Aharon Helman

Capicua DNA-binding sites are general response elements for RTK signaling in Drosophila

RTK/Ras/MAPK signaling pathways play key functions in metazoan development, but how they control expression of downstream genes is not well understood. In Drosophila, it is generally assumed that most transcriptional responses to RTK signal activation depend on binding of Ets-family proteins to specific cis-acting sites in target enhancers. Here, we show that several Drosophila RTK pathways control expression of downstream genes through common octameric elements that are binding sites for the HMG-box factor Capicua, a transcriptional repressor that is downregulated by RTK signaling in different contexts. We show that Torso RTK-dependent regulation of terminal gap gene expression in the early embryo critically depends on Capicua octameric sites, and that binding of Capicua to these sites is essential for recruitment of the Groucho co-repressor to the huckebein enhancer in vivo. We then show that subsequent activation of the EGFR RTK pathway in the neuroectodermal region of the embryo controls dorsal-ventral gene expression by downregulating the Capicua protein, and that this control also depends on Capicua octameric motifs. Thus, a similar mechanism of RTK regulation operates during subdivision of the anterior-posterior and dorsal-ventral embryonic axes. We also find that identical DNA octamers mediate Capicua-dependent regulation of another EGFR target in the developing wing. Remarkably, a simple combination of activator-binding sites and Capicua motifs is sufficient to establish complex patterns of gene expression in response to both Torso and EGFR activation in different tissues. We conclude that Capicua octamers are general response elements for RTK signaling in Drosophila.

p16Ink4a-induced senescence of pancreatic beta cells enhances insulin secretion

Cellular senescence is thought to contribute to age-associated deterioration of tissue physiology. The senescence effector p16Ink4a is expressed in pancreatic beta cells during aging and limits their proliferative potential; however, its effects on beta cell function are poorly characterized. We found that beta cell–specific activation of p16Ink4a in transgenic mice enhances glucose-stimulated insulin secretion (GSIS). In mice with diabetes, this leads to improved glucose homeostasis, providing an unexpected functional benefit. Expression of p16Ink4a in beta cells induces hallmarks of senescence—including cell enlargement, and greater glucose uptake and mitochondrial activity—which promote increased insulin secretion. GSIS increases during the normal aging of mice and is driven by elevated p16Ink4a activity. We found that islets from human adults contain p16Ink4a-expressing senescent beta cells and that senescence induced by p16Ink4a in a human beta cell line increases insulin secretion in a manner dependent, in part, on the activity of the mechanistic target of rapamycin (mTOR) and the peroxisome proliferator-activated receptor (PPAR)-γ proteins. Our findings reveal a novel role for p16Ink4a and cellular senescence in promoting insulin secretion by beta cells and in regulating normal functional tissue maturation with age.

Multiple RTK pathways downregulate Groucho-mediated repression in Drosophila embryogenesis.

RTK pathways establish cell fates in a wide range of developmental processes. However, how the pathway effector MAPK coordinately regulates the expression of multiple target genes is not fully understood. We have previously shown that the EGFR RTK pathway causes phosphorylation and downregulation of Groucho, a global co-repressor that is widely used by many developmentally important repressors for silencing their various targets. Here, we use specific antibodies that reveal the dynamics of Groucho phosphorylation by MAPK, and show that Groucho is phosphorylated in response to several RTK pathways during Drosophila embryogenesis. Focusing on the regulation of terminal patterning by the Torso RTK pathway, we demonstrate that attenuation of Grouchos repressor function via phosphorylation is essential for the transcriptional output of the pathway and for terminal cell specification. Importantly, Groucho is phosphorylated by an efficient mechanism that does not alter its subcellular localisation or decrease its stability; rather, modified Groucho endures long after MAPK activation has terminated. We propose that phosphorylation of Groucho provides a widespread, long-term mechanism by which RTK signals control target gene expression.

Capicua integrates input from two maternal systems in Drosophila terminal patterning

In Drosophila, the maternal terminal system specifies cell fates at the embryonic poles via the localised stimulation of the Torso receptor tyrosine kinase (RTK). Signalling by the Torso pathway relieves repression mediated by the Capicua and Groucho repressors, allowing the restricted expression of the zygotic terminal gap genes tailless and huckebein. Here we report a novel positive input into tailless and huckebein transcription by maternal posterior group genes, previously implicated in abdomen and pole cell formation. We show that absence of a subset of posterior group genes, or their overactivation, leads to the spatial reduction or expansion of the tailless and huckebein posterior expression domains, respectively. We demonstrate that the terminal and posterior systems converge, and that exclusion of Capicua from the termini of posterior group mutants is ineffective, accounting for reduced terminal gap gene expression in these embryos. We propose that the terminal and posterior systems function coordinately to alleviate transcriptional silencing by Capicua, and that the posterior system fine‐tunes Torso RTK signalling output, ensuring precise spatial domains of tailless and huckebein expression.

The Genetic Program of Pancreatic β-Cell Replication In Vivo.

The molecular program underlying infrequent replication of pancreatic β-cells remains largely inaccessible. Using transgenic mice expressing green fluorescent protein in cycling cells, we sorted live, replicating β-cells and determined their transcriptome. Replicating β-cells upregulate hundreds of proliferation-related genes, along with many novel putative cell cycle components. Strikingly, genes involved in β-cell functions, namely, glucose sensing and insulin secretion, were repressed. Further studies using single-molecule RNA in situ hybridization revealed that in fact, replicating β-cells double the amount of RNA for most genes, but this upregulation excludes genes involved in β-cell function. These data suggest that the quiescence-proliferation transition involves global amplification of gene expression, except for a subset of tissue-specific genes, which are “left behind” and whose relative mRNA amount decreases. Our work provides a unique resource for the study of replicating β-cells in vivo.

Phosphorylation of Groucho Mediates RTK Feedback Inhibition and Prolonged Pathway Target Gene Expression

BACKGROUND Signaling by receptor tyrosine kinase (RTK) pathways plays fundamental roles in processes of cell-fate determination, often through the induction of specific transcriptional responses. Yet it is not fully understood how continuous target gene expression, required for irreversible cell-fate specification, is preserved after RTK signaling has ended. Here we address this question using the Drosophila embryo, a model system that has been instrumental in elucidating the developmental functions of RTK signal transduction. RESULTS The Groucho corepressor is phosphorylated and downregulated in response to RTK signaling. Here we show that RTK pathways use Groucho phosphorylation as a general mechanism for inducing expression of pathway target genes encoding cell-fate determinants as well as feedback antagonists, indicating that relief of Groucho-dependent repression is an integral element of RTK signaling networks. We further demonstrate that after mitogen-activated protein kinase (MAPK) has been deactivated, sustained phosphorylation of Groucho is essential for persistent RTK-induced target gene expression and cell-fate determination in several developmental contexts. CONCLUSIONS Phosphorylation of Groucho by MAPK plays a dual role in the regulation of RTK responses: (1) it mediates rapid feedback inhibition, and (2) it provides a stable memory mechanism of past MAPK activity. We propose that, in this manner, phosphorylation of Groucho enables transiently active RTK pathways to fix the spatiotemporal expression profiles of downstream targets over time.

RTK signaling modulates the Dorsal gradient

The dorsoventral (DV) axis of the Drosophila embryo is patterned by a nuclear gradient of the Rel family transcription factor, Dorsal (Dl), that activates or represses numerous target genes in a region-specific manner. Here, we demonstrate that signaling by receptor tyrosine kinases (RTK) reduces nuclear levels and transcriptional activity of Dl, both at the poles and in the mid-body of the embryo. These effects depend on wntD, which encodes a Dl antagonist belonging to the Wingless/Wnt family of secreted factors. Specifically, we show that, via relief of Groucho- and Capicua-mediated repression, the Torso and EGFR RTK pathways induce expression of WntD, which in turn limits Dl nuclear localization at the poles and along the DV axis. Furthermore, this RTK-dependent control of Dl is important for restricting expression of its targets in both contexts. Thus, our results reveal a new mechanism of crosstalk, whereby RTK signals modulate the spatial distribution and activity of a developmental morphogen in vivo.

Effects of ageing and senescence on pancreatic β-cell function

Ageing is generally associated with deterioration of organ function and regenerative potential. In the case of pancreatic β‐cells, an age‐related decline in proliferative potential is well documented, and was proposed to contribute to the increased prevalence of type 2 diabetes in the elderly. The effects of ageing on β‐cell function, namely glucose‐stimulated insulin secretion (GSIS), have not been studied as extensively. Recent work revealed that, surprisingly, β‐cells of mature mice and humans secrete more insulin than young β‐cells in response to high glucose concentrations, potentially serving to counteract age‐related peripheral insulin resistance. This functional change appears to be orchestrated by p16Ink4A‐driven cellular senescence and downstream remodelling of chromatin structure and DNA methylation, enhancing the expression of genes controlling β‐cell function. We propose that activation of the cellular senescence program drives life‐long functional maturation of β‐cells, due to β‐cell hypertrophy, enhanced glucose uptake and more efficient mitochondrial metabolism, in parallel to locking these cells in a non‐replicative state. We speculate that the beneficial aspects of this process can be harnessed to enhance GSIS. Other age‐related mechanisms, which are currently poorly understood, act to increase basal insulin secretion levels also in low glucose conditions. This leads to an overall reduction in the amplitude of insulin secretion between low and high glucose at old age, which may contribute to a deterioration in metabolic control.

Dynamics of senescent cell formation and retention revealed by p14ARF induction in the epidermis.

Cellular senescence, a state of cell-cycle arrest accompanied by dramatic morphologic and metabolic changes, is a central means by which cells respond to physiologic stress and oncogene activity. Senescence is thought to play important roles in aging and in tumor suppression, yet the dynamics by which senescent cells are formed, their effects on tissue function and their eventual fate are poorly understood. To study cellular senescence within an adult tissue, we developed transgenic mice inducibly expressing p14(ARF) (human ortholog of murine p19(ARF)), a central activator of senescence. Induction of p14(ARF) in the epidermis rapidly led to widespread apoptosis and cell-cycle arrest, a stage that was transient, and was followed by p53-dependent cellular senescence. The endogenous Cdkn2a products p19(ARF) and p16(Ink4a) were activated by the transgenic p14(ARF) through p53, revealing a senescence-promoting feed-forward loop. Commitment of cells to senescence required continued p14(ARF) expression, indicating that entry into this state depends on a persistent signal. However, once formed, senescent cells were retained in the epidermis, often for weeks after transgene silencing, indicating an absence of an efficient rapidly acting mechanism for their removal. Stem cells in the hair follicle bulge were largely protected from apoptosis upon p14(ARF) induction, but irreversibly lost their ability to proliferate and initiate follicle growth. Interestingly, induction of epidermal hyperplasia prevented the appearance of senescent cells upon p14(ARF) induction. Our findings provide basic insights into the dynamics of cellular senescence, a central tumor- suppressive mechanism, and reveal the potential for prolonged retention of senescent cells within tissues.

Detection of RTK pathway activation in Drosophila using anti-dpERK immunofluorescence staining.

In Drosophila, like in other metazoans, receptor tyrosine kinase (RTK) signaling pathways control diverse cellular processes such as migration, growth, fate determination, and differentiation (Shilo, Development 132:4017-4027, 2005). Activation of RTKs by their extracellular ligands triggers a signal transduction cascade, mediated by the Ras/Raf/MEK cassette, which ultimately leads to dual phosphorylation and activation of the mitogen-activated protein kinase/extracellularly regulated kinase (MAPK/Erk). Once active, MAPK/Erk phosphorylates its cytoplasmic and nuclear substrates, consequently modulating (i.e., stimulating or inhibiting) their biological function (Murphy and Blenis, Trends in Biochemical Sciences 31:268-275, 2006). The currently available antibody specific for the doubly phosphorylated form of MAPK/Erk (dpERK) (Yung et al., FEBS Letters 408:292-296, 1997) provides a valuable readout for RTK signaling: it enables the spatiotemporal detection of RTK pathway activity in the developing organism, in situ (Gabay et al., Development 124:3535-3541, 1997; Gabay et al., Science 277:1103-1106, 1997). Here, we present a detailed protocol for anti-dpERK immunofluorescent staining that can be applied to the analysis of MAPK/Erk signaling in Drosophila embryogenesis.