic insight into the vital functions of Notch in the aetiology of several common forms of paediatric and adult cardiac disease.

Endothelial expression of the Notch ligand Jagged1 is required for vascular smooth muscle development

The Notch ligand Jagged1 (Jag1) is essential for vascular remodeling and has been linked to congenital heart disease in humans, but its precise role in various cell types of the cardiovascular system has not been extensively investigated. We show that endothelial-specific deletion of Jag1 results in embryonic lethality and cardiovascular defects, recapitulating the Jag1 null phenotype. These embryos show striking deficits in vascular smooth muscle, whereas endothelial Notch activation and arterial-venous differentiation appear normal. Endothelial Jag1 mutant embryos are phenotypically distinct from embryos in which Notch signaling is inhibited in endothelium. Together, these results imply that the primary role of endothelial Jag1 is to potentiate the development of neighboring vascular smooth muscle.

Gene Silencing Quantitatively Controls the Function of a Developmental trans-Activator

How a single cell gives rise to progeny with differing fates remains poorly understood. We examined cells lacking methyl CpG binding domain protein-2 (MBD2), a molecule that has been proposed to link DNA methylation to silent chromatin. Helper T cells from Mbd2(-/-) mice exhibit disordered differentiation. IL-4, the signature of a restricted set of progeny, is expressed ectopically in Mbd2(-/-) parent and daughter cells. Loss of MBD2-mediated silencing renders the normally essential activator, Gata-3, dispensable for IL-4 induction. Gata-3 and MBD2 act in competition, wherein each factor independently, and quantitatively, regulates the binary choice of whether heritable IL-4 expression is established. Gata-3 functions, in part, to displace MBD2 from methylated DNA. These results suggest that activating and silencing signals integrate to provide spatially and temporally restricted patterns of gene activity.

An essential role for Notch in neural crest during cardiovascular development and smooth muscle differentiation

The cardiac outflow tract develops as a result of a complex interplay among several cell types, including cardiac neural crest cells, endothelial cells, and cardiomyocytes. In both humans and mice, mutations in components of the Notch signaling pathway result in congenital heart disease characterized by cardiac outflow tract defects. However, the specific cell types in which Notch functions during cardiovascular development remain to be defined. In addition, in vitro studies have provided conflicting data regarding the ability of Notch to promote or inhibit smooth muscle differentiation, while the physiological role for Notch in smooth muscle formation during development remains unclear. In this study, we generated mice in which Notch signaling was specifically inactivated in derivatives of the neural crest. These mice exhibited cardiovascular anomalies, including aortic arch patterning defects, pulmonary artery stenosis, and ventricular septal defects. We show that Notch plays a critical, cell-autonomous role in the differentiation of cardiac neural crest precursors into smooth muscle cells both in vitro and in vivo, and we identify specific Notch targets in neural crest that are implicated in this process. These results provide a molecular and cellular framework for understanding the role of Notch signaling in the etiology of congenital heart disease.

Murine Jagged1/Notch signaling in the second heart field orchestrates Fgf8 expression and tissue-tissue interactions during outflow tract development

Notch signaling is vital for proper cardiovascular development and function in both humans and animal models. Indeed, mutations in either JAGGED or NOTCH cause congenital heart disease in humans and NOTCH mutations are associated with adult valvular disease. Notch typically functions to mediate developmental interactions between adjacent tissues. Here we show that either absence of the Notch ligand Jagged1 or inhibition of Notch signaling in second heart field tissues results in murine aortic arch artery and cardiac anomalies. In mid-gestation, these mutants displayed decreased Fgf8 and Bmp4 expression. Notch inhibition within the second heart field affected the development of neighboring tissues. For example, faulty migration of cardiac neural crest cells and defective endothelial-mesenchymal transition within the outflow tract endocardial cushions were observed. Furthermore, exogenous Fgf8 was sufficient to rescue the defect in endothelial-mesenchymal transition in explant assays of endocardial cushions following Notch inhibition within second heart field derivatives. These data support a model that relates second heart field, neural crest, and endocardial cushion development and suggests that perturbed Notch-Jagged signaling within second heart field progenitors accounts for some forms of congenital and adult cardiac disease.

Nuclear repositioning marks the selective exclusion of lineage-inappropriate transcription factor loci during T helper cell differentiation

To address how heritable patterns of gene expression are acquired during the differentiation of Th1 and Th2 cells, we analyzed the nuclear position of lineage‐restricted cytokine genes and their upstream regulators by 3‐dimensional fluorescence in situ hybridization. During Th1 differentiation, GATA‐3 and c‐maf loci, which encode upstream regulators of Th2 cytokines, were progressively repositioned to centromeric heterochromatin as defined by a γ‐satellite repeat probe and/or the nuclear periphery, compartments that have been associated with transcriptional repression. A third transcription factor locus, T‐bet, which controls Th1‐specific programs, was subject to de novo CpG methylation in a Th2 cell clone. In contrast, we did not find repositioning of the cytokine gene loci IL‐2, IL‐3, IL‐4 or IFN‐γ during T helper cell differentiation. Instead, IFN‐γ was constitutively associated with the nuclear periphery, even when primed for expression in Th1 cells. Our results suggest that Th1/Th2 lineage commitment and differentiation involve repositioning of the regulators of cytokine expression, rather than the cytokine genes themselves.

Notch Activation of Jagged1 Contributes to the Assembly of the Arterial Wall

Background— Notch signaling in vascular smooth muscle precursors is required for smooth muscle differentiation. Jagged1 expression on endothelium activates Notch in vascular smooth muscle precursors including those of neural crest origin to initiate the formation of a smooth muscle layer in a maturing blood vessel. Methods and Results— Here, we show that Jagged1 is a direct Notch target in smooth muscle, resulting in a positive feedback loop and lateral induction that propagates a wave of smooth muscle differentiation during aortic arch artery development. In vivo, we show that Notch inhibition in cardiac neural crest impairs Jagged1 messenger RNA expression and results in deficient smooth muscle differentiation and resultant aortic arch artery defects. Ex vivo, Jagged1 ligand activates Notch in neural crest explants and results in activation of Jagged1 messenger RNA, a response that is blocked by Notch inhibition. We examine 15 evolutionary conserved regions within the Jagged1 genomic locus and identify a single Notch response element within the second intron. This element contains a functional Rbp-J binding site demonstrated by luciferase reporter and chromatin immunoprecipitation assays and is sufficient to recapitulate aortic arch artery expression of Jagged1 in transgenic mice. Loss of Jagged1 in neural crest impairs vascular smooth muscle differentiation and results in aortic arch artery defects. Conclusions— Taken together, these results provide a mechanism for lateral induction that allows for a multilayered smooth muscle wall to form around a nascent arterial endothelial tube and identify Jagged1 as a direct Notch target.

Cell cycle controlling the silencing and functioning of mammalian activators

Naïve CD4(+) helper T (T(H)) cells respond to stimulation by terminally differentiating into two mature classes, T(H)1 cells, which express interferon gamma (IFN-gamma), and T(H)2 cells, which express interleukin 4 (IL-4). The transcriptional activators T-bet and Gata-3 mediate commitment to the T(H)1 and T(H)2 fates, respectively, including chromatin remodeling of signature genes. The cytokine IL-12 fosters growth of committed T(H)1 cells, while IL-4 fosters growth of committed T(H)2 cells. IL-12 and IL-4 also play critical roles in commitment by promoting transcriptional silencing of Gata-3 and T-bet, respectively. We now show that both T-bet and Gata-3 are induced in a cell cycle-independent manner in bipotent progenitor cells. In contrast, both lineage-restricted gene induction by the activator proteins and heritable silencing of the transcription of each activator, the hallmarks of terminal differentiation, are cell cycle dependent. We found that cells that cannot cycle remain uncommitted and bipotent in response to the most polarizing signals for maturation. These results provide mechanistic insight into a mammalian model of terminal differentiation by illustrating that cell cycle-coupled epigenetic effects, as originally described in yeast, may represent an evolutionarily conserved strategy for organizing signaling and cell fate.

Cutting Edge: Innate Production of IFN-γ by NK Cells Is Independent of Epigenetic Modification of the IFN-γ Promoter

The ability of NK and T cells to produce IFN-γ is critical for resistance to numerous intracellular pathogens but the kinetics of these responses differ. Consistent with this is a requirement for naive T cells to become activated and undergo proliferation-dependent epigenetic changes to the IFN-γ locus that allow them to produce IFN-γ. The data presented here reveal that unlike T cells, murine NK cells produce IFN-γ under conditions of short-term cytokine stimulation, and these events are independent of proliferation and cell cycle progression. Furthermore, analysis of the IFN-γ locus in NK cells reveals that this locus is constitutively demethylated. The finding that NK cells do not need to remodel the IFN-γ locus to produce IFN-γ, either because they do not exhibit epigenetic repression or they have undergone prior remodeling during development, provides a molecular basis for the innate and adaptive regulation of the production of this cytokine.