The repressor element 1-silencing transcription factor (REST) was first identified as a protein
that binds to a 21-bp DNA sequence element (known as repressor element 1 (RE1)) resulting in
transcriptional repression of the neural-specific genes [Chong et al., 1995; Schoenherr and
Anderson, 1995]. The original proposed role for REST was that of a factor responsible for
restricting neuronal gene expression to the nervous system by silencing expression of these genes
in non-neuronal cells. Although it was initially thought to repress neuronal genes in non-neuronal
cells, the role of REST is complex and tissue dependent.
In this study I investigated any role played by REST in the induction and patterning of
differentiation of SH-SY5Y human neuroblastoma cells exposed to IGF-I. and phorbol 12-
myristate 13-acetate (PMA) To down-regulate REST expression we developed an antisense (AS)
strategy based on the use of phosphorothioate oligonucleotides (ODNs). In order to evaluate
REST mRNA levels, we developed a real-time PCR technique and REST protein levels were
evaluated by western blotting.
Results showed that nuclear REST is increased in SH-SY5Y neuroblastoma cells cultured in SFM
and exposed to IGF-I for 2-days and it then declines in 5-day-treated cells concomitant with a
progressive neurite extension. Also the phorbol ester PMA was able to increase nuclear REST
levels after 3-days treatment concomitant to neuronal differentiation of neuroblastoma cells,
whereas, at later stages, it is down-regulated. Supporting these data, the exposure to PKC
inhibitors (GF10923X and Gö6976) and PMA (16nM) reverted the effects observed with PMA
alone. REST levels were related to morphological differentiation, expression of growth coneassociated
protein 43 (GAP-43; a gene not regulated by REST) and of synapsin I and βIII tubulin
(genes regulated by REST), proteins involved in the early stage of neuronal development. We
observed that differentiation of SH-SY5Y cells by IGF-I and PMA was accompanied by a
significant increase of these neuronal markers, an effect that was concomitant with REST
decrease.
In order to relate the decreased REST expression with a progressive neurite extension, I
investigated any possible involvement of the ubiquitin–proteasome system (UPS), a multienzymatic
pathway which degrades polyubiquinated soluble cytoplasmic proteins [Pickart and
Cohen, 2004]. For this purpose, SH-SY5Y cells are concomitantly exposed to PMA and the
proteasome inhibitor MG132. In SH-SY5Y exposed to PMA and MG 132, we observed an
inverse pattern of expression of synapsin I and β- tubulin III, two neuronal differentiation
markers regulated by REST. Their cytoplasmic levels are reduced when compared to cells
exposed to PMA alone, as a consequence of the increase of REST expression by proteasome
inhibitor. The majority of proteasome substrates identified to date are marked for degradation by
polyubiquitinylation; however, exceptions to this principle, are well documented [Hoyt and
Coffino, 2004]. Interestingly, REST degradation seems to be completely ubiquitin-independent.
The expression pattern of REST could be consistent with the theory that, during early neuronal
differentiation induced by IGF-I and PKC, it may help to repress the expression of several genes
not yet required by the differentiation program and then it declines later. Interestingly, the
observation that REST expression is progressively reduced in parallel with cell proliferation
seems to indicate that the role of this transcription factor could also be related to cell survival or
to counteract apotosis events [Lawinger et al., 2000] although, as shown by AS-ODN
experiments, it does not seem to be directly involved in cell proliferation. Therefore, the decline
of REST expression is a comparatively later event during maturation of neuroroblasts in vitro.
Thus, we propose that REST is regulated by growth factors, like IGF-I, and PKC activators in a
time-dependent manner: it is elevated during early steps of neural induction and could contribute
to down-regulate genes not yet required by the differentiation program while it declines later for
the acquisition of neural phenotypes, concomitantly with a progressive neurite extension. This
later decline is regulated by the proteasome system activation in an ubiquitin-indipendent way
and adds more evidences to the hypothesis that REST down-regulation contributes to
differentiation and arrest of proliferation of neuroblastoma cells.
Finally, the glycosylation pattern of the REST protein was analysed, moving from the observation
that the molecular weight calculated on REST sequence is about 116 kDa but using western
blotting this transcription factor appears to have distinct apparent molecular weight (see Table
1.1): this difference could be explained by post-translational modifications of the proteins, like
glycosylation. In fact recently, several studies underlined the importance of O-glycosylation in
modulating transcriptional silencing, protein phosphorylation, protein degradation by proteasome
and protein–protein interactions [Julenius et al., 2005; Zachara and Hart, 2006].
Deglycosilating analysis showed that REST protein in SH-SY5Y and HEK293 cells is Oglycosylated
and not N-glycosylated. Moreover, using several combination of deglycosilating
enzymes it is possible to hypothesize the presence of Gal-β(1-3)-GalNAc residues on the
endogenous REST, while β(1-4)-linked galactose residues may be present on recombinant REST
protein expressed in HEK293 cells. However, the O-glycosylation process produces an immense
multiplicity of chemical structures and monosaccharides must be sequentially hydrolyzed by a
series of exoglycosidase. Further experiments are needed to characterize all the post-translational
modification of the transcription factor REST
