The E2F1 transcription factor modulates neuronal apoptosis induced by staurosporine, DNA damage and b-amyloid. We demonstrate E2F1 involvement in neuronal death induced by the more physiological oxygen-glucose deprivation (OGD) in mouse cortical cultures and by anoxia in mouse hippocampal slices. E2F1(1/1) and (2/2) cultures were comparable, in that they contained similar neuronal densities, responded with similar increases in intracellular calcium concentration ([Ca 21 ] i ) to glutamate receptor agonists, and showed similar NMDA receptor subunit mRNA expression levels for NR1, NR2A and NR2B. Despite these similarities, E2F1(2/2) cultures were signi®cantly less susceptible to neuronal death than E2F1(1/1) cultures 24 and 48 h following 120±180 min of OGD. Furthermore, the absence of E2F1 signi®cantly improved the ability of CA1 neurons in hippocampal slices to recover synaptic transmission following a transient anoxic insult in vitro. These results, along with our ®nding that E2F1 mRNA levels are signi®cantly increased following OGD, support a role for E2F1 in the modulation of OGD-and anoxia-induced neuronal death. These ®ndings are consistent with studies showing that overexpression of E2F1 in postmitotic neurons causes neuronal degeneration and the absence of E2F1 decreases infarct volume following cerebral ischemia.

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Although a great deal has been elucidated concerning the mechanisms regulating muscle differentiation, little is known about transcription factor-specific gene regulation. Our understanding of the genetic mechanisms regulating cell differentiation is quite limited. Much of what has been defined centers on regulatory signaling cascades and transcription factors. Surprisingly few studies have investigated the association of genes with specific transcription factors. To address these issues, we have utilized a method coupling chromatin immunoprecipitation and CpG microarrays to characterize the genes associated with MEF2 in differentiating C2C12 cells. Results demonstrated a defined binding pattern over the course of differentiation. Filtered data demonstrated 9 clones to be elevated at 0 h, 792 at 6 h, 163 by 1 day, and 316 at 3 days. Using unbiased selection parameters, we selected a subset of 291 prospective candidates. Clones were sequenced and filtered for removal of redundancy between clones and for the presence of repetitive elements. We were able to place 50 of these on the mouse genome, and 20 were found to be located near well-annotated genes. From this list, previously undefined associations with MEF2 were discovered. Many of these genes represent proteins involved in neurogenesis, neuromuscular junctions, signaling and metabolism. The remaining clones include many full-length cDNA and represent novel gene targets. The results of this study provides for the first time, a unique look at gene regulation at the level of transcription factor binding in differentiating muscle.

The phosphatidylinositol 3-kinase (PI3K) signaling pathway has been associated with a variety of cellular functions ranging from cell cycle regulation to tissue development. Although years of research have extensively characterized this signaling pathway, little is known as to how specific cellular events are coordinated by its activation. Here we demonstrate that Dapr (differentiation-associated protein), a novel protein, appears to focus one aspect of this pathway by acting as a putative scaffold protein during skeletal muscle differentiation. We present for the first time a description of this protein using in silico analysis. dapr was discovered through a previous study employing chromatin immunoprecipitation and CpG microarray analysis experiments as being regulated by myocyte-enhancing factor 2, a key transcription factor involved in the differentiation of skeletal muscle tissue. In this study we show that during the course of differentiation, Dapr binds to the PI3K signaling pathway member protein kinase B (PKB). In C 2 C 12 myoblast cells before differentiation Dapr is localized to the cytosol, migrating with PKB to the membrane after initiation of muscle differentiation. Knockdown of Dapr by RNAi resulted in inhibition of myotube formation. Our findings indicate that Dapr is a key component required by myoblasts for orchestrating their differentiation during myogenesis. Furthermore, it appears that Dapr is involved in the PI3K signaling cascade, potentially acting as a scaffold protein for PKB and coordinating its compartmentalization during differentiation. In the transformation of myoblasts into myotubes, PI3K2 signaling is one of the key pathways (1). Activation of this pathway typically begins with signaling through the activated surface receptor by insulin growth factor 1 (2). Downstream, PI3K-targeted phosphorylation produces the lipid products phosphatidylinositol 3,4-diphosphate and phosphatidylinositol 3,4,5-trisphosphate. These phosphoinositides provide membrane anchoring points for pleckstrin homology domain-containing proteins with binding motifs to them. Protein kinase B (PKB, also known as AKT) is one such protein (3). The targeted localization of PKB and other kinases to the plasma membrane is an important part of their activation (4, 5). Indeed, the compartmentalization of kinases within the cell has come to be known as a key regulatory mechanism of signaling cascades. A kinase-associated protein (6) is an example of one of the first proteins identified to function in this manner. Not only does A kinase-associated protein localize kinases to specific parts of the cell, it also brings proteins together within proximity of one another; in this case, protein phosphatase 2 and protein kinase A (7).Among its many important roles as a signal transducer, including insulin-mediated signaling, PKB has also been shown to play a pivotal role in cellular differentiation (1,8,9). Its role in the development of myotubes has been well established. Initial work demonstrated that PI3K inhibition causing ...