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MOLECULAR CELL
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A molecular cell is the fundamental unit of life. It's a tiny, self-contained structure that performs essential functions necessary for the existence of an organism.
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Molecular Cell Molecular CellmiR-211 Inhibits Stress-Dependent chop Expression miR-211 Inhibits Stress-Dependent chop Expression
nucleotide 2,820 of the adjacent) or second exon-intron (nucleo-
tide 46 of exon 2 to 1,561 of the adjacent intron) junctions. In
unstressed cells, no nascent transcript was detected using
either primer set (data not shown). In contrast, following ER
stress, RNA spanning the first exon-intron junction of chop
nascent transcript was precipitated (Figure 4B).
The proximal chop promoter has one perfect miR-211 seed
(site 2) and a second (site 1) with two mismatches (Figure S2C).
Critically, these sites are conserved in chop sequences from
multiple species (human, mouse, hamster). To evaluate the
potential role of these sequences, we utilized a luciferase
reporter, wherein the hamster chop promoter region drives
expression of the luciferase reporter gene; this hamster chop-
reporter has been widely used to dissect regulatory elements
in the chop promoter (Fawcett et al., 1999; Luethy et al., 1990;
Ma et al., 2002; Yoshida et al., 2000). Cotransfection of
miR-211 reduced chop-luciferase 6-fold but had no effect on
the luciferase lacking the chop promoter (Figure 4C). To assess
the role of the putative 211 seed sites, we generated constructs
where site 1 or site 2 was deleted. Deletion of either site abol-
ished miR-211-dependent repression, suggesting direct regula-
tion (Figure 4C). Importantly, mutant luciferase constructs
showed similar expression as that of wild-type, indicating that
seed matches do not fall within key regulatory regions.
We next determined the impact of mutation of these
sequences on ER stress-dependent chop expression. Consis-
tent with site 1 and 2 mediating expression, disruption of site 1
increased stress-dependent expression 2-fold while mutation
of site 2 increased expression nearly 7-fold (Figure 4D). To
further address whether regulation is direct, we generated
a reporter wherein either miR-211 site 1 or 2 was replaced with
Figure 2. MiR-211 Induction Depends on eIF2a Phosphorylation seed matches for let-7, which, like miR-211, has nuclear func-
and Expression of ATF4 tions (Benhamed et al., 2012). Consistent with direct regulation,
(A) qRT-PCR analysis of PERK+/+ (WT), eIF2aA/A, and ATF4/ (KO) MEFs coexpression of let-7 repressed chop expression in a site-
treated with thapsigargin (500 nM) for 5 hr.
specific manner (Figure 4E). As an independent measure, we
(B) HeLa cells were transfected with either empty, ATF4, or Nrf2-encoding
vectors. Cells transfected with empty vector were treated with thapsigargin also generated a chop reporter construct wherein site 1 or site
(500 nM) as a positive control or not treated (negative control). After 24 hr, total 2 was replaced with the sequence matching the scrambled
RNA was isolated and miR-211 levels were assessed by qRT-PCR. sequence of our miR-211 control. The scrambled miR-211
(C) Cells in (B) were also assessed for chop expression by qRT-PCR to confirm repressed chop-luciferase expression in a site-specific manner
functional ATF4. All panels provide values that are the average of at least (Figure S2D).
three independent experiments, and error bars indicate standard deviation
between experimental replicates.
MiR-211 Induces Chromatin Modifications
at the chop Locus
Figure 1. PERK-Dependent Expression of miR-211 S2A and S2B). Therefore, we focused on potential nuclear regu- MiRNA-dependent transcriptional silencing in mammalian cells
(A) Heatmap representation of ER-stress-dependent microRNA expression. lation of chop by miR-211. If miR-211-dependent regulation of has been associated with stalled RNA polymerase elongation
(B) qRT-PCR Analysis of PERK+/+ (WT) and PERK/ (KO) treated with thapsigargin (500 nM) or starved for glucose. Values are the average of at least three chop is direct, we reasoned that we should be able to detect (Kim et al., 2006), increased trimethylation of Lys27 of histone
independent experiments, and error bars indicate standard deviation between experimental replicates.
miRNA-promoter RNA complexes. NIH 3T3 cells transfected 3 (H3K27me3; Kim et al., 2006), and Argonaut recruitment to
(C) Induction of CHOP and eIF2a phosphorylation in response to thapsigargin and glucose starvation.
(D and E) qRT-PCR analysis of trpm1 or pri-211 in wild-type or PERK/ MEFs treated with thapsigargin (500 nM) for the indicated intervals. Values are the
with biotin-fused miR-211 or a scramble control were subjected the promoter (Kim et al., 2006; Verdel et al., 2004). We therefore
average of at least three independent experiments, and error bars indicate standard deviation between experimental replicates. to precipitation using streptavidin beads. Primers spanning evaluated the effect of miR-211 on each of these processes
regions from 619 to 503 or 34 to 135 were used to quantify by chromatin immunoprecipitation (ChIP). Expression of A211
chop promoter (RNA) levels in the pull-down. Chop RNA was en- increased RNA polymerase occupancy following exposure of
ER stress-dependent chop expression (Figure 3E). These results whether regulation is direct. Given the strong affect on chop riched in biotin-211 precipitations relative to mutant 211 (Fig- cells to ER stress for either 5 or 8 hr (Figure 5A) relative to
reveal a relationship between miR-211 expression and CHOP mRNA accumulation by both the antagomir and miR-211 ex- ure 4A). Equivalent amounts of biotin-labeled mut-211 and scramble control. In contrast, expression of the miR-211 sup-
accumulation following ER stress. pression along with the absence of high-relevance seed miR-211 were pulled down as determined by qRT-PCR of pressed RNA polymerase occupancy by 4-fold compared to
matches for miR-211 in the 30 UTR of chop, we anticipated that precipitates (Figure 4A). We surmised that if miR-211 is pulling scrambled RNA and 6-fold compared to A211 after 8 hr of
MiR-211-Dependent Silencing of chop translational regulation through the 30 UTR might not contribute down promoter RNA, it should be able to precipitate nascent ER stress (Figure 5A). The absence of significant suppression
While the above results suggest that miR-211 can regulate chop significantly. Indeed, a luciferase reporter fused with the 30 UTR chop RNA as well. To evaluate this hypothesis, we designed at 5 hr by miR-211 presumably reflects low RNA polymerase
expression during an ER stress response, they do not address of chop failed to respond to thapsigargin or miR-211 (Figures primers, which either span the first (nucleotide 118 of exon 1 to occupancy at that time point.
Molecular Cell 48, 353–364, November 9, 2012 ª2012 Elsevier Inc. 355 356 Molecular Cell 48, 353–364, November 9, 2012 ª2012 Elsevier Inc.
, Molecular Cell Molecular Cell
miR-211 Inhibits Stress-Dependent chop Expression miR-211 Inhibits Stress-Dependent chop Expression
Figure 3. MiR-211 Regulates Stress-Depen-
dent CHOP Expression
(A) The recognition site of miR-211 was cloned in
the 30 UTR of the luciferase gene (211-Luc); empty
vector or 211-Luc was expressed in PERK+/+ or
PERK/ MEFs and treated with thapsigargin.
Error bars represent standard deviation for three
independent experiments.
(B) Scrambled (control) or A211 was introduced
into NIH 3T3 cells, treated with thapsigargin for the
indicated intervals, and levels of CHOP and ATF4
assessed by western.
(C) Scramble (control) or a miR-211 was intro-
duced into NIH 3T3; treated with thapsigargin
for the indicated intervals; and levels of phospho-
PERK, CHOP, and eIF4E assessed by immuno-
blot.
(D) qRT-PCR analysis of chop mRNA in NIH 3T3
cells transfected with A211 and challenged
with thapsigargin. The difference between un-
transfected and A211-transfected cells challenged
with thapsigargin is significant (p = 0.023). Error
bars indicate the standard deviation of three
independent experiments.
(E) qRT-PCR assessment of chop mRNA in cells
challenged with thapsigargin ± miR-211. Values
are the average of at least three independent
experiments, and error bars indicate standard
deviation.
We next determined whether ER stress or miR-211 expression death following exposure of cells to ER stressing agents. Indeed,
was associated with increased deposition of the heterochro- expression of the miR-211 antagomir (A211) resulted in acceler-
matin mark, H3K27me3, as this modification is induced by ated accumulation of cleaved caspase-3 (Figure 6A). Consistent
other miRNAs that have nuclear functions (Kim et al., 2006). with increased caspase-3 cleavage, cells expressing A211 ex-
Transfection of miR-211 enhanced thapsigargin-induced hibited an increase in the kinetics of cell death (Figure 6B). To
H3K27me3 at the chop promoter (Figure 5B). Conversely, evaluate whether chop is a significant target, we compared the
H3K27me3 deposition at the chop promoter in response to ER sensitivity of wild-type versus chop/ MEFs to A211 expression
stress was suppressed by A211 (Figure 5B). In contrast, no and low-dose thapsigargin treatment. A211-transfected wild-
specific increase in H3K9me2 at the chop promoter was type cells exhibited accelerated kinetics of caspase activation
detected following introduction of miR-211 (Figure S3). The and cell death compared to scramble RNA-transfected cells
H3K27 methylation of the chop promoter is specific, as no (Figures 6C and 6D). In contrast, chop/ cells were refractory
such modification was induced by miR-211 at the promoters of to the effects of A211 expression with or without thapsigargin,
neighboring genes, dctn2 or gli1, which lack potential miR- consistent with chop being a primary target in cells exposed to
211-interacting sites (Figure 5C). ER stress.
Finally, we assessed whether Argonaut proteins were re-
cruited to the chop promoter by ChIP analysis. Because suitable MiR-211 Is Overexpressed in Mammary Carcinoma
antisera could not be identified for endogenous Ago proteins, in a PERK-Dependent Manner
HeLa cells were transfected with myc-tagged Ago1 along with Recent work has demonstrated that PERK provides critical Figure 4. MiR-211 Regulates Stress-Dependent CHOP Accumulation through Binding Sites in Promoter
(A) MicroRNA-211 pulls down chop promoter RNA. Biotinylated wild-type miR-211 or control scramble were introduced into NIH 3T3 cells (mut-211). Cells were
either miR-211 or A211. A 3-fold increase in Ago1 recruitment prosurvival signals that permit tumor cells to evade a hostile
processed essentially for chromatin IP, except that RNA:RNA duplexes were collected on streptavidin beads. Precipitation of chop with miR-211 was analyzed by
to the chop promoter was observed following induction of microenvironment. Because miR-211 functions to mediate qRT-PCR using primers scanning region 619 to 503. (Right) Equivalent amounts of biotinylated microRNAs were present in precipitates.
miR-211. Conversely, we observed a 2-fold suppression of PERK-dependent cell survival in cultured cells, we reasoned (B) NIH 3T3 cells transfected with biotinylated microRNAs were stressed for 8 hr and then precipitated with streptavidin beads. Precipitates were analyzed for the
Ago1 recruitment in A211-transfected cells (Figure 5D). We that miR-211 accumulation would likely be apparent in primary presence of RNA spanning the first exon-intron junction (118 exonic–2820 intronic). Data represents the average of three experiments.
were unable to detect Ago1 at neighboring promoters such as tumors. We focused on mammary tumors, given that PERK (C) MiR-211 regulates chop promoter activity through its recognition sites at nucleotide 121 (site 1) and 141 (site 2). NIH 3T3 cells were transfected with either
dctn2 or gli1 (negative data not shown). Collectively, these potentiates mammary tumorigenesis (Bobrovnikova-Marjon a promoterless luciferase reporter, with the wild-type, or with indicated mutant chop promoter (mut1 = site 1; mut2 = site 2). In addition, each transfected
construct was supplemented with or without mimic miR-211. Error bars show values for three independent experiments.
data are consistent with a direct mode of miR-211-dependent et al., 2010). We assessed miR-211 levels in a panel of nine
(D) MiR-211 site deletion mutants in the CHOP promoter abrogates regulation by ER stress. Cells expressing the wild-type chop-luc or the indicated mutants
regulation of chop expression. tumors isolated from mice wherein mammary carcinoma was were challenged with thapsigargin for 8 hr. Values are the average of at least three independent experiments.
driven by MMTV-D1T286A (Lin et al., 2008). This analysis (E) NIH 3T3 cells expressing chop-luc reporter constructs containing let-7 seed matches at either site 1 or site 2 were challenged with thapsigargin following
Suppression of miR-211 Regulates ER Stress- revealed accumulation of miR-211 in tumor tissue relative expression of a let-7 miR where indicated. The error bars provided in each panel indicate standard deviation.
Dependent Apoptosis to normal mammary epithelium (Figure 7A). Because miR-
If chop is a significant and primary target of miR-211, then 211 regulates chop accumulation, we expected an inverse expression of miR-211, we noted a significant inverse correlation (197, 1848, 1967), miR-211 expression were low. We also
antagonizing its function should be associated not only with correlation between miR-211 and chop expression. In tumors with chop expression that reached significance (Figure 7B; p = assessed miR-211 expression in a panel of MMTV-Neu tumors
increased CHOP accumulation but also with accelerated cell 149, 179, 266, 1,846, 1,886, and 355, which exhibited the highest 0.03; pearson coefficient = 0.71). In the remaining three tumors that were derived in mice with either wild-type PERK or from
Molecular Cell 48, 353–364, November 9, 2012 ª2012 Elsevier Inc. 357 358 Molecular Cell 48, 353–364, November 9, 2012 ª2012 Elsevier Inc.
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