Molecular and cellular mechanisms of memory allocation in neuronetworks.

Determining how neuronal networks encode memories is a key goal of neuroscience. Although neuronal circuit processes involved in encoding, storing and retrieving memory have attracted a great deal of attention, the processes that allocate individual memories to specific neurons within a network have remained elusive. Recent findings unraveled the first insights into the processes that modulate memory allocation in neuronetworks. They showed that neurons in the lateral amygdala compete to take part in auditory fear conditioned memory traces and that the levels of the transcription factor CREB (cAMP-response element binding protein) can affect the probability of a neuron to be recruited into a given memory representation. CREB-mediated transcriptional regulation involves several signaling pathways, known to mediate nuclear responses to diverse behavioral stimuli, along with coordinated interactions with multiple other transcription activators, coactivators and repressors. Moreover, activation of CREB triggers an autoinhibitory feedback loop, a metaplastic process that could be used to allocate memories away from cells that have been recently involved in memory. Beyond CREB, there may be a host of other processes that dynamically modulate memory allocation in neuronetworks by shaping cooperation and competition among neurons.

Histone modifications and transcription factor binding on chromatin ChIP-PCR assays.

Chromatin, the eukaryotic template of genetic information, is subject to a diverse array of posttranslational modifications that largely impinge on the N-termini of histones, such as acetylation, methylation, phosphorylation, and ubiquitination. Distinct histone modifications generate synergistic or antagonistic interaction affinities for novhistone proteins, which in turn dictate dynamic transitions between transcriptionally active or silent states of chromatin. Besides transcription, numerous biological processes, including DNA replication, DNA repair, and recombination, are regulated by chromatin-associated factors. The chromatin immunoprecipitation (ChIP) technique provides us with an exquisite tool to investigate the interplay between the structural or regulatory proteins and DNA and its role in regulating diverse cellular processes in vivo by formaldehyde crosslinking of proteins to proteins and proteins to DNA, followed by immunoprecipitation of the fixed material and detection of the associated DNA. Here we illustrate the overall experimental procedure by taking ChIP analysis of the human telomerase reverse transcriptase promoter as an example.

Diclofenac attenuates Wnt/beta-catenin signaling in colon cancer cells by activation of NF-kappaB.

The dysregulation of Wnt/beta-catenin signaling and subsequent upregulation of beta-catenin response transcription (CRT) occur frequently in colon cancer cells. Non-steroidal anti-inflammatory drugs (NSAIDs) can repress CRT in colorectal cancer, but little is known about the mechanism of action. We show that the NSAID diclofenac inhibits Wnt/beta-catenin signaling without altering the level of beta-catenin protein and reduces the expression of beta-catenin/TCF-dependent genes. Diclofenac induced the degradation of IkappaBalpha, which increased free nuclear factor kappaB (NF-kappaB) in cells. Also, the ectopic expression of p65, which is a component of NF-kappaB, suppressed CRT. Our findings suggest that diclofenac inhibits Wnt/beta-catenin signaling via the activation of NF-kappaB in colon cancer cells.

Opposing regulatory roles of E2F in human telomerase reverse transcriptase (hTERT) gene expression in human tumor and normal somatic cells.

Telomerase activity is closely correlated with cellular proliferative activity in human tissues. Human cells with high proliferative potential, such as tumor cells or stem cells, exhibit telomerase activity, whereas most normal human somatic cells do not. Telomerase activity is tightly regulated by the expression of its catalytic subunit human telomerase reverse transcriptase (hTERT). Through an expression cloning approach, we identified E2F-1 as a repressor of the hTERT gene in human tumor cells. Ectopic expression of E2F-1 repressed hTERT promoter activity by inhibiting Sp1 activation of the hTERT promoter. In contrast to the repressor function of E2F-1 in human tumor cells, we demonstrated that E2F-1 is an activator of the hTERT gene in normal human somatic cells. Ectopically expressed E2F-1 activated the hTERT promoter through a noncanonical DNA binding site. E2F-1, E2F-2, and E2F-3 (but not E2F-4 and E2F-5) repressed hTERT promoter activity in human tumor cells, whereas they activated it in normal somatic cells. These contrasting effects of E2F transcription factors on the hTERT promoter could underlie the paradoxical biological activities of E2F, which can both promote and inhibit cellular proliferation and tumorigenesis.

CREB regulates excitability and the allocation of memory to subsets of neurons in the amygdala.

The mechanisms that determine how information is allocated to specific regions and cells in the brain are important for memory capacity, storage and retrieval, but are poorly understood. We manipulated CREB in a subset of lateral amygdala neurons in mice with a modified herpes simplex virus (HSV) and reversibly inactivated transfected neurons with the Drosophila allatostatin G protein-coupled receptor (AlstR)/ligand system. We found that inactivation of the neurons transfected with HSV-CREB during training disrupted memory for tone conditioning, whereas inactivation of a similar proportion of transfected control neurons did not. Whole-cell recordings of fluorescently tagged transfected neurons revealed that neurons with higher CREB levels are more excitable than neighboring neurons and showed larger synaptic efficacy changes following conditioning. Our findings demonstrate that CREB modulates the allocation of fear memory to specific cells in lateral amygdala and suggest that neuronal excitability is important in this process.

Hexachlorophene inhibits Wnt/beta-catenin pathway by promoting Siah-mediated beta-catenin degradation.

Aberrant activation of Wnt/beta-catenin signaling and subsequent up-regulation of beta-catenin response transcription (CRT) is a critical event in the development of human colon cancer. Thus, Wnt/beta-catenin signaling is an attractive target for the development of anticancer therapeutics. In this study, we identified hexachlorophene as an inhibitor of Wnt/beta-catenin signaling from cell-based small-molecule screening. Hexachlorophene antagonized CRT that was stimulated by Wnt3a-conditioned medium by promoting the degradation of beta-catenin. This degradation pathway is Siah-1 and adenomatous polyposis colidependent, but glycogen synthase kinase-3beta and F-box beta-transducin repeat-containing protein-independent. In addition, hexachlorophene represses the expression of cyclin D1, which is a known beta-catenin target gene, and inhibits the growth of colon cancer cells. Our findings suggest that hexachlorophene attenuates Wnt/beta-catenin signaling through the Siah-1-mediated beta-catenin degradation.

Protein-kinase-C-mediated beta-catenin phosphorylation negatively regulates the Wnt/beta-catenin pathway.

Normally, the Wnt/beta-catenin pathway controls developmental processes and homeostasis, but abnormal activation of this pathway is a frequent event during the development of cancer. The key mechanism in regulation of the Wnt/beta-catenin pathway is the amino-terminal phosphorylation of beta-catenin, marking it for proteasomal degradation. Here we present small-molecule-based identification of protein kinase C (PKC)-mediated beta-catenin phosphorylation as a novel mechanism regulating the Wnt/beta-catenin pathway. We used a cell-based chemical screen to identify A23187, which inhibits the Wnt/beta-catenin pathway. PKC was activated by A23187 treatment and subsequently phosphorylated N-terminal serine (Ser) residues of beta-catenin, which promoted beta-catenin degradation. Moreover, the depletion of PKCalpha inhibited the phosphorylation and degradation of beta-catenin. Therefore, our findings suggest that the PKC pathway negatively regulates the beta-catenin level outside of the Wnt/beta-catenin pathway.

Small molecule-based reversible reprogramming of cellular lifespan.

Most somatic cells encounter an inevitable destiny, senescence. Little progress has been made in identifying small molecules that extend the finite lifespan of normal human cells. Here we show that the intrinsic 'senescence clock' can be reset in a reversible manner by selective modulation of the ataxia telangiectasia-mutated (ATM) protein and ATM- and Rad3-related (ATR) protein with a small molecule, CGK733. This compound was identified by a high-throughput phenotypic screen with automated imaging. Employing a magnetic nanoprobe technology, magnetism-based interaction capture (MAGIC), we identified ATM as the molecular target of CGK733 from a genome-wide screen. CGK733 inhibits ATM and ATR kinase activities and blocks their checkpoint signaling pathways with great selectivity. Consistently, siRNA-mediated knockdown of ATM and ATR induced the proliferation of senescent cells, although with lesser efficiency than CGK733. These results might reflect the specific targeting of the kinase activities of ATM and ATR by CGK733 without affecting any other domains required for cell proliferation.

A magnetic nanoprobe technology for detecting molecular interactions in live cells.

Technologies to assess the molecular targets of biomolecules in living cells are lacking. We have developed a technology called magnetism-based interaction capture (MAGIC) that identifies molecular targets on the basis of induced movement of superparamagnetic nanoparticles inside living cells. Efficient intracellular uptake of superparamagnetic nanoparticles (coated with a small molecule of interest) was mediated by a transducible fusogenic peptide. These nanoprobes captured the small molecule's labeled target protein and were translocated in a direction specified by the magnetic field. Use of MAGIC in genome-wide expression screening identified multiple protein targets of a drug. MAGIC was also used to monitor signal-dependent modification and multiple interactions of proteins.

Small-molecule-based identification of dynamic assembly of E2F-pocket protein-histone deacetylase complex for telomerase regulation in human cells.

Activation of telomerase is crucial for cells to gain immortality. Most normal human somatic cells have a limited proliferative life span, and expression of the rate-limiting telomerase catalytic subunit, known as human telomerase reverse transcriptase (hTERT), has been believed to be tightly repressed. This model of hTERT regulation is challenged by the recent identification of the induction of hTERT in normal cycling human fibroblasts during their transit through S phase. Here we show the small-molecule-based identification of the assembly and disassembly of E2F-pocket protein-histone deacetylase (HDAC) complex as a key mechanistic basis for the repression and activation of hTERT in normal human cells. A cell-based chemical screen was used to identify a small molecule, CGK1026, that derepresses hTERT expression. CGK1026 inhibits the recruitment of HDAC into E2F-pocket protein complexes assembled on the hTERT promoter. Chromatin immunoprecipitation analysis reveals dynamic alterations in hTERT promoter occupancy by E2F and pocket proteins according to the cell cycle-dependent regulation of hTERT. Dominant-negative or protein-knockout strategies to disrupt the assembly of E2F-pocket protein-HDAC complex derepress hTERT and telomerase activity. Taken together with the results on the regulatory function of these complexes in cellular senescence and tumorigenesis, our findings suggest that dynamic assembly of E2F-pocket protein-HDAC complex plays a central role in the regulation of hTERT in a variety of proliferative conditions (e.g., normal cycling, senescent, and tumor cells).

Sp1 and Sp3 recruit histone deacetylase to repress transcription of human telomerase reverse transcriptase (hTERT) promoter in normal human somatic cells.

Activation of telomerase is crucial for cells to gain immortality. In human cells, telomerase activity is tightly regulated by the expression of its catalytic subunit, human telomerase reverse transcriptase (hTERT). In most normal human somatic cells, hTERT is not expressed, and its suppression acts as an important gatekeeper against tumorigenesis. Here we describe the systematic analyses of hTERT promoter to understand the transcriptional repression mechanism of the hTERT gene in normal human somatic cells. Through the serial deletion analysis of hTERT promoter in normal human fibroblasts, we identified a critical repressive element on the hTERT promoter. The repressive element formed DNA-protein complexes with Sp1 and Sp3 in nuclear extracts. Using formaldehyde cross-linked chromatin immunoprecipitation analysis, we found that Sp1 and Sp3 were associated with the endogenously repressed hTERT promoter in human fibroblasts. Furthermore, Sp1 and Sp3 interacted with histone deacetylase (HDAC) in these cells. Overexpression of dominant-negative mutants of Sp1 and Sp3, which contained mainly the HDAC2-binding domain, relieved the HDAC-mediated repression of the hTERT promoter. Taken together, these results suggest that Sp1 and Sp3 associate with the hTERT promoter, recruiting HDAC for the localized deacetylation of nucleosomal histones and transcriptional silencing of the hTERT gene in normal human somatic cells.