Faculty Bibliography

E2A and HEB are basic helix-loop-helix transcription factors that play important roles in T cell development. Expression of Id1, one of their inhibitors, severely impairs T cell development in transgenic mice. Aberrant activation of NF-kappaB transcription factors has been shown to contribute to the developmental defects, but it is not clear whether NF-kappaB activation is directly due to Id1 expression or is secondary to an abnormal thymic environment in Id1 transgenic mice. Here, by using a T cell line model, we demonstrate that Id1 expression stimulates basal levels of NF-kappaB activity and further enhances NF-kappaB activation upon T cell receptor (TCR) signaling achieved by anti-CD3 and anti-CD28 stimulation. Activation of NF-kappaB is partially mediated by the classical pathway involving the interaction between the regulatory subunit, NF-kappaB essential modulator (NEMO), and the catalytic subunit, IkappaB kinase beta. However, a NEMO-independent pathway also appears to be at play. Id1-potentiated activation of NF-kappaB leads to overproduction of cytokines such as tumor necrosis factor alpha and interferon-gamma in a T cell line as well as in thymocytes. Among members of the NF-kappaB family, c-Rel appears to be preferentially activated by Id1, especially during TCR stimulation. Consistently, c-rel deficiency diminishes tumor necrosis factor alpha and interferon-gamma expression induced by Id1 and TCR signaling.

BACKGROUND:While developmental processes such as axon pathfinding and synapse formation have been characterized in detail, comparatively less is known of the intrinsic developmental mechanisms that regulate transcription of ion channel genes in embryonic neurons. Early decisions, including motoneuron axon targeting, are orchestrated by a cohort of transcription factors that act together in a combinatorial manner. These transcription factors include Even-skipped (Eve), islet and Lim3. The perdurance of these factors in late embryonic neurons is, however, indicative that they might also regulate additional aspects of neuron development, including the acquisition of electrical properties. RESULTS:To test the hypothesis that a combinatorial code transcription factor is also able to influence the acquisition of electrical properties in embryonic neurons we utilized the molecular genetics of Drosophila to manipulate the expression of Eve in identified motoneurons. We show that increasing expression of this transcription factor, in two Eve-positive motoneurons (aCC and RP2), is indeed sufficient to affect the electrical properties of these neurons in early first instar larvae. Specifically, we observed a decrease in both the fast K+ conductance (IKfast) and amplitude of quantal cholinergic synaptic input. We used charybdotoxin to pharmacologically separate the individual components of IKfast to show that increased Eve specifically down regulates the Slowpoke (a BK Ca2+-gated potassium channel), but not Shal, component of this current. Identification of target genes for Eve, using DNA adenine methyltransferase identification, revealed strong binding sites in slowpoke and nAcRalpha-96Aa (a nicotinic acetylcholine receptor subunit). Verification using real-time PCR shows that pan-neuronal expression of eve is sufficient to repress transcripts for both slo and nAcRalpha-96Aa. CONCLUSION/CONCLUSIONS:Taken together, our findings demonstrate, for the first time, that Eve is sufficient to regulate both voltage- and ligand-gated currents in motoneurons, extending its known repertoire of action beyond its already characterized role in axon guidance. Our data are also consistent with a common developmental program that utilizes a defined set of transcription factors to determine both morphological and functional neuronal properties.

Ionizing radiation causes DNA damage that elicits a cellular program of damage control coordinated by the kinase activity of ataxia telangiectasia mutated protein (ATM). Transforming growth factor beta (TGFbeta)-1, which is activated by radiation, is a potent and pleiotropic mediator of physiologic and pathologic processes. Here we show that TGFbeta inhibition impedes the canonical cellular DNA damage stress response. Irradiated Tgfbeta1 null murine epithelial cells or human epithelial cells treated with a small-molecule inhibitor of TGFbeta type I receptor kinase exhibit decreased phosphorylation of Chk2, Rad17, and p53; reduced gammaH2AX radiation-induced foci; and increased radiosensitivity compared with TGFbeta competent cells. We determined that loss of TGFbeta signaling in epithelial cells truncated ATM autophosphorylation and significantly reduced its kinase activity, without affecting protein abundance. Addition of TGFbeta restored functional ATM and downstream DNA damage responses. These data reveal a heretofore undetected critical link between the microenvironment and ATM, which directs epithelial cell stress responses, cell fate, and tissue integrity. Thus, Tgfbeta1, in addition to its role in homoeostatic growth control, plays a complex role in regulating responses to genotoxic stress, the failure of which would contribute to the development of cancer; conversely, inhibiting TGFbeta may be used to advantage in cancer therapy

During embryogenesis, the lateral cortical stream (LCS) emerges from the corticostriatal border (CSB), the boundary between the developing cerebral cortex and striatum. The LCS is comprised of a mix of pallial- and subpallial-derived neural progenitor cells that migrate to the developing structures of the basal telencephalon, most notably the piriform cortex and amygdala. Using a combination of in vitro and in vivo approaches, we analyzed the timing, composition, migratory modes, origin, and requirement of the homeodomain-containing transcription factor Gsh2 (genomic screened homeobox 2) in the development of this prominent migratory stream. We reveal that Pax6 (paired box gene 6)-positive pallial-derived and Dlx2 (distal-less homeobox 2)-positive subpallial-derived subpopulations of LCS cells are generated in distinct temporal windows during embryogenesis. Furthermore, our data indicate the CSB border not only is comprised of separate populations of pallial- and subpallial-derived progenitors that contribute to the LCS but also a subpopulation of cells coexpressing Pax6 and Dlx2. Moreover, despite migrating along a route outlined by a cascade of radial glia, the Dlx2-positive population appears to migrate primarily in an apparent chain-like manner, with LCS migratory cells being generated locally at the CSB with little contribution from other subpallial structures such as the medial, lateral, or caudal ganglionic eminences. We further demonstrate that the generation of the LCS is dependent on the homeodomain-containing gene Gsh2, revealing a novel requirement for Gsh2 in telencephalic development

Newly designed norbornene-appended hexabenzocoronene 1 self-assembles, upon diffusion of an Et(2)O vapor into its CH(2)Cl(2) solution, to form either graphitic nanocoils or nanotubes, depending on the self-assembling conditions. The coiled assembly, selectively formed at 15 degrees C, is a kinetic intermediate for the tubular assembly and transforms into nanotubes on standing at 25 degrees C. However, post-ring-opening metathesis polymerization of the norbornene pendants of 1 enhances the thermal stability of the coiled assembly as well as the tubular one and disables a thermodynamic coil-to-tube transition. The polymerized nanocoils show an electroconductivity of 1 x 10(-)(4) S cm(-)(1) upon doping with I(2), while the nonpolymerized nanocoils are disrupted upon being doped.

Early in development, primordial germ cells (PGCs) are set aside from somatic cells and acquire a unique gene-expression program . The mechanisms underlying germline-specific gene expression are largely unknown. Nanos expression is required during germline development and is posttranscriptionally restricted to PGCs . Here we report that the microRNA miR-430 targets the 3' untranslated region (UTR) of nanos1 during zebrafish embryogenesis. A miR-430 target site within the nanos1 3' UTR reduces poly(A) tail length, mRNA stability, and translation. Repression is disrupted in maternal-zygotic dicer mutants (MZdicer), which lack mature miRNAs , and is restored by injection of processed miR-430. Although miR-430 represses other genes equally in germline and soma, specific regions in the nanos1 3' UTR compensate for microRNA-mediated repression in PGCs and allow germline-specific expression. We show that the 3' UTR of an additional PGC-specific gene, TDRD7, is also targeted by miR-430. These results indicate that miR-430 targets the 3' UTRs of germline genes and suggest that differential susceptibility to microRNAs contributes to tissue-specific gene expression.

Fish and amphibian hearts are known to regenerate after partial resection, but the molecular mechanisms underlying this process remain unclear. In this issue of Cell, Lipilina et al. analyze regeneration in the zebrafish heart. Their work indicates that new cardiomyocytes originate from undifferentiated progenitor cells and reveals a critical role for the epicardium, the cellular layer that covers the heart.

We previously compared by microarray analysis gene expression in rheumatoid arthritis (RA) and osteoarthritis (OA) tissues. Among the set of genes identified as a molecular signature of RA, clusterin (clu) was one of the most differentially expressed. In the present study we sought to assess the expression and the role of CLU (mRNA and protein) in the affected joints and in cultured fibroblast-like synoviocytes (FLS) and to determine its functional role. Quantitative RT-PCR, Northern blot, in situ hybridization, immunohistochemistry, and Western blot were used to specify and quantify the expression of CLU in ex vivo synovial tissue. In synovial tissue, the protein was predominantly expressed by synoviocytes and it was detected in synovial fluids. Both full-length and spliced isoform CLU mRNA levels of expression were lower in RA tissues compared with OA and healthy synovium. In synovium and in cultured FLS, the overexpression of CLU concerned all protein isoforms in OA whereas in RA, the intracellular forms of the protein were barely detectable. Transgenic overexpression of CLU in RA FLS promoted apoptosis within 24 h. We observed that CLU knockdown with small interfering RNA promoted IL-6 and IL-8 production. CLU interacted with phosphorylated IkappaBalpha. Differential expression of CLU by OA and RA FLS appeared to be an intrinsic property of the cells. Expression of intracellular isoforms of CLU is differentially regulated between OA and RA. We propose that in RA joints, high levels of extracellular CLU and low expression of intracellular CLU may enhance NF-kappaB activation and survival of the synoviocytes.

The Rag proteins carry out V(D)J recombination through a process mechanistically similar to cut-and-paste transposition. Specifically, Rag complexes form DNA hairpins through direct transesterification, using a catalytic Asp-Asp-Glu (DDE) triad in Rag1. How is sufficient DNA distortion introduced to allow hairpin formation? We hypothesized that, like certain transposases, the Rag proteins might use aromatic amino acid residues to stabilize a flipped-out base. Through in vivo and in vitro experiments and structural predictions, we identified residues in Rag1 crucial for hairpin formation. One of these, a conserved tryptophan (Trp893), probably participates in base-stacking interactions near the cleavage site, as do Trp298, Trp265 and Trp319 in the Tn5, Tn10 and Hermes transposases, respectively. Other residues surrounding the catalytic glutamate (YKEFRK) may share functional similarities with the YREK motif in IS4 family transposases