Morgan Sheng

The regulation and function of c-fos and other immediate early genes in the nervous system

Morgan Sheng and Michael E. Greenberg Department of Microbiology and Molecular Genetics Harvard Medical School Boston. Massachusetts 02115 Introduction The unique morphological and excitable properties of nerve cells endow them with specialized properties that permit the reception, transmission, and storage of information. It has long been recognized that trans- synaptic signals cause rapid responses in neurons. These occur over a time frame ranging from millise- conds (e.g., opening of ligand-gated channels) to sec- onds and minutes (e.g., second messenger-mediated events). Recent studies, however, have revealed that trans.synaptic activation also elicits slower, long-term responses in neural cells that are correlated with, and in some cases shown to be dependent on, the induc- tion of new programs of gene expression (reviewed in Goelet et al., 1986; Black et al., 1987; Comb et al., 1987; Morgan and Curran, 1988). Neuronal gene ex- pression can be modulated by neurotransmitters, mem- brane electrical activity, and neurotrophic growth fac- tors and is likely to play an important role both in the development and in the adaptive plasticity of the ner- vous system. Many of the long-term consequences of trans-synaptic stimulation may therefore be mediated by changes in gene expression. These include altera- tions in neuronal sprouting or synaptic density and changes in the level of expression of neurotransmit- ters, receptors, and ion channel proteins. In inver- tebrates, the activation of new gene expression in neurons has been shown to be critical for the devel- opment of a learning-related long-term facilitation (Montarolo et al., 1986). Substantial progress has been made over the last 5 years in identifying the genes that are responsive to trans-synaptic stimulation and membrane electrical activity in neural cells. These genes fall into two gen- eral classes: genes whose transcription is activated rapidly and transiently within minutes of stimulation (Greenberg et al., 1985; Morgan and Curran, 1986; Bartel et al., 1989; Barzilai et al., 1989), termed the cel- lular immediate early genes (IEGs), and the late re- sponse genes (Merlie et al., 1984; Castellucci et al., 1988; Goldman et al., 1988; Barzilai et al., 1989; Offord and Catterall, 1989; Klarsfeld et al., 1989), whose ex- pression is induced (or repressed) more slowly, over a time frame of hours, via a mechanism that is gener- ally dependent on new protein synthesis. While the distinction between the IECs and the late response genes is not always clear-cut, it has been proposed that IEGs encode regulatory proteins that control the expression of late response genes. The products of the late response genes are then thought to serve more specific effector functions in the neuronal re- sponse. Recent studies, which are the subject of this review, have provided increasing support for this idea. Many IEGs have been shown to encode transcription factors. By directing specific programs of late gene ex- pression, the induction of these proteins could there- fore mediate many of the long-term responses of the neuron to trans-synaptic signals. What Are Immediate Early Genes? The activation of IECs by extracellular stimuli is not specific to neuronal cells. The IEGs were first charac- terized in nonneuronal cells through efforts to iden- tify growth factor-responsive genes that might con- trol the reentry of Go resting cells into cell cycle. This work resulted in the discovery of a class genes whose transcription is activated within minutes after addition of a growth factor. The c-fos and c-myc proto- oncogenes were among the first IECs to be identified (Kelly et 1983; Greenberg and Ziff, 1984) are prototypic members of this family. c-fos transcription- al activation occurs within a few minutes of growth factor stimulation and precedes the activation of c-myc (Greenberg and Ziff, 1984; Kruijer et al., Muller et al., 1984). The induction of these genes is transient; the level of c-fos transcription is once again undetect- able 30 min after growth factor treatment. The obser- vation that growth factors activate the transcription of two genes whose mutation or deregulated expression can lead to cell transformation suggested that Fos and Myc proteins could have important regulatory func- tions during cell proliferation. Indeed, studies using c-fos anti-sense RNA or anti-c-fos antibodies indicate that the activation of c-fos is critical for reentry of qui- escent fibroblasts into the cell cycle (Nishikura and Murray, 1987; Riabowol et al., 1988). In addition to c-fos and c-myc, a large number of other IEGs have been identified by the differential screening of cDNA libraries from growth factor-stimulated cells (Coch- ran et al., 1983; Lau and Nathans, 1985, 1987; Almen- dral et al., 1988; Lim 1987; Kujubu 1987). The total number of IECs is now thought to be close to 100, though relatively few of these genes have been extensively characterized. In general, IEGs share the following characteristics: their expression is low or undetectable in quiescent cells, but is rapidly induced at the transcriptional level within minutes of extracellular stimulation; this tran- scriptional induction is transient and independent of new protein synthesis; the subsequent shut-off of transcription requires new protein synthesis; the mRNAs transcribed from these genes often have a very short half-life (in the case of c-fos, approximately

Membrane depolarization and calcium induce c-fos transcription via phosphorylation of transcription factor CREB

The mechanism by which the calcium influx signal, triggered by membrane depolarization, is transduced to the nucleus to activate c-fos proto-oncogene transcription has been characterized. A calcium response element (CaRE) that is indistinguishable from a cAMP response element (CRE) mediates transcriptional inducibility by depolarization. Its cognate transcription factor CREB is the target for both calcium and cAMP signals. CREB is rapidly phosphorylated in response to depolarization or cAMP, at a site known to be important for the transcriptional activating function of this protein. The convergent effects of calcium and cAMP on CREB activation are mediated by distinct protein kinase signaling pathways. CREB and its binding site, the Ca/CRE, can thus function as a regulatory element that integrates both calcium and cAMP signals in the control of gene expression.

Calcium and growth factor pathways of c-fos transcriptional activation require distinct upstream regulatory sequences.

Transcription of the c-fos proto-oncogene is rapidly induced in the rat pheochromocytoma PC12 cell line by a wide variety of stimuli, including polypeptide growth factors, phorbol esters, and calcium ion fluxes. We have mapped the upstream sequence requirements for this activation in PC12 cells by analysis of promoter deletion mutants in a transient expression assay. Two distinct pathways of c-fos induction are defined that differ in their requirement for cis-acting DNA sequences. Calcium activation of c-fos transcription is dependent on a DNA element located approximately 60 base pairs upstream of the transcription start site. This region is highly conserved between human, mouse, and chicken c-fos genes and contains a sequence that resembles the consensus for a cyclic AMP response element. The dyad symmetry element at position -300, which is necessary for serum responsiveness of c-fos, appears to be unimportant for calcium activation of the gene. The dyad symmetry element is, however, an essential cis-acting sequence for c-fos inducibility by nerve growth factor, epidermal growth factor, fibroblast growth factor, and the phorbol ester 12-O-tetradecanoyl phorbol-13-acetate. Studies in vivo and in vitro with various mutants of the dyad symmetry element indicate that c-fos activation by polypeptide growth factors and 12-O-tetradecanoyl activation by polypeptide growth factors and 12-O-tetradecanoyl phorbol-13-acetate is mediated by a common transcription factor, and that this factor is identical to the previously described serum response factor. In vitro DNA-binding assays suggest that the quantity of serum response factor-binding activity remains unchanged during c-fos transcriptional activation.

Characterization of the Shank Family of Synaptic Proteins MULTIPLE GENES, ALTERNATIVE SPLICING, AND DIFFERENTIAL EXPRESSION IN BRAIN AND DEVELOPMENT

Shank1, Shank2, and Shank3 constitute a family of proteins that may function as molecular scaffolds in the postsynaptic density (PSD). Shank directly interacts with GKAP and Homer, thus potentially bridging the N-methyl-d-aspartate receptor-PSD-95-GKAP complex and the mGluR-Homer complex in synapses (Naisbitt, S., Kim, E., Tu, J. C., Xiao, B., Sala, S., Valtschanoff, J., Weinberg, R. J., Worley, P. F., and Sheng, M. (1999) Neuron 23, 569–582; Tu, J. C., Xiao, B., Naisbitt, S., Yuan, J. P., Petralia, R. S., Brakeman, P., Doan, A., Aakalu, V. K., Lanahan, A. A., Sheng, M., and Worley, P. F. (1999) Neuron 23, 583–592). Shank contains multiple domains for protein-protein interaction including ankyrin repeats, an SH3 domain, a PSD-95/Dlg/ZO-1 domain, a sterile α motif domain, and a proline-rich region. By characterizingShank cDNA clones and RT-PCR products, we found that there are four sites for alternative splicing in Shank1 and another four sites in Shank2, some of which result in deletion of specific domains of the Shank protein. In addition, the expression of the splice variants is differentially regulated in different regions of rat brain during development. Immunoblot analysis of Shank proteins in rat brain using five different Shank antibodies reveals marked heterogeneity in size (120–240 kDa) and differential spatiotemporal expression. Shank1 immunoreactivity is concentrated at excitatory synaptic sites in adult brain, and the punctate staining of Shank1 is seen in developing rat brains as early as postnatal day 7. These results suggest that alternative splicing in the Shank family may be a mechanism that regulates the molecular structure of Shank and the spectrum of Shank-interacting proteins in the PSDs of adult and developing brain.

Calcium regulation of immediate early gene transcription

Cellular immediate early genes (IEGs) are a class of genes whose transcription is transiently activated within minutes of exposure of cells to a wide range of extracellular stimuli. In mature neurons IEG expression can be triggered by a variety of neutrotransmitters and neurotrophic factors. The IEGs, many of which encode transcription factors, are believed to control the physiological response of the cells to the initial stimulation event by activating secondary programs of gene expression. The mechanism by which membrane depolarization/Ca2+ influx trigger the activation of one IEG, c-fos, has been characterized in PC12 cells. In these cells, the cAMP response element-binding protein (CREB) functions as a Ca2+ regulated transcription factor. In addition, CREB is an in vitro substrate for several Ca2+ calmodulin-dependent protein kinases (CaM kinases). These results suggest a model whereby activation of voltage sensitive Ca2+ channels stimulates CaM kinase activation leading to CREB phosphorylation and c-fos transcriptional activation.

Antibodies in haystacks: how selection strategy influences the outcome of selection from molecular diversity libraries

Antibodies against most antigens can be isolated from high quality phage antibody libraries. However, not all antibodies binding a particular antigen are necessarily found when standard selections are performed. Here we investigate the effect of two different selection strategies on the isolation of antibodies against a number of different antigens, and find that these different strategies tend to select different antibodies, with little overlap between them. This indicates that the full diversity of these libraries is not tapped by a single selection strategy and that each selection strategy imposes different selective criteria in addition to that of antigen binding. To fully exploit such libraries, therefore, many different selection strategies should probably be employed for each antigen. The use of alternative strategies should be considered when selection apparently fails, or when the number of different antibodies recognizing an antigen needs to be maximised. Furthermore, the microtitre selection strategy developed is likely to prove useful in the application of phage antibody libraries to the human genome project, allowing the high throughput selection of antibodies against multiple antigens simultaneously.