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The Skaggs Institute for Chemical Biology
Scientific Report 1999-2000


Regulation, Function, and Signaling Mechanisms of Cell Adhesion Molecules


G.M. Edelman

The fundamental processes of embryogenesis have evolved to translate the linear genetic code reproducibly into a 3-dimensional organism. Early in development, dividing cells derived from the fertilized egg adhere into specific aggregates. The gene program of each aggregate is then altered, moving the cells down differentiation pathways leading to specific tissues and organs. My colleagues and I have identified and characterized adhesion proteins on the surfaces of cells that allow aggregation, and we have shown that different tissues have different combinations of these cell adhesion molecules or CAMs. In previous studies, we defined signals that regulate the expression of gene programs when cells aggregate via specific CAMs and identified CAM promoters as targets for transcription factors of the Hox and Pax gene families. In more recent studies, we followed up on this work and also began analyzing the fundamental mechanisms in the control of transcription and translation.

The neural cell adhesion molecule N-CAM is a transmembrane glycoprotein of the immunoglobulin superfamily that is expressed by both neurons and astrocytes. N-CAM levels increase during embryogenesis and after injury, suggesting that regulation of N-CAM expression is important in both neural development and nerve regeneration. In previous studies, we showed that N-CAM binding inhibited astrocyte proliferation in vitro and in vivo after an injury and altered cell signaling and gene expression. We recently turned our attention to the effects of N-CAM on progenitor or stem cells in the nervous system.

Neural stem cells have received increased attention because of their potential use in cell replacement therapy in CNS injury and disease. Addition of soluble N-CAM to cultured rat and mouse hippocampal progenitor cells reduced cell proliferation and enhanced differentiation of the cells to the neuronal lineage, as indicated by a 2-fold increase in the number of cells expressing neuronal markers. Experiments with cells from mice that lack N-CAM indicated that N-CAM on the cell surface is not required for these effects, suggesting that added N-CAM induces a signal by binding to a molecule other than N-CAM itself. Overall, the findings suggest that N-CAM and its ligands play a role in controlling stem cell proliferation and differentiation.

One of the postulated functions of CAMs is participation in the formation of neuronal synapses and in dynamic changes that occur in the plasticity of the synapses. Excitation of synapses can lead to elevations of calcium in synaptic structures called dendritic spines, and this elevation in turn activates a calcium-dependent protease called calpain. To study synaptic changes, we developed a fluorescent reagent that can be used to detect dendritic spines in which calpain has been activated. A fusion protein was expressed that contained enhanced yellow fluorescent protein and enhanced cyan fluorescent protein linked by a peptide that included the µ-calpain cleavage site from α-spectrin. The fusion protein exhibited fluorescence resonance energy transfer (FRET) from enhanced cyan fluorescent protein to enhanced yellow fluorescent protein. The diminution of FRET after proteolysis (and separation of these fluorescent markers) was used to localize calpain activity in situ by fluorescence microscopy.

In neurons, FRET was diminished when intracellular calcium levels were increased by treatment with an ionophore or with neurotransmitter agonists. Immunostaining of cultured neurons with antibodies to spectrin products produced by calpain-mediated digestion indicated that the amount of FRET present at postsynaptic elements was inversely related to the concentration of spectrin breakdown products. Thus, the FRET method can be used to detect sites of synaptic activity and should be particularly useful in analyzing synapses undergoing changes in efficacy. These changes are thought to be critical to learning and memory, and they may be correlated with changes in CAMs at these synapses.

We have continued studies of regulation of the expression of CAM genes. Genes are controlled through regions of the DNA called promoters, and transcription factors can bind to specific parts of promoter DNA to activate or repress gene expression. We found that the promoters of 2 related CAMs restricted to the nervous system, Ng-CAM and L1, contain a 21-bp negative regulatory element known as the neural restrictive silencer element (NRSE). The NRSE and its binding factor, REST/NRSF, silence the expression of Ng-CAM and L1 in nonneural tissues.

Early in development, NRSF/REST is expressed in nonneural cells and silences the expression of neuronally expressed genes. During postnatal development, it is expressed in neurons during the excitation of neurons and the refinement of their connections, suggesting that the gene for NRSF/REST may be regulated by neuronal plasticity. To investigate factors that regulate these 2 contexts of NRSF/REST expression, we identified 3 separate promoters, 6 enhancer regions, and 2 repressor regions within the mouse gene for NRSF/REST. The ultimate goal of these studies is to connect neurotrophin signaling, transcriptional regulation by NRSF/REST, and the control of CAM expression during neuronal plasticity.

Our studies of the transcriptional regulation of CAMs led us to a fundamental analysis of the organization of promoter activity. We designed a set of methods to create, select, and vary the order of promoter elements in order to examine the activity of the elements in various mammalian cells. For example, we recently developed a method for isolating highly active synthetic promoters from a pool of random double-stranded 18mer oligonucleotide segments that are linked to a minimal promoter. Using fluorescence-activated cell sorting in conjunction with the new technique, synthetic promoter construction method (SPCM), we identified 133 synthetic promoters active in the neuroblastoma cell line Neuro2A. A major element of the analysis was a software package called RIGHT that was developed in collaboration with G. Reeke, the Neurosciences Institute, San Diego.

Analyses of the synthetic promoters revealed a predominance of 8 known components of promoters and enhancers in eukaryotic genomes: AP2, CEBP, GRE, E-box, ETS, CREB, AP1, and SP1/MAZ. Up to 10% of the 133 active synthetic promoters had no match in currently available databases, suggesting that this 10% may be new DNA regulatory sequence motifs.

On the basis of these initial promising results, we are modifying SPCM in order to identify endogenous promoters and enhancers in actual genomic DNA. Using these modifications to SPCM, we hope to identify both synthetic and naturally occurring promoters and enhancers that function in different differentiated cells. These and other applications of SPCM open up a new horizon for investigating the anatomy and specificity of promoters and should enhance our understanding of the mechanisms of eukaryotic transcriptional regulation during development and cell differentiation.

In addition to gene transcription, cell differentiation during development can be influenced by controlling the translation of mRNA. For many eukaryotic genes, mRNA levels are often poorly correlated with protein expression because events such as translation that occur after transcription are subject to regulation. In earlier studies, we observed that large numbers of eukaryotic mRNAs contain sequences resembling segments contained within the 18S or 28S RNA of the ribosome. We hypothesized that these rRNA-like sequences would interact with ribosomes, and indeed recent experiments indicated that mRNA-rRNA interactions have direct effects on the efficacy of translation.

Our most recent studies address how complementarity between mRNA and rRNA affects initiation of translation. In eukaryotes, initiation is thought to occur primarily by a cap-binding/scanning mechanism. However, evidence also exists for a cap-independent mechanism in which 40S ribosomal subunits are recruited at sequences referred to as internal ribosome entry sites (IRESs).

We noted that cellular IRESs contain numerous segments that are complementary to different regions of the 18S rRNA, suggesting that such IRESs recruit ribosomes by base pairing to 18S rRNA. To test this hypothesis, we examined 2 cellular mRNAs that contain segments with complementarity to 18S rRNA. These mRNAs encode 2 proteins: the Gtx homeodomain protein and Rbm3, a cold stress-induced RNA-binding protein. We showed that both of these RNAs contain IRESs and that both IRESs are composed of numerous smaller sequences that can function independently (so-called IRES modules).

In the case of Gtx, a small (9-nucleotide) segment had IRES activity. The sequence of the segment is 100% complementary to a region of 18S rRNA. We found that increasing the number of linked copies of this IRES module greatly enhanced internal initiation. These findings are important because they allow the construction of synthetic IRESs with powerful effects on translation that may be applicable to gene therapy.

Our ongoing studies indicate that base pairing to the 18S rRNA may be a novel mechanism for regulating translation of some eukaryotic mRNAs. We expect that mRNA-rRNA interactions will have a general role during the initiation of translation by affecting how efficiently mRNAs can compete for the translation machinery of the cell.

Publications

Amoureux, M.C., Cunningham, B.A., Edelman, G.M., Crossin, K.L. N-CAM binding inhibits the proliferation of hippocampal progenitor cells and promotes their differentiation to a neuronal phenotype. J. Neurosci. 20:3631, 2000.

Chappell, S.A., Edelman, G.M., Mauro, V.P. A 9-nt segment of a cellular mRNA can function as an internal ribosome entry site (IRES) and when present in linked multiple copies greatly enhances IRES activity. Proc. Natl. Acad. Sci. U. S. A. 97:1536, 2000.

Edelman, G.M., Meech, R., Owens, G.C., Jones, F.S. Synthetic promoter elements obtained by nucleotide sequence variation and selection for activity. Proc. Natl. Acad. Sci. U. S. A. 97:3038, 2000.

Koenigsberger, C., Chicca, J.J. II, Amoureux, M.C., Edelman, G.M., Jones, F.S. Differential regulation by multiple promoters of the gene encoding the neuron-restrictive silencer factor. Proc. Natl. Acad. Sci. U. S. A. 97:2291, 2000.

Vanderklish, P.W., Krushel, L.A., Holst, B.H., Gally, J.A., Crossin, K.L., Edelman, G.M. Marking synaptic activity in dendritic spines with a calpain substrate exhibiting fluorescence resonance energy transfer. Proc. Natl. Acad Sci. U. S. A. 97:2253, 2000.

 

 







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