News and Publications
The Skaggs Institute for Chemical Biology
Scientific Report 1999-2000
Regulation, Function, and Signaling Mechanisms of Cell Adhesion Molecules
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
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
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.
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,
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.