REST levels decline during differentiation of embryonic stem cells to neural stem and progenitor cells5
, consistent with a role for REST in restraining neuronal gene expression programs. This decrease results from a 3-fold reduction in REST half-life (), suggesting that a regulatory pathway controls REST degradation during early neural differentiation. To determine whether ubiquitination is involved, REST was evaluated for ubiquitin-modification in vivo
. Immunoprecipitation of HA-ubiquitin revealed slower migrating species of REST suggestive of polyubiquitination (, lane 3). REST also precipitated with an HA-ubiquitin mutant lacking all lysines except K48 (, lane 4), suggesting REST is K48 polyubiquitinated which promotes degradation.
Identification of βTRCP and FBW4 ubiquitin ligases as regulators of REST stability
To search for the E3 ubiquitin ligase for REST, we began with the SCF superfamily of ligases6
. Each SCF family contains a common Cullin scaffold that is required for ligase function. Notably, coexpression of a dominant negative Cullin-1 (Cul1) mutant resulted in a dramatic increase (11-fold) in REST levels (Supp. Fig. 1b
), indicating that one or more Cul1-containing ligases negatively regulate REST abundance.
F-box proteins act as substrate receptors for the SCF7,8
. To determine which F-box proteins are required for REST turnover, we established a system for monitoring REST abundance in a high-throughput manner using an mRFP-REST fusion protein. Similar to endogenous REST, mRFP-REST was unstable, and its abundance increased upon inhibition of Cul-1 (Supp. Fig. 2a
). To identify the F-box proteins regulating REST, individual siRNAs targeting each F-box protein (4 siRNAs/gene) were cotransfected with a plasmid encoding mRFP-REST, and changes in cellular fluorescence were monitored by flow cytometry (Supp. Fig. 2b
). siRNAs that increased fluorescence >2 standard deviations from the mean were retested in triplicate for their effects on both mRFP and mRFP-REST to identify siRNAs that specifically alter REST stability (). This approach identified FBW4 and βTRCP2. Notably, multiple siRNAs targeting additional sequences within FBW4 and βTRCP2 also increased mRFP-REST abundance (), confirming the specificity of the siRNAs. Supporting this conclusion, coexpression of a dominant negative βTRCP mutant (lacking the F-box) also increased REST levels (Supp. Fig. 4a-b
βTRCP2 and FBW4 may control REST abundance by direct ubiquitination of REST or by modulating upstream regulators of REST. βTRCP2, but not FBW4, was capable of binding REST in cells (), suggesting that FBW4-mediated regulation is indirect. The highly homologous βTRCP1 also interacted with REST ( and Supp. Fig. 3a
), consistent with previous reports that βTRCP1 and βTRCP2 have similar substrate specificities and frequently function redundantly9,10
. Importantly, endogenous βTRCP and REST interact in cells (Supp. Fig. 3b
), and REST was polyubiquitinated by SCFβTRCP1 in vitro
(), suggesting that SCFβTRCP
regulates REST by direct ubiquitination. In agreement, stable expression of βTRCP-shRNA (targeting βTRCP1 and βTRCP2) in both human mammary epithelial cells (HMECs) and NIH3T3 cells resulted in a moderate but reproducible increase in REST protein abundance and half-life (, lanes 2 and 3, and Supp. Fig. 4c
), indicating that endogenous REST is regulated by βTRCP. These data indicate that SCFβTRCP
controls REST by ubiquitin-mediated destabilization.
binds substrates in a phosphorylation-dependent manner6,10
. Consistent with this, λ-phosphatase treatment abolished the interaction between REST and βTRCP and this was prevented by λ-phosphatase inhibitors (). Notably, a dominant negative frame-shift mutant of REST found in human colon cancer cells4
failed to interact with βTRCP and exhibited substantially increased stability in cells (Supp. Fig. 6a
), indicating the c-terminal half of REST is required for βTRCP recognition. Analysis of this region revealed a sequence highly similar to the phosphodegron found in Cdc25A, a well documented βTRCP-substrate11,12
(). This putative degron includes a conserved DpSG motif that constitutes a critical interaction element within phosphodegrons for βTRCP15
. Mass spectrometry was used to examine phosphorylation of REST within this region. To enable tryptic digestion of the peptide of interest, a N1022R substitution was introduced into REST that does not alter interaction with βTRCP or protein stability in cells (Supp. Figs. 5a-b
). His-tagged RESTN1022R
was co-expressed with dominant-negative Cul1 in 293T cells and purified under denaturing conditions (Supp. Fig. 5c
). Analysis of phosphopeptides in RESTN1022R
demonstrated that S1027 and S1030 within the MSEGS
GLHGARPVPQESSR peptide are phosphorylated both singly and in combination (Supp. Figs. 5c-g
A conserved phosphodegron in REST is required for regulation by βTRCP
To test the ability of the candidate REST-degron to interact with βTRCP, peptides spanning the degron were synthesized with phosphates at serines 1024, 1027, and 1030 alone or in combination. Individual serine-phosphorylation facilitated weak (S1030) or no interaction (S1024 or S1027) with βTRCP (Supp. Fig. 7
). In contrast, peptides phosphorylated in combination at S1027+S1030 or S1024+S1027+S1030 associated with βTRCP (but not Fbw4) with an efficiency comparable to that of the well-established IκB phosphodegron peptide ( and Supp. Fig. 7
). Mutation of each serine to alanine in the context of full-length REST resulted in decreased binding to βTRCP, and combined mutation of these critical serines completely abrogated the interaction with βTRCP ( and Supp. Fig. 6b
). Notably, degron-mutant REST was substantially more stable than wild-type REST in cells (). These data support the hypothesis that phosphorylation of the REST degron primes ubiquitination by SCFβTRCP
, thereby promoting REST degradation.
The role of βTRCP in degradation of the REST tumor suppressor predicts that βTRCP overproduction might transform human cells. To examine this prediction, HMECs stably expressing human telomerase catalytic subunit (hTERT) and the SV40 LT oncogene (“TLM-HMECs,”16
) were transduced with a control or GFP-βTRCP1-expressing retrovirus. Stable ectopic expression of βTRCP1 resulted in reduced REST abundance () and robust anchorage-independent proliferation (), thus phenocopying REST loss-of-function4
. This is consistent with a transgenic mouse model in which ectopic βTRCP1 expression in the mammary gland produced advanced breast cancer17
. To determine whether REST degradation is critical for βTRCP1-mediated transformation, TLM-HMECs stably expressing βTRCP1 were transduced with retroviruses expressing wild-type or degron-mutant REST. Exogenous REST expression did not alter proliferation on an adhesive cell culture surface (). In contrast, βTRCP1-induced anchorage-independent proliferation was severely impaired by restoring REST expression (). Consistent with its increased stability, degron-defective REST suppressed βTRCP1-transformation more efficiently ( and Supp. Fig. 8
). These data implicate REST as an essential target in βTRCP-driven oncogenic transformation.
βTRCP targets REST during oncogenic transformation
While REST is a well documented regulator of neuronal gene expression and has been proposed to restrain several steps in neurogenesis (reviewed in 3
), its role in neurogenesis has not been tested genetically. Thus, we used embryonic stem (ES) cells to genetically examine the roles of REST and βTRCP in the differentiation program of neural stem and progenitor cells (reviewed in 18
). For this we employed ES cells in which eGFP was recombined into the Sox1 locus19,20
(“46c cells”), a well characterized marker of early neural differentiation in vitro
and in vivo
We first confirmed that endogenous REST stability is regulated during neural differentiation of 46c cells. As shown in , REST half-life declined 2-fold in differentiated cells, consistent with the decreased REST stability observed in homogeneous neural stem cells (). This decrease may be driven, in part, by a concomitant 13-fold increase in βTRCP1 expression (). To test the role of REST and βTRCP in this differentiation program, 46c cells were transfected with control, REST, or βTRCP1 targeting siRNAs alone or in combination, and subsequently cultured in differentiation media and analyzed for neural differentiation by flow cytometric analysis of Sox1:eGFP fluorescence. Inactivation of REST promoted differentiation, correlating with the efficiency of REST knockdown ( and Supp. Figs. 10a-b
), thus providing the first genetic evidence that REST negatively regulates early neural differentiation. Conversely, siRNAs that suppress βTRCP1 expression >90% (Supp. Fig. 11c
) attenuate differentiation into the neural lineage (). These results were confirmed in multiple time points (data not shown) and with multiple siRNAs (Supp. Figs. 10b,d
). Importantly, simultaneous REST+βTRCP1 knockdown increased Sox1:eGFP-positive cells >5-fold relative to βTRCP1-siRNA alone (), showing REST reduction restores neural differentiation in the absence of βTRCP. Similar results were observed by measuring the abundance of an independent neuronal marker, TUBB3 (). Thus, down regulation of REST is a critical function of βTRCP during early neural differentiation.
The βTRCP-REST pathway controls neural differentiation
These data support the model that βTRCP regulates neural differentiation by facilitating REST degradation and predicts that a non-degradable REST would impede neural differentiation. To test this, we first examined the stability of wild-type or degron-mutant REST expressed in the context of neural differentiation. In this experiment, REST transgenes were expressed at levels much lower than endogenous REST to prevent alterations in differentiation kinetics (see below). Notably, the stability of endogenous REST and wild-type exogenous REST decreased similarly during neural differentiation (Supp. Fig. 10e
and ). In contrast, degron-mutant REST was stable regardless of the cellular differentiation status (Supp. Fig. 10e
). To test whether REST stabilization alters neural differentiation, 46c cells were transduced with high-titer retroviruses expressing wild-type or degron-mutant REST, resulting in a 1.5- and 2.6-fold increase in total REST (). Notably, both transgenes attenuated neural differentiation, with the degron-mutant REST eliciting a more dramatic phenotype ().
To further demonstrate REST's role in neural differentiation, we employed an independent neural differentiation assay. ES cells stably expressing wild-type or degron-mutant REST were differentiated by formation of embryoid bodies followed by stimulation with retinoic acid, a protocol routinely used to differentiate ES cells into the neuronal lineage21
. In this context, non-degradable REST suppressed differentiation >5-fold as measured by mRNA expression of Sox1 (). Collectively, these observations strongly link βTRCP function and REST degradation in controlling neural differentiation.
Here we demonstrate that REST is a labile protein targeted for ubiquitin-dependent proteasomal degradation by SCFβTRCP
through a phospho-degron on REST. We show SCFβTRCP
is a critical regulator of both physiologic and pathologic REST activities, constituting a new pathway controlling neural differentiation and cellular transformation (see Supp. Fig. 11
). We provide the first genetic evidence that REST and SCFβTRCP
regulate an early stage in neural specification. Our data are consistent with a model in which developmental cues induce degradation of REST, resulting in the derepression of proneural REST targets. The ability of REST to inhibit terminal differentiation of neurons also predicts that REST may promote proliferative properties in the neuronal lineage when overproduced or inappropriately stabilized. Consistent with this notion, REST is overexpressed in human medulloblastoma and ectopic REST expression in v-myc
-immortalized neural stem cells promotes medulloblastoma formation in mice 22,23
. Thus, the contrasting roles of REST as an oncogene and tumor suppressor are highly dependent on the developmental lineages.
βTRCP is overexpressed and oncogenic in epithelial cancers17,24,25
and we identified REST as a key target in this context. This suggests that pharmacologic inhibition of βTRCP may provide a means to restore REST tumor suppressor function in human cancer. The presence of a phosphodegron motif within REST suggests a role for upstream kinase(s) and/or phosphatase(s) that control REST degradation. We propose a model in which differentiation into the neural state is induced by this yet to be discovered signal transduction cascade that targets REST for degradation by SCFβTRCP
, acting cooperatively with induction of βTRCP expression during neural differentiation. Conversely, hyperactivation of such pathway(s) priming REST degradation may be oncogenic in epithelial tissues and thus serve as new therapeutic targets in cancers with compromised REST function. Thus, exploration of these pathways will likely provide new opportunities for modulating neural stem cell and cancer cell behavior.