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First published online February 6, 2009
doi: 10.1242/10.1242/dev.028548
Research Report |
1 MRC Clinical Sciences Centre, Imperial College School of Medicine, Hammersmith
Hospital Campus, Du Cane Road, London W12 0NN, UK.
2 Departments of Anesthesiology, Psychiatry, Developmental Biology, Washington
University School of Medicine Pain Center, St Louis, MO 63110, USA.
* Authors for correspondence (e-mails: helle.jorgensen{at}csc.mrc.ac.uk; amanda.fisher{at}csc.mrc.ac.uk)
Accepted 5 January 2009
SUMMARY
REST is a transcriptional repressor that targets a group of neuronal genes in non-neuronal cells. In embryonic stem (ES) cells, REST has been implicated in controlling the expression of transcription factor genes that are crucial for lineage determination and for maintaining ES cell potential. Here, we asked whether REST directly regulates neural-specifying genes in mouse ES cells using siRNA-mediated REST knockdown and ES cells that lack functional REST protein as a result of gene targeting. Loss of REST did not affect the expression of any of ten transcription factor genes known to promote neural commitment and did not affect the expression of several microRNAs, including miR-21, a putative REST target in ES cells. REST-deficient ES cells retained the ability to self-renew and to undergo appropriate differentiation towards mesoderm, endoderm and ectoderm lineages upon LIF withdrawal. Genome-wide expression profiling showed that genes that were deregulated in the absence of REST were preferentially expressed in the brain and highly enriched for the presence of canonical REST binding sites (RE1). Chromatin immunoprecipitation studies confirmed these genes as direct targets of REST in ES cells. Collectively, these data show that REST selectively silences a cohort of neuronal genes in ES cells.
Key words: REST (NRSF), Embryonic stem cells, Gene silencing, Neurogenesis
INTRODUCTION
Neural fate specification is controlled by the interplay of transcription
factors and signalling networks that cooperate to establish a temporal and
spatial identity of cells in the nervous system
(Guillemot, 2007
;
Levine and Brivanlou, 2007).
Many aspects of neurogenesis can be recapitulated in vitro using mouse or
human embryonic stem (ES) cell differentiation systems
(Eiraku et al., 2008;
Giadrossi et al., 2007). This
includes the correct induction of proneural bHLH transcription factors such as
MASH1 (ASCL1 - Mouse Genome Informatics), NEUROG1/2 (NGN1/2) and MATH1 (ATOH1)
and of other key transcription factors (e.g. PAX6, SOX1) that are crucial for
neural patterning, commitment and differentiation
(Bertrand et al., 2002). In
undifferentiated ES cells, the genes encoding these factors appear to be
functionally `primed' (reviewed by
Spivakov and Fisher, 2007)
such that phosphorylated RNA polymerase II is bound throughout the promoter
and coding regions but the genes are prevented from being productively
expressed by the action of repressors, including those of the Polycomb group
family (Guenther et al., 2007;
Stock et al., 2007).
The neuronal repressor REST (RE1-silencing transcription factor, also known
as NRSF) has been proposed to negatively regulate lineage-specific gene
expression in undifferentiated ES cells
(Ballas et al., 2005;
Singh et al., 2008). REST is
abundant in ES cells, where its expression is regulated by pluripotency
factors such as OCT4 (POU5F1) and NANOG
(Boyer et al., 2005;
Kim et al., 2008;
Loh et al., 2006). The REST
protein binds in a sequence-specific manner to a 21-bp motif referred to as an
RE1 element (Chong et al.,
1995; Schoenherr and Anderson,
1995). Using the RE1 consensus sequence, REST binding sites have
been predicted computationally (Bruce et
al., 2004; Wu and Xie,
2006), and REST binding close to many genes that are expressed by
mature neurons has been demonstrated by genome-wide chromatin
immunoprecipitation analysis as well as by candidate studies in mouse and
human cells (Johnson et al.,
2007; Johnson et al.,
2008; Otto et al.,
2007; Sun et al.,
2005). Despite this, the function of REST at individual sites in
ES cells remains largely unresolved, as does the question of whether
tissue-specific occupancy of RE1 sites accounts for a selective function for
REST in different cell types (Johnson et
al., 2008; Sun et al.,
2005).
REST was initially described as a transcriptional repressor in non-neuronal
tissues (Chong et al., 1995;
Schoenherr and Anderson,
1995). Subsequent biochemical studies revealed that REST interacts
with several different co-repressors, implying that REST might mediate
transcriptional repression by a variety of distinct mechanisms
(Ballas and Mandel, 2005). A
biological function for REST during embryonic development has been implied
from the analysis of Rest-null mice, in which development appears
largely normal until embryonic day (E) 9, when forebrain malformation becomes
evident and the embryos die (at E9.5-10.5) from unidentified causes
(Chen et al., 1998). In ES
cells, REST has been implicated in diverse functions, including the repression
of lineage-specific genes [e.g. Mash1, Ngn2, brachyury (Bry,
T), Gata4, Sox18, Calb (Calb1)], microRNA genes
(Ballas et al., 2005;
Singh et al., 2008), and in
maintaining the expression of pluripotency genes in undifferentiated ES cells
(Singh et al., 2008). To
clarify the role of REST in ES cells we have used homozygously targeted
Rest mutant ES cells and RNAi-mediated REST knockdown. We show that
lowering REST levels in ES cells results in the derepression of a subset of
neuronal genes that are highly enriched for the canonical RE1 elements and
that directly bind REST protein in wild-type ES cells. By contrast, the
expression of genes crucial for neural determination, or that regulate stem
cell potential, was unaffected in REST-depleted ES cells.
MATERIALS AND METHODS
Cells and antibodies
Wild-type, Rest+/- and Rest-/- ES
cells (see Jorgensen et al.,
2009) were cultured on a layer of mitotically inactivated
embryonic fibroblasts in the presence of LIF (1000 U/ml). Karyotype analysis
of wild-type and Rest-/- ES cells showed normal chromosome
content (2n=40). For embryoid body differentiation,
7x106 cells were plated in non-adherent plates in ES cell
medium without LIF, with or without retinoic acid (5 µM) from day 4.
Wild-type ES cell lines used for RNAi experiments [46C
(Ying et al., 2003) and OS25
(Billon et al., 2002)] were
cultured on gelatinised plates without feeder cells as described
(Jorgensen et al., 2007). For
knockdown by siRNA or shRNA and transfection of Flag-REST, see Figs S1 and S5
in the supplementary material. For the heterokaryon reprogramming assays,
human B lymphocytes were fused with mouse ES cells and analysed as described
(Pereira et al., 2008).
Antibodies used were as follows. For ChIP: anti-IgG (DAKO, Z0259); anti-Histone H3 (Abcam, ab-1791-100); anti-H3K9ac (Upstate/Millipore, 07-352); anti-H3K4me2 (Upstate/Millipore, 07-030); anti-H3K4me3 (Abcam, ab-8580-50); anti-H3K27me3 (Upstate/Millipore, 07-449). For western blot: anti-lamin B (Santa Cruz, C-20 sc-6216); anti-goat-HRP (Santa Cruz, sc-2020); anti-rabbit-HRP (GE Healthcare, NA934V). For ChIP and western blot: anti-REST (Upstate/Millipore, 07-579). For FACS: anti-SSEA1-APC (R&D, FAB2155A); anti-B220-APC (BD Pharmingen, RA 3-6B2); anti-goat-Alexa568 (anti-Oct4 staining; Invitrogen/Molecular Probes, A11057). For western blot and FACS: anti-Oct4 (Santa Cruz, N-19 sc-8628).
Expression analysis
RNA was isolated using the RNeasy Kit (Qiagen, Crawley, West Sussex, UK)
and either reverse transcribed [using SuperScriptIII as recommended by the
manufacturer (Invitrogen)] and analysed by real-time PCR as described
(Azuara et al., 2006;
Jorgensen et al., 2007), or
labelled (using 8 µg RNA with the One-Cycle cDNA Synthesis Kit and IVT
Labelling Kit) and hybridised to Mouse 430 2.0 Arrays (all from Affymetrix).
For analysis of microarray data, see Fig. S4 in the supplementary material;
primer sequences for the RT-PCR analysis are available upon request. To
analyse microRNA levels, RNA was extracted using the mirVANA Kit
(Ambion, Warrington, UK), reverse transcribed and analysed using miRNA assays
as described by the provider (Applied Biosystems, Foster City, CA, USA).
Epigenetic profiling and 3D FISH analysis
The replication timing analysis was carried out as described
(Azuara, 2006).
Three-dimensional (3D) FISH analysis was performed using a BAC probe spanning
the Mash1 locus [RP24-130P7, prepared and labelled as described
(Williams et al., 2006)].
Cells were trypsinised, washed in PBS and left to attach onto
poly-L-lysine-coated coverslips. Fixation, denaturation,
hybridisation and washing were as described
(Brown et al., 1997). After
mounting, nuclei were viewed with a Leica TCS SP5 laser-scanning confocal
microscope fitted with a 63x oil-immersion objective. Optical sections
through the nuclei were captured with a LAS AF 6000 camera every 0.24 µm to
create z-stacks for analysis. The position of Mash1 loci
relative to the nuclear periphery was determined on single focal plane
sections using ImageJ. For each allele, the focal plane where the FISH signal
was most intense was selected for measurements and the distance d=nuclear
centre to FISH signal was divided by the distance r=nuclear centre to
periphery; FISH signals with a d/r-ratio 
0.80 were considered peripheral
(Kosak et al., 2002). Only
nuclei containing two visible Mash1 alleles were scored (36 cells for
REST wild-type, 33 for Rest-/- ES cells, 29 for
undifferentiated 46C ES cells, and 18 for 46C-derived neural stem cells).
Chromatin immunoprecipitation (ChIP) analyses were performed as described
(Azuara et al., 2006) using 100
µg of chromatin per sample. Primer sequences are available upon
request.
RESULTS AND DISCUSSION
ES cells lacking REST appropriately repress neural determinants
Loss of REST function in the mouse results in embryonic death around day 10
of gestation, but does not appear to affect early developmental processes such
as gastrulation and body axis formation
(Chen et al., 1998). To assess
whether REST is required to repress the expression of neural-specifying genes
in pluripotent ES cells, we analysed the mRNA levels of transcription factors
known to promote neural commitment, in ES cells that lack REST.
Fig. 1A shows a comparative
analysis of Sox1, Math1, Mash1, Ngn1, Ngn2, Pax3, Pax6, Pax7, Msx1
and Nkx2-2 gene expression in mouse ES cells homozygous for
a targeted REST allele (Rest-/-) (upper panel) or in
wild-type ES cells in which REST protein levels were substantially reduced by
RNAi-mediated knockdown, relative to matched controls (lower panel and see
Fig. S1A in the supplementary material). As anticipated, undifferentiated ES
cells expressed very low levels of each of these genes as compared with
control tissue (quantitative RT-PCR, see Table S1 in the supplementary
material). In REST-deficient ES cells, expression of neural-specifying genes
was either comparable to that in the wild type (9/10) or slightly reduced
(Ngn1). Similarly, RNAi-mediated REST knockdown did not significantly
enhance the expression of Sox1, Mash1, Math1, Ngn1, Ngn2, Pax3, Pax6,
Pax7, Msx1 or Nkx2-2 in ES cells
(Fig. 1A, lower panel). Two
established REST target genes, Syt4 and Calb
(Ballas et al., 2005), were, by
contrast, consistently upregulated both in Rest-/- ES
cells and following RNAi-mediated REST knockdown
(Fig. 1A), a result that is
consistent with REST-mediated derepression. Collectively, these data suggest
that in ES cells, REST is not required to silence crucial transcription factor
genes known to promote neural commitment.
As REST was previously implicated in the silencing of Mash1 in ES
cells by binding to a putative RE1 element located 49 kb downstream of the
transcription start site (Fig.
1B, top panel) (Ballas et al.,
2005; Wu and Xie,
2006), we examined whether the epigenetic status of the
Mash1 locus was altered in REST-deficient ES cells. In earlier
studies, we showed that the Mash1 locus replicates late in S-phase in
wild-type ES cells, preferentially localises to the nuclear periphery and is
hypoacetylated at the promoter (features that are consistent with a repressed
chromatin state), whereas the locus switches to earlier replication, becomes
acetylated and relocates to the nuclear interior when the Mash1 gene
is productively transcribed upon neural induction
(Williams et al., 2006). As
shown in Fig. 1B, we found that
Mash1 alleles had a similar propensity to localise at the nuclear
periphery in wild-type and REST-deficient ES cells (middle panel), and that
REST-deficiency did not alter the timing of Mash1 locus replication
in ES cells (bottom panel and see Fig. S2 in the supplementary material).
Likewise, we did not detect any differences in the levels of active or
repressive histone modifications at the Mash1 promoter between
REST-deficient and wild-type ES cells (see Fig. S3 in the supplementary
material). These data indicate that REST is required neither to silence nor to
maintain the repressive epigenetic environment of the Mash1 locus in
undifferentiated ES cells.
As regulation of microRNAs has been proposed as an alternative mechanism
underlying REST-mediated gene repression in ES cells
(Singh et al., 2008), we asked
whether the expression of a selected panel of microRNAs was significantly
altered in Rest-/- and Rest+/- ES
cells, as compared with wild-type cells. As shown in
Table 1, expression of
miR-30 and miR-16, two ubiquitously expressed microRNA
species (Landgraf et al.,
2007), was similar in REST-deficient and wild-type ES cells.
Likewise, the brain-specific microRNAs miR-9, miR-124a
(Chen et al., 2007),
miR-152 [upregulated upon ES cell differentiation
(Chen et al., 2007)] and
miR-21 [which has been suggested to be a target of REST in ES cells
(Singh et al., 2008)] were
detected at similar levels in REST-deficient and wild-type ES cells
(Table 1). This analysis does
not, therefore, provide any evidence of a generalised role for REST in
regulating microRNA expression in ES cells.
|
|
), this
was extended to include >60% of all upregulated genes. By contrast, no REST
binding was detected at the negative control provided by the Math1
promoter or at the putative REST binding site downstream of the Mash1
gene (Fig. 2C). Most
differentially expressed genes (84/87) and all RE1-containing genes (25/25)
identified in our analysis were upregulated in the absence of REST. Real-time
RT-PCR analysis confirmed the upregulation of the neuronal REST target genes
Scg3, Stmn3, Celsr3, Syp, Cplx1 and Syt4, and not of the
proneural genes Mash1 and Math1, in REST knockout
(Fig. 2D) and REST knockdown
cells (Fig. 2E). Transfection
experiments in which we reconstituted REST knockout ES cells with Flag-tagged
REST provided additional evidence that REST directly represses Scg3,
Stmn3, Celsr3, Syp, Cplx1 and Syt4
(Fig. 2F and see Fig. S5 in the
supplementary material). The elevated expression of each of these candidate
genes in the absence of REST (Fig.
2F white bars, Rest-/- Vector) was at least
partially reduced by transfection of Flag-REST (blue bars), whereas
Mash1 and Math1 expression remained unaffected.
|
; Kim et al.,
2008; Loh et al.,
2006). Although it has been claimed that REST itself may regulate
the expression of Nanog, Oct4 and Sox2 in ES cells
(Singh et al., 2008), we have
recently shown that normal levels of these transcripts are present in
Rest-/- mutant ES cells and in siREST-transfected ES cell
lines (Jorgensen et al.,
2009). Consistently, REST-deficient ES cells expressed the stem
cell markers OCT4 and SSEA1 (FUT4) (detected by FACS analysis, see Fig. S6 in
the supplementary material) and had similar morphology, growth rate and
colony-forming characteristics as their wild-type counterparts (data not
shown) (Jorgensen et al.,
2009). To investigate whether REST-deficient ES cells were capable
of inducing mesoderm, endoderm and ectoderm lineage differentiation, we
cultured Rest-/- ES cells under conditions that allow the
formation of embryoid bodies. Upon LIF withdrawal, REST-deficient and
wild-type control ES cells showed declining levels of Oct4 (and of
Nanog) transcripts, and a progressive increase in the expression of
neural genes such as Sox1 (and Pax6, Ngn1) that was enhanced
by the addition of retinoic acid (Fig.
3A and see Fig. S7 in the supplementary material). Likewise,
withdrawal of LIF resulted in an upregulation of Bry (and
Mixl1) and Gata4 (and Sox17) in both REST-deficient
and wild-type samples (Fig. 3A
and see Fig. S7 in the supplementary material), reflecting the induction of
mesoderm and endoderm lineages, respectively. These results indicate that
REST-deficient ES cells can appropriately upregulate markers of each of the
three germ layers and suggest that the multi-lineage potential of ES cells is
not critically dependent on the REST repressor.
|
), we compared the reprogramming capacity of
Rest-/- and wild-type ES cells when fused with human B
cells in experimental heterokaryons (Fig.
3B). This assay tests the ability of stem cells to redirect the
fate of differentiated cells (lymphocytes in this example), and successful
reprogramming is indicated by the upregulation of the human genes OCT4,
NANOG, CRIPTO (TDGF1 - HUGO) and DNMT3B as described
previously (Pereira et al.,
2008). Dominant reprogramming of this nature is a feature of
pluripotent stem cell lines and previous studies have shown that mouse ES
cells lacking crucial pluripotency factors, such as OCT4, are unable to
reprogram lymphocytes in heterokaryon assays
(Maherali et al., 2007;
Pereira et al., 2008). By
comparing the kinetics of induction of human pluripotency-associated
transcripts in heterokaryons formed between human B cells and either wild-type
or REST-deficient ES cells (Fig.
3B), we found that REST-deficient ES cells were capable of
dominantly reprogramming lymphocytes to a similar extent as their wild-type
counterparts. These results show that ES cells lacking REST retain the
capacity for multi-lineage differentiation and dominant reprogramming of
somatic cells, two functional properties that are associated with pluripotency
(Jaenisch and Young,
2008).
REST function in ES cells
Here we show that REST directly represses a subset of RE1-containing
neuronal genes in ES cells: in the absence of REST, a cohort of genes
important for the terminal differentiation and function of neuronal cells are
inappropriately expressed. This derepression does not appear to be a
consequence of unscheduled neural differentiation because we found no evidence
that REST regulates the expression of genes encoding any of the transcription
factors thought to be crucial for promoting neural commitment in ES cells.
These include Mash1, a proneural factor that was previously thought
to be a REST target in ES cells (Ballas et
al., 2005), and Ngn2, also purported to be regulated by
REST (Singh et al., 2008).
Recent studies in which a dominant-negative form of REST was used to inhibit
REST function support the idea that only a proportion of RE1-containing genes
are in fact REST-dependent in ES cells
(Johnson et al., 2008), and
many of those identified overlap with target genes defined here. Importantly,
our results show that misexpression of these brain-specific genes (including
Scg3, Cplx1 and Stmn3) by REST-deficient ES cells does not
appear to abrogate stem cell function: REST-deficient ES cells express the
same level of many pluripotency-associated genes [Oct4, Nanog and
others (Jorgensen et al.,
2009)] and display similar functional properties, including
multipotency and reprogramming capacity, as their wild-type counterparts.
Recently, REST ablation has been shown to compromise the generation of neurons
from ES cells through dysregulation of laminin genes
(Sun et al., 2008).
Collectively, these studies argue that REST might be important for the correct
execution of neuronal differentiation programmes, but is not required for
neural commitment per se or for maintaining the multipotent status of ES
cells.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/136/5/715/DC1
Footnotes
We thank Natalie Ryan for excellent technical assistance; Eric O'Connor and Eugene Ng for FACS; Mikhail Spivakov for advice on statistics; Luca Mazzarella for providing reagents; and Maria Dvorkina for advice and suggestions on the manuscript. Hybridisation to Affymetrix arrays was performed by the Clinical Sciences Centre/Imperial College Microarray Centre. This work was funded by the MRC. Deposited in PMC for release after 6 months.
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