3.3.5 Molecular mechanism of REST/NRSF action
Biochemical analysis begins to illuminate the mechanism by which such gene regulation may be accomplished. The important observation that REST/NRSF binds the corepressor Sin3 and recruits histone deacetylase (HDAC) into a complex (Naruse et al. 1999; Huang et al. 1999a; Roopra et al. 2000) suggests that REST/NRSF-mediated repression involves histone deacetylation. Indeed, REST/NRSF binding to RE1/NRSE is accompanied by a decrease of histone acetylation around the RE1/NRSE motif and inhibition of histone deacetylation leads to expression of neuron-specific genes in non-neuronal cells. Whereas the corepressor mSin3A/B interacts with the N terminus of REST/NRSF, another co-repressor, CoRest, interacts with the C-terminal repressor domain of REST/NRSF (Andres et al. 1999). Significantly, CoRest is tightly associated with HDAC1/2 and the combination of REST/NRSF, CoRest and HDAC2 may repress the induction of type II sodium channel expression by nerve growth factor in PC12 cells (Ballas et al. 2001). These observations point out the recruitment of histone deacatylating activity by REST/NRSF and the role of corepressor proteins. On the other hand, due to the involvement of different corepressors binding to different repressor domains on REST/NRSF, the possibility arises that the successive recruitment of different corepressor complexes may help to explain dynamic versus stable, long-lasting regulation of neuronal gene expression (Griffith et al. 2001).
3.3.6 Conclusions
Currently, we have no complete picture of the functional role of the NRSE/RE1 motif and its binding factor REST/NRSF in neuron-specific gene expression. It may rather serve as a model to illustrate the complexity of protein–protein and protein–DNA interactions involved in gene regulation.
A plausible hypothesis for the role of REST/NRSF could be an early differentiation of neuronal and non-neuronal lineages and an additional, later action in differentiated neurons. Its role in non-neuronal cells may rest on an interaction with other transcrip-tion factors which differs between different genes and their regulatory regions. This may explain the non-neuronal expression of genes which despite containing the NRSE/RE1 motif are not neuron-specific.
While REST/NRSF has been analysed in vertebrates, tramtrack, another zinc finger transcription factor found in Drosophila represses neuroblast-specific genes and controls glial development (Badenhorst et al. 1996; Badenhorst 2001). This further underscores an early role of zinc finger transcription factors in lineage decisions. The comparison of vertebrate and invertebrate REST/NRSF and tramtrack homologues will show whether there is a conserved interaction or succession of such transcription factors during the early division of neuronal and non-neuronal development.
3.4
The regulation of neuronal gene expression by
for a number of activating promoter elements and the respective binding factors.
These include factors which are available in many different cell types, not only neurons. And, more interestingly, transcription factors expressed specifically in neurons, and, to be more precise, in neuronal subpopulations.
The characterization of Arix/Phox2 transcription factors will be discussed as a pro-totypic example for a neuron population-specific activator regulating the expression of the neurotransmitter synthesizing enzyme dopamine β-hydroxylase (DBH). It is particularly interesting as the characterization went all the way from the determination of critical promoter regions and expression cloning of binding factors to the analysis of effects of overexpression or mutational inactivation in vivo. As a result, a family of transcription factors is described which, across different vertebrate taxa, are involved in the induction of DBH expression as part of a synexpression group of transmitter-synthesizing enzymes.
3.4.1 Dopamine ββ-hydroxylase — expression cloning of a
population-specific homeobox-containing transcription activator Dopamine β-hydroxylase (DBH) is the enzyme required for norepinephrine synthesis in noradrenergic and adrenergic neurons and endocrine cells. Analysing the regulatory elements involved in the specific expression of this transmitter-synthesizing enzyme led to the characterization of a homeobox-containing transcription factor family required for the development of certain peripheral and central neuron populations (Table 3.1).
Expression of a lacZ reporter under the control of 5.8 kb 5′flanking sequence of the human DBH gene in transgenic mice resulted in reporter gene activity in nor-adrenergic and nor-adrenergic neurons in addition to few other sites (Mercer et al. 1991).
In noradrenergic sympathetic ganglia, transgene expression was detectable at around the time when endogenous noradrenergic properties become induced (Kapur et al.
1991). This indicates that the promoter region tested is sufficient to generate a spatial and temporal expression pattern roughly equal to that of endogenous DBH. Analysing promoter fragments after transfection into cell lines demonstrates distinct activating and repressing elements involved in cell type-selective expression of the reporter (Shaskus et al. 1992, 1995). With expression cloning using the activating element DB1, a homeodomain-containing transcription factor called Arix was isolated (Zellmer et al.
1995). Earlier, the same transcription factor was isolated with an expression screen using a fragment of the NCAM promoter and called Phox2a (Valarché et al. 1993).
A closely related family member called Phox2b (Pattyn et al. 1997) has been character-ized and both Arix/Phox2a and 2b are expressed in adrenergic and noradrenergic neu-rons and endocrine cells. The DBH promoter contains multiple Arix/Phox2 binding sites and both Phox2a and 2b activate DBH transcription in vitro (Zellmer et al. 1995;
Kim et al. 1998; Yang et al. 1998). The tightly coupled expression of Phox2 transcrip-tion factors and DBH in differentiating sympathetic neurons strongly suggests that these transcription factors are required for induction of the noradrenergic transmitter phenotype (Ernsberger et al. 2000). This is confirmed by the development of excess
DBH-expressing cells after retrovirally mediated overexpression of Phox2a or 2b in chick embryos (Stanke et al. 1999) and the lack of DBH expression in developing sym-pathetic ganglia of Phox2b mutant mice (Pattyn et al. 1999). In addition, all noradren-ergic centers in the brain are missing in these mice (Pattyn et al. 2000a). In the case of the Phox2a knockout, the noradrenergic cells in the locus coeruleus do not form (Morin et al. 1997).
These studies demonstrate that the homeodomain-containing Phox2 transcription factors can specifically activate DBH expression in noradrenergic and adrenergic neurons. The absence of these cells in the peripheral and the central nervous system of Phox2b mutant mice makes Phox2 transcription factors master regulators of neurons with these transmitter properties. Comparison of different species suggests that this developmental function is conserved throughout different classes of vertebrates (Guo et al. 1999; Ernsberger 2000).
The expression of Phox2 transcription factors also in cholinergic sympathetic neu-rons at later developmental stages (Ernsberger et al. 2000), the induction of cholinergic
Table 3.1 Characterization of Phox2 transcription factors and their role in the regulation of DBH expression
Analysis performed Results obtained
DBH promoter/reporter analysis reproduction of the spatial and temporal in transgenic mice expression pattern of the endogenous gene DBH promoter/reporter analysis characterization of activating and repressing
in cell lines promoter elements
electrophoretic mobility shift analysis of characterization of cell type-specific nuclear an activating DBH enhancer fragment DB1 proteins binding to the DB1 fragment expression cloning with the DB1 characterization of Arix cDNAs
promoter fragment and their identity with Phox 2a
homology screen characterization of Phox 2b
comparison of DBH and Phox 2a and 2b evidence for a role of Phox 2a and 2b in the expression patterns in adult nervous tissue specification of the noradrenergic phenotype comparison of DBH and Phox 2a and 2b evidence for a role of Phox 2a and 2b in the expression patterns during development induction of the noradrenergic phenotype overexpression of Phox 2a and 2b in evidence that Phox 2a and 2b are sufficient precursor cells in the chick embryo to induce noradrenergic differentiation mutational inactivation of the Phox 2b demonstration of the necessity of Phox2b for
gene in mice DBH expression in various types of neurons
analysis of Phox2a mutations in mice demonstration of the necessity of Phox2a for
and zebrafish noradrenergic locus coeruleus neurons
comparison of observations in fish, chick evidence for an evolutionary conserved role of
and mouse embryos Phox 2a and 2b in the development of
noradrenergic neurons
traits upon misexpression of Phox2b (Stanke et al. 1999; Dubreuil et al. 2000), and the lack of autonomic neurons irrespective of transmitter phenotype in Phox2b knockout mice (Pattyn et al. 1999) indicate that these transcription factors have other functions in addition to the regulation of DBH expression. Apart from a role in the development of cholinergic neurons which is not characterized in detail, Phox2 transcription factors are involved in the acquisition of general neuronal properties. Phox2b inactivation leads to disruption of the general neuronal differentiation program in certain popula-tions of motoneurons (Pattyn et al. 2000b). Overexpression of Phox2a and 2b induces the development of ectopic neurons expressing different general neuronal genes such as neurofilament M or synaptotagmin I (Stanke et al. 1999; Patzke et al. 2001). As Phox2a binds to elements in the NCAM promoter (Valarché et al. 1993), the question arises whether corresponding binding elements in other neuronal genes such as those coding for neurofilaments or synaptotagmins may allow direct regulation by Phox2s. Thus, Phox2 transcription factors are not exclusively in charge of regulating a very modular aspect of neuronal differentiation, the expression of DBH. They also contribute to other aspects of differentiation in a restricted set of neuronal precursor populations. The molecular substrate of this coordinated expression of general neuronal properties and subpopulation-specific features remains to be worked out.
3.4.2 Transmitter release-related protein and neurotrophin receptor gene promoters — more evidence for regulation by subpopulation-specific transcription factors
The SNAP-25 gene encodes a protein involved in neurotransmitter release and is expressed in many neuron populations (Oyler et al. 1989). Interestingly, POU domain proteins of the Brn3 subfamily, which are restricted to certain neuronal subpopula-tions (Lillicrop et al. 1992; Xiang et al. 1993; Turner et al. 1994), can regulate SNAP-25 expression Cotransfection of a CAT reporter driven by some 2.1 kb of 5′flanking region of the SNAP-25 gene with a Brn3a expression vector in ND7 cells stimulates reporter gene expression as compared to an expression vector without Brn3a coding sequence (Lakin et al. 1995). In addition to Brn3a, the closely related Brn3c is able to activate this promoter, whereas Brn3b represses activity (Morris et al. 1996). The promoter of the gene encoding the synaptic vesicle protein synapsin I is activated by all three Brn3 factors. The important topic highlighted by these studies is the role of neu-ronal subpopulation-specific transcription factors for the expression of genes not restricted to certain neuronal subpopulations. Evidence for a role of other transcrip-tion factors with restricted expression pattern, neurogenin1, Phox2a and Phox2b, for the regulation of SNAP-25, synaptotagmin I, and neurexin I expression has been obtained after overexpression of the transcription factors in Xenopus and chick embryos (Olson et al. 1998; Patzke et al. 2001). From these studies the question arises whether the expression of widely expressed neuron-specific genes is regulated by different transcription factors in distinct neuronal subpopulations in vivo.
Trk neurotrophin receptors are not expressed as widely as the transmitter release-related proteins but can be detected in a variety of neuron subpopulations. In Brn3a mutant mice, Trk expression is affected in a neuron subpopulation and stage-specific manner resulting in the death of certain populations of sensory neurons such as trigeminal neurons whereas dorsal root ganglion neurons are unaffected (Huang et al. 1999b). The analysis of the trkA/NGF receptor promoter demonstrates a region conserved between mammals and birds that is sufficient to direct reporter gene expres-sion in transgenic mice with the appropriate timing (Ma et al. 2000). Mutation of distinct transcription factor binding motifs in this region demonstrates different binding sites to be required for expression in trigeminal, dorsal root and sympathetic ganglia.
These studies support the crucial role of neuronal subpopulation-specific transcrip-tion factors for neuron-specific genes widely expressed in different neuron popula-tions. As the expression patterns of many neuronal genes do not respect the boundaries of expression of individual subpopulation-specific transcription factors, widely expressed neuron-specific genes may be regulated under the regime of different transcription factors in different neuron populations.
3.4.3 Conclusions
So far, the analysis of activating elements in the regulatory regions of neuron-specific genes has demonstrated transcriptional activation at a level specific for neuronal sub-populations and at a level which is not restricted to neurons at all. What has not been found to date is an activating counterpart to REST, namely a transactivating factor generally expressed in neurons but not non-neuronal cells. Such a factor may still appear with ongoing genomic analysis. If this will not be the case, a combination of neuron-restrictive silencing, general, not neuron-restrictive activation, and neuron subpopulation-specific activation is sufficient to drive the appropriate levels of expres-sion for neuron-specific genes in the different neuron populations.
Importantly, neuron subpopulation-specific activation of gene expression is confirmed in vivo in mutant mice for different classes of homeobox-containing transcription factors and for neuron-specific genes as diverse as those coding for transmitter synthesis enzymes or neurotrophin receptors. These transcription factors belong to different families such as paired homeobox or POU proteins. Certain transcription factors or promoter elements may be important only during restricted periods in a neuron’s lifespan. Consequently, the question arises what developmental cascades of sequential transcription factor action are required for neuronal differentiation and specification.
3.5