An SREBP-Responsive microRNA Operon
Contributes to a Regulatory Loop
for Intracellular Lipid Homeostasis
Tae-Il Jeon,1,3,6Ryan M. Esquejo,1,6Manuel Roqueta-Rivera,1Peter E. Phelan,1Young-Ah Moon,4
Subramaniam S. Govindarajan,2Christine C. Esau,5and Timothy F. Osborne1,*
1Metabolic Signaling and Disease Program and Diabetes and Obesity Center
2Analytical Genomics Core Facility
Sanford-Burnham Medical Research Institute, Orlando, Florida 32827, USA
3Department of Animal Science, College of Agriculture & Life Science, Chonnam National University, Gwanju 500-757, South Korea
4Department of Molecular Genetics, University of Texas Southwestern Medical Center, Dallas, Texas 75235, USA
5Regulus Therapeutics, San Diego, California 92121, USA
6These authors contributed equally to this work
Sterol regulatory element-binding proteins (SREBPs)
have evolved as a focal point for linking lipid synthe-
sis with other pathways that regulate cell growth and
survival. Here, we have uncovered a polycistrionic
microRNA (miRNA) locus that is activated directly
by SREBP-2. Two of the encoded miRNAs, miR-182
and miR-96, negatively regulate the expression of
Fbxw7 and Insig-2, respectively, and both are known
to negatively affect nuclear SREBP accumulation.
Direct manipulation of this miRNA pathway alters
nuclear SREBP levels and endogenous lipid synthe-
sis. Thus, we have uncovered a mechanism for the
regulation of intracellular lipid metabolism mediated
by the concerted action of a pair of miRNAs that
are expressed from the same SREBP-2-regulated
miRNA locus, and each targets a different protein
of the multistep pathway that regulates SREBP func-
tion. These studies reveal an miRNA ‘‘operon’’ analo-
gous to the classic model for genetic control in
bacterial regulatory systems.
lian cells, and both are critical membrane components that are
continuously required for maintaining cell integrity and support-
ing optimal growth. These lipids are also utilized for more
specialized roles that rely on their unique physical properties to
influence diverse biological processes. Over the last several de-
cades, major advances in understanding the regulation of lipid
metabolism that have been fueled by parallel advances in cell,
molecular, and genomic sciences have occurred, and these
advances continue to revolutionize biomedical research. The
pioneering studies from Brown and Goldstein (2009) have pro-
vided many of the elegant advances in cell cholesterol (Ch) regu-
lation, including the discovery of a pathway for Ch uptake
through the low-density lipoprotein (LDL) receptor, which is
regulated in balance with an endogenous Ch production
pathway centered on two endoplasmic reticulum (ER) mem-
brane proteins, HMG CoA reductase and sterol regulatory
element-binding proteins (SREBPs).
Mammalian SREBPs regulate the genes of both Ch and fatty
acid metabolism, and recent studies have shown that they link
lipid metabolism to cell growth and survival through the direct
activation of additional key target genes of other cellular pro-
cesses (Jeon and Osborne, 2012). Synthesized as ?125 KDa
precursors, SREBPs are composed of an amino-terminal tran-
scription factor domain connected to a membrane localization
regulation domain. Two closely spaced membrane hydrophobic
forms a complex with the SREBP cleavage-activating protein
(SCAP) (Sakai et al., 1997). ER localized SCAP interacts with a
third ER membrane protein called INSIG, and the SCAP-INSIG
association effectively anchors the precursor SREBP in the ER
ditions arise where increased nuclear SREBP levels are required
(Jeon and Osborne, 2012), key signaling pathways decrease the
SCAP-INSIG interaction. Then, the COPII trafficking system es-
corts the SCAP-SREBP complex to the Golgi apparatus where
two resident proteases sequentially cleave the SREBP precur-
sor, leaving the membrane anchor linked to the Golgi membrane
and releasing the mature soluble SREBP transcription factor that
is rapidly targeted to the nucleus (Sun et al., 2007).
INSIG proteins also interact directly with the ER membrane-
localized HMG CoA reductase enzyme, which catalyzes a key
early step in the endogenous synthesis pathway for Ch (Sever
et al., 2003). INSIG directs HMG CoA reductase into a proteoso-
mal degradation pathway so that, when new Ch synthesis is
required, the INSIG-reductase interaction is disfavored, leading
to a rapid increase in Ch synthesis. Thus, the connection
between the rapid regulation of Ch biosynthesis through the
stabilization of HMG CoA reductase with the slow-to-develop
mechanism through SREBP-dependent activation of gene
expression is coordinately integrated through protein-protein
interactions with INSIGs.
Cell Metabolism 18, 51–61, July 2, 2013 ª2013 Elsevier Inc. 51
Once in the nucleus, SREBPs activate the expression of many
the LDL receptor and HMG CoA reductase. Recent advances in
genomic technologies have allowed the comprehensive interro-
gation of transcription factors at a genome-wide level, and, for
SREBPs, these studies have definitively shown their direct roles
set of generic transcription factor partners (Reed et al., 2008;
Seo et al., 2009; Seo et al., 2011). Additionally, these global
studies have also provided evidence of a broader role for
SREBPs in physiology and metabolism (Seo et al., 2011).
Genome-wide analyses of RNA transcription patterns have
uncovered an extensive network of noncoding RNAs, including
small microRNAs (miRNAs) (Bartel, 2004). miRNAs are pro-
cessed from longer transcripts into mature ?22- to 24-nucleo-
tide single-stranded RNAs. They are incorporated into RNA
protein complexes and decrease messenger RNA (mRNA)
stability and/or translation efficiency of target genes through
base pair interactions between the miRNA and target mRNA.
Recent estimates suggest there are approximately 1,000
miRNAs peppered throughout the mammalian genome, of which
approximately half are encoded from their own transcriptional
regions of primary host protein coding mRNAs (Small and Olson,
2011). Because the embedded miRNAs are processed from the
host primary transcript, the expression of the miRNA is depen-
dent on the same transcriptional regulatory mechanisms that
govern the expression of the host gene. In contrast, the nonem-
bedded miRNAs are uniquely expressed through regulatory in-
teractions that specifically target their own promoters.
Recent studies have uncovered a pair of miRNAs, miR-33a
and miR-33b, that are encoded within introns of the human
SREBF-2 and SREBF-1 genes, respectively (Gerin et al., 2010;
Horie et al., 2010; Marquart et al., 2010; Najafi-Shoushtari
et al., 2010; Rayner et al., 2010). However, only miR-33a is
conserved in the mouse genome. These two miRNAs have iden-
tical seed regions and, therefore, are predicted to inhibit expres-
sion from many of the same genes. One conserved miR-33
target gene encodes the ABCA1 transporter, which plays an
important role in modulating intracellular Ch metabolism by
effluxing free Ch to extracellular Ch carriers such as HDL. This
pathway plays a key role in regulating reverse Ch transport
from macrophages and is also part of the interactive mechanism
for controlling intracellular Ch balance in many other cell
types (Ferna ´ndez-Hernando and Moore, 2011; Rottiers et al.,
in miR-33a and SREBP-2 provides two complementary mecha-
nisms whereby increased SREBP-2 transcription increases
The SREBF-2 gene is autoregulated, and the magnitude of
autoinduction is relatively mild at 2- to 3-fold, which is similar
to the magnitude for miR-33a induction by sterol depletion in
macrophages (Rayner et al., 2010). In contrast, other target
genes show much more robust induction by SREBP (Horton
et al., 2003; Yokoyama et al., 1993). Because intracellular Ch
levels are tightly controlled (Goldstein and Brown, 1990), we
reasoned that additional miRNAs might be involved in regulating
intracellular Ch, possibly being more robustly activated by
SREBPs than miR-33 and having unique target genes involved
in the multistep SREBP regulatory pathway. In the current study,
we performed a genome-wide analysis searching for miRNAs
that are differentially expressed in the livers of mice fed a normal
plemented with a combination of lovastatin plus ezetimibe (LE).
Variations of this dietary regimen have been used for three de-
cades to analyze the hepatic regulatory pathway for Ch meta-
bolism (Bennett et al., 2008; Liscum et al., 1983; Seo et al.,
2011; Sheng et al., 1995). The LE combination inhibits both
endogenous Ch synthesis and dietary absorption of Ch, and,
when combined with the Ch-supplemented group, the diets
represent homeostatic extremes for hepatic Ch overload versus
We identified 30 differentially expressed miRNAs, 21 that
were expressed at higher levels in the LE-supplemented group,
and 9 that were more abundant in the Ch-fed sample. At the
extreme, miR-182 was expressed at 80-fold higher levels in
the LE versus the Ch group. miR-182, along with its two miRNA
siblings, miR-96 and miR-183, is expressed from a unique pri-
mary transcript (Xu et al., 2007) at an miRNA locus on mouse
chromosome 6, and we show that the promoter for this locus
is a direct target for SREBP activation. We also demonstrate
that miR-182 and miR-96 negatively regulate the expression of
Fbxw7 and Insig-2, respectively; two proteins that are known
to negatively influence the levels of nuclear SREBPs. Further-
more, we show that this regulatory pathway is conserved in
human cells and that the direct manipulation of miR-182 and
miR-96 expression leads to changes in nuclear SREBPs as
well as alterations in endogenous lipid synthesis. Thus, we
have uncovered a mechanism for the regulation of intracellular
Ch metabolism mediated by the concerted action of a pair of
miRNAs. Importantly, both miRNAs are expressed from the
same SREBP-2-regulated miRNA transcription unit, and each
miRNA targets a different protein in the multistep pathway that
regulates SREBP action. Thus, this regulatory system is analo-
gous to the classic operon mechanism for genetic regulation in
bacterial systems where gene products that function together
in a common biological pathway are coordinately expressed
from the same primary transcript and from a single promoter
that is regulated by the biological pathway associated with the
operon (Jacob and Monod, 1961).
investigated in rodent models by combining dietary manipula-
tions with statin supplementation for over three decades (Ben-
nett et al., 2008; Liscum et al., 1983; Seo et al., 2011; Sheng
et al., 1995). As novel methods, reagents, and molecular path-
programs have been very useful in applying new principles
to further understand the regulatory mechanism for Ch meta-
bolism. For example, we recently uncovered an unexpected
connection between autophagy and Ch regulation by combining
a chromatin immunoprecipitation (ChIP) sequencing approach
for the genome-wide localization of SREBP-2 in hepatic chro-
matin from mice fed a chow diet supplemented with LE to inhibit
hepatic Ch synthesis and limit dietary absorption of Ch (Seo
et al., 2011). In the current studies, we have used this dietary
An miRNA Operon Regulates Mammalian SREBP Pathway
52 Cell Metabolism 18, 51–61, July 2, 2013 ª2013 Elsevier Inc.
comparison to analyze differences in expression for the individ-
Identification of 30 Ch-Regulated miRNAs in the Mouse
We fed mice a regular chow diet supplemented with either
patic miRNAs from each group using a mouse tiling low-density
PCR array (TLDA). A heat map for the data is presented in Fig-
ure S1 (available online) along with control measurements
showing the LE-diet-induced expression of HMG CoA reductase
mRNA and protein as well as the mature nuclear form of
SREBP-2. The heat map emphasizes that there is a range in
expression for individual miRNAs that includes some that are
highly induced by the LE diet and some that were expressed at
higher levels in the Ch-supplemented samples. In further
analyzing the data, we focused on miRNAs that were expressed
with a Ct value % 35 with a differential expression of 2-fold or
There were 30 miRNAs that met these stringent criteria, and they
ranged from miR-182, which was expressed at 80-fold higher
levels in the LE-treated livers, to miR-455, which was expressed
with the TLDA kit do not accurately measure the expression of
miR-33a, which is encoded within the Srebf-2 host gene and is
known to be autoregulated. However, this analysis did identify
the complementary strand miRNA miR-33* as being increased
2- to 3-fold by LE supplementation.
miR-182 is expressed from a miRNA island locus on mouse
chromosome 6 that also encodes miR-96 and miR-183. Interest-
ingly, all three miRNAs are transcribed from a single promoter
(Chien et al., 2011) and are part of the same primary transcript
(Xu et al., 2007) (Figure 1, bottom). It was previously shown
that the expression of this miRNA locus is activated during T
helper cell clonal expansion (Stittrich et al., 2010) and light-
dark transition in the retina (Krol et al., 2010). Interestingly, in
both cases, miR-182 was more robustly expressed than the
To begin to evaluate the potential role for these miRNAs in regu-
lating hepatic Ch metabolism, we analyzed the expression of
each one separately with specific quantitative PCR (qPCR) ana-
lyses, and all three miRNA siblings were robustly induced by LE
treatment (Figure 1A). We also measured miR-33 directly and
confirmed that it was also induced by LE feeding as expected.
In evaluating the TLDA data, the Ct values for miR-96 and miR-
183 were below the 35-cycle cutoff in the Ch-fed sample and
did not meet the stringent criteria we used to prepare the list in
Table1.Theexpression ofthesethreemiRNAswas alsorobustly
increased in the livers of transgenic mice overexpressing
SREBP-1a or SREBP-2, but not bySREBP-1c (Figure S2). These
results suggest that the miRNA locus may be directly activated
The miR-96/182/183 Locus Is Regulated by SREBP-2
On the basis of the above data, we reasoned that the promoter
lated by SREBP-2. Computational methods have been used to
predict putative miRNA promoters throughout the human
genome by combining sequence analysis with epigenetic signa-
tures and mapping short promoter-proximal RNA transcripts
(Chien et al., 2011). This analysis predicted a putative promoter
for the human miR-96/182/183 cluster. A sequence alignment
with the corresponding region of the mouse genome revealed
a high level of conservation between the two species (Fig-
ure S3A). It is noteworthy that there are conserved putative
binding sites for SREBPs, as well as for other more generic tran-
scription factors, such as Sp-1, NF-Y, and CREB and ATF, that
have been shown to interact with SREBPs for efficient promoter
activation (Osborne and Espenshade, 2009). Additionally, the
expression of these human miRNAs were increased similarly to
miR-33a in RNAs isolated from two different human hepatoma
Table 1. Hepatic miRNA Differential Expression Profiles from
Mice Fed a Normal Chow Diet Supplemented with a Mixture of
Lovastatin plus Ezetimibe versus Cholesterol
miRNAFold Change (LE/Ch)
microRNA expression profiling for total RNAs pooled from six C57BL/6
mouse livers for each feeding condition. Expression was analyzed by
TLDA profiling, as described in the Experimental Procedures. miRNA
expression is displayed as the fold change from the lovastatin plus eze-
tially expressed by R2-fold with a p < 0.05 with a Ct value % 35 are
See also Figure S1.
An miRNA Operon Regulates Mammalian SREBP Pathway
Cell Metabolism 18, 51–61, July 2, 2013 ª2013 Elsevier Inc. 53
cells infected with an adenovirus expressing the mature form of
SREBP-2 (Figure S3B). Thus, the human miR-96/182/183 locus
is also most likely regulated directly by SREBPs.
This putative promoter region is schematically represented as
a gray box in the diagram at the bottom of Figure 1. To determine
whether the predicted SREBP sitesare functional, weperformed
a ChIP study to evaluate SREBP-2 binding in chromatin pre-
pared from the LE versus Ch samples. The results in Figure 1B
demonstrate that SREBP-2 binds to this predicted promoter re-
gion in the LE chromatin (detected by primer pair 1F and 1R in
Figure 1B), but not to the coding regions of the miRNA locus,
which were analyzed as negative controls.
A cartoon diagram of the promoter region from Figure S3 is
presented at the top of Figure 2 highlighting the E box sites,
which are classic recognition elements for bHLH proteins, such
as SREBPs, along with the predicted binding sites for the more
generic transcription factors Sp1 and NF-Y, which are preferen-
tially coenriched in SREBP target promoters (Seo et al., 2011).
Next, we fused the putative promoter DNA from the mouse
genome to luciferase and showed that luciferase expression
was enhanced in a dose-dependent fashion by the cotransfec-
tion of an expression vector encoding the nuclear-targeted
SREBP-2 protein (Figure 2, bottom). When the two predicted
SREBP-binding E box elements were mutated, SREBP-2 activa-
tion was significantly reduced. Along with the ChIP studies,
these results provide compelling evidence that the expression
of the miR-96/182/183 locus is directly regulated by SREBP-2.
miR-96 and miR-182 Decrease the Expression of Insig-2
and Fbxw7, Two Proteins that Negatively Regulate
Nuclear Levels of SREBP-2
miRNAs regulate gene expression through putative base-pair
interactions with target mRNAs as part of the RNA-induced
Figure 2. SREBP-2 Activates the miR-96/182/183 Promoter
The putative promoter region shown in gray at the bottom of Figure 1 was
cloned upstream of luciferase in the control luciferase reporter, as shown and
described in the Experimental Procedures. Key putative transcription-factor-
binding sites that are conserved between mice and humans (Figure S3) are
noted on the diagram of the sequence.Top, there are two Ebox motifs that are
putative SREBPresponseelements,and pointmutationswereengineeredinto
each separately or in combination, as noted by the X. Bottom, wild-type and
the indicated mutant promoters were transfected into human embryonic kid-
ney 293T cells along with increasing amounts of an SREBP-2 expression
vector, as described in the Experimental Procedures. The negative and pos-
itive control promoters analyzed in parallel are shown as pSynTLuc and
pSynSRELuc and are described elsewhere (Dooley et al., 1998). Luciferase
activities were normalized to b-galactosidase that was expressed from an
internal control cotransfected cytomegalovirus b-galactosidase plasmid.
Data are represented as mean ± SEM. See also Figure S3.
Figure 1. The miR-96/182/183 Locus Is Directly Regulated by
(A) qPCR analysis of miR-96, miR-182, and miR-183 in RNA from mice fed
chow (N), chow supplemented with cholesterol (Ch), or chow supplemented
with lovastatin plus ezetimibe (LE). Samples were normalized to sno202 RNA
samples analyzed in parallel. Data are plotted relative to normalized values
from the chow group set at 1.0.
(B) ChIP analysis for SREBP-2 binding in hepatic chromatin from Ch- versus
LE-supplemented mice. The three regions in the miR-96/182/183 locus that
were analyzed for SREBP-2 association are shown by the location of forward
(F) and reverse (R) primer pairs 1, 2, or 3 used for the qPCR analysis as indi-
cated. The thick gray box denotes the putative promoter region for the locus
interrogated by primer pair 1, as discussed in the Results.
Data are represented as mean ± SEM. See also Figure S2.
An miRNA Operon Regulates Mammalian SREBP Pathway
54 Cell Metabolism 18, 51–61, July 2, 2013 ª2013 Elsevier Inc.
silencing complex (RISC) and reduce the expression of the
encoded proteins by inhibiting translation and/or increasing
mRNA degradation (Bartel, 2004). We hypothesized that,
because the miR-96/182/183 locus is directly activated by
SREBP-2, these miRNAs might target proteins involved in the
SREBP regulatory pathway. Thus, the TargetScan program
was used to identify putative target mRNAs for miR-96, miR-
182, and miR-183, and this list was cross-matched for proteins
that are known to be involved in the complex sterol-regulated
SREBP proteolytic maturation pathway, which is partially
diagrammed in Figure 3A. Lists of the highest-scoring putative
gene targets predicted by TargetScan for miR-182 and miR-96
are shown in Table S1 and S2, respectively. This analysis pre-
gets of miR-96 and miR-182, respectively, across several
mammalian species (Figures 3 and S4). In fact, there are two
putative miR-182 sites within the FBXW7 30untranslated region
complex in the ER (Yabe et al., 2002) (Figure 3A), and FBXW7 is
the E3 ubiquitin ligase that targets nuclear SREBPs for proteaso-
mal degradation (Sundqvist et al., 2005) (Figure 3A). Because
INSIGs and FBXW7 both limit the accumulation of nuclear
SREBPs, the elevated expression of miRNAs that target these
proteins would be predicted to increase nuclear SREBP-2,
which is a signature hepatic response of the LE dietary supple-
mentation. To test this prediction, we measured Insig-2 and
Fbxw7 mRNA and protein levels in extracts from Ch- or LE-
treated mice. The results demonstrate that Insig-2 and Fbxw7
protein levels were both significantly lower in the LE- versus
Ch-fed samples (Figure3B).Interestingly, thiswas accompanied
icantly altered. These results are consistent with miR-96 and
miR-182 targeting Insig-2 and Fbxw7, respectively.
Additional studies were focused on Fbxw7, given that the Ct
value for miR-182 suggested that it was more highly expressed
after LE treatment than miR-96 and miR-183 in the liver. We
reasoned that, if miR-182 repression of Fbxw7 was crucial for
increasing SREBP-2 levels in response to LE feeding, then the
addition of Ch after LE induction would coordinately suppress
SREBP-2 and miR-182 while reciprocally increasing the expres-
sion of Fbxw7 back to the level observed in control animals. The
resultsin Figures 4Aand 4Bshow thattheincrease inexpression
of both SREBP-2 and miR-182 in response to LE supplementa-
tion was significantly suppressed after 1 day of Ch supplemen-
tation. Additionally, the low levels of Fbxw7 protein observed
after LE supplementation increased steadily as miR-182 levels
declined over the course of 3 days of Ch feeding. We also
measured the expression of SREBP-2 mRNA along with its
embedded miRNA, miR-33a. As predicted, the expression of
SREBP-2 and miR-33a were induced similarly by LE and sup-
pressed in parallel by Ch addition (Figure S5).
To directly determine whether the increase in miR-182
following LE treatment contributes to the increased nuclear
Figure 3. miR-96 and miR-182 Target Key Proteins of the SREBP
(A) A cartoon depicting key molecules and trafficking of the SREBP maturation
pathway ispresentedalongwiththeputativetarget genes formiR-96and miR-
182 as Insig and Fbxw7, respectively.
(B) Immunoblotting and qPCR analyses for Insig-2 and Fbxw7 in extracts from
the livers of Ch- and LE-treated mice.
Figure 4. Coordinate and Reciprocal Regulation of nSREBP-2 with
miR-182 or Fbxw7, Respectively
(A and B) Mice were fed chow (N) supplemented with Ch, LE, or LE followed by
were analyzed for nSREBP-2, Fbxw7, and control proteins, as indicated by
immunoblotting (A), and results were quantified and plotted as relative ex-
pression (B) along with the relative expression of miR-182 analyzed by qPCR.
(C) Hepatic protein from three individual mice was analyzed as in (A). Where
indicated, a control anti-miRNA (A-miR-Con) or an anti-miRNA designed to
target miR-182 (A-miR-182) were injected (30 mg/kg) on days 3, 4, and 5 of LE
diet supplementation, and mice were sacrificed on day 7.
Data are represented ad mean ± SEM. See also Figure S5.
An miRNA Operon Regulates Mammalian SREBP Pathway
Cell Metabolism 18, 51–61, July 2, 2013 ª2013 Elsevier Inc. 55
accumulation of SREBP-2, we treated mice with LE and injected
them with an anti-miRNA designed to pair with and inactivate
miR-182. The results show that anti-miR-182 treatment blunted
the induction of SREBP-2 by LE treatment (Figures 4C and S6).
The expression of some SREBP target genes, such as SREBP-2
itself, were also reduced (Figure S6), whereas others, such as
Hmgcr, were minimally affected (Figure S6). This is consistent
with many other observations that demonstrate that individual
SREBP target genes are differentially affected by direct changes
in SREBP levels (Osborne and Espenshade, 2009).
Fbxw7 protein levels were only minimally affected by the anti-
miRNA-182 treatment (Figures 4C and S6), suggesting there are
other miR-182 target genes involved in regulating nuclear
SREBP levels. Mice were also injected with anti-miRNAs target-
ing either miR-96 or miR-183, and neither miRNA resulted in a
decrease in nuclear SREBP-2 (Figure S6). Altogether, the results
suggest that miR-182 plays a dominant role in the regulation of
SREBP-2 under these conditions, which is consistent with its
more robust induction relative to the other two miRNAs in
response to the LE diet challenge.
miRNA Regulation of SREBPs through FBXW7 Is
Conserved in Human Cells
To analyze the role of miR-182 in regulating human SREBPs
through FBXW7, we compared the effects of direct small inter-
fering RNA (siRNA) targeting of Fbxw7 to miR-182 on nuclear
SREBP levels in HeLa cells (Figure 5A). Treatment of HeLa cells
with siRNA targeting FBXW7 resulted in an increase in nuclear
SREBP-1, and this was similar to the samples treated with pre-
miR-182. The magnitude of the induction of nuclear SREBP-1
was similar to that obtained by sterol depletion, which is the
classic treatment for inducing nuclear SREBP accumulation in
cultured cells (Brown and Goldstein, 1986, 1999). Interestingly,
although siRNA targeting resulted in parallel reduction in
FBXW7 mRNA and protein, the pre-miR-182 treatment resulted
in a decrease in FBXW7 protein without a change in RNA. This is
consistent with the effects observed for the LE diet treatment on
Fbxw7 levels in mice (Figure 3).
Increase in Nuclear SREBP-2 Mediated by miR-182
Transfection Is Reversed by the Reintroduction of
Ectopic FBXW7 and Is Sensitive to the 30UTR
To directly analyze the effects of miR-182 on FBXW7 protein
expression, we transfected HeLa cells with an expression vector
encoding the full-length SREBP-2 protein, including its carboxy-
terminal membrane-targeting domain and a FLAG epitope tag at
the amino terminus. Where indicated, cells were cotransfected
vector encoding FBXW7 (without its native 30UTR) was added as
shown in Figure S6A. In this study, the increase in nuclear
SREBP-2 that resulted from pre-miR-182 addition was similar
to that observed when cells were depleted of endogenous ste-
rols. Importantly, the effect was reversed when the FBXW7
expression vector was also included, which was consistent
with FBXW7 being the major target of miR-182 for the regulation
of nuclear SREBP levels under these conditions.
To determine whether the effect of ectopic FBXW7 was sensi-
tive to miR-182 targeting the predicted miR-182-binding sites in
the FBXW7 30UTR, we performed two experiments. First, we ex-
pressed FBXW7 protein from a constitutive mRNA that contains
either its natural 30UTR or a mutant version where both predicted
miR-182-targeting sites (Figure S4) were changed to destroy
complementarity (Figure 5B). We also showed that FBXW7 pro-
tein was expressed at similar levels from both constructs in
transfected cells (Figure S6). When we cotransfected a pre-
miR-182 along with the two FBXW7 expression constructs,
only the one with the mutated miR-182-targeted sites was able
to decrease the nuclear SREBP-1. In a separate experiment,
we also inserted the wild-type and mutant FBXW7 mRNA
30UTR regions downstream from the luciferase coding sequence
Figure 5. Regulation of SREBPs by the miR-
182 Locus through FBXW7 Is Conserved in
(A) HeLa cells were transfected with siRNA or
pre-miRNAs as indicated (10 nM, Ambion) in
antibiotic-free medium as described in Materials
and Methods. After 24 hr, the dishes were
switched to DMEM containing 5% lipoprotein-
deficient serum, 12 mg/ml Ch, and 1 mg/ml 25-hy-
droxycholesterol and incubated for 24 hr at 37?C.
Also shown are qPCR for FBXW7 and immuno-
blotting for FBXW7, SREBP-1, and b-actin.
(B) The full-length FBXW7 coding sequence was
cloned downstream of the constitutive RPL10
promoter with the natural FBXW7 30UTR intact.
We also prepared a version where the two pre-
dicted miR-182 targeting sites were mutated to
decrease the predicted complementarity. These
constructs were transfected into HeLa cells in
cultured as described in the Experimental Pro-
cedures and in the figure diagram. Quantitation
from a scanned image of the immunoblot is pre-
sented at the top.
Data are represented as mean ± SD. See also
An miRNA Operon Regulates Mammalian SREBP Pathway
56 Cell Metabolism 18, 51–61, July 2, 2013 ª2013 Elsevier Inc.
ure S6). When these two luciferase expression constructs were
transfected into mammalian cells, luciferase expressed from
the FBXW7 construct containing the native FBXW7 30UTR was
suppressed by the addition of miR-182, whereas luciferase ex-
pressed from the 30UTR mutant construct was not affected.
We also performed a similar experiment to analyze miR-96 tar-
geting of INSIG-2 (Figure S6). In this experiment, the reduction
in luciferase expression in response to miR-96 cotransfection
was abrogated when the bases complementary to the miRNA
seed sequences in the corresponding 30UTRs (Figure S4) were
mutated (Figure S6). Altogether, the results from the cotransfec-
miR-182 directly targets the FBXW7 30UTR and that miR-96
directly targets the INSIG 2 30UTR.
miR-96, miR-182, and miR-183 Regulate Lipid Synthesis
through the Modulation of Nuclear SREBP Levels
The miR-96/182/183 locus is conserved in humans, and
sequence alignment predicts that human INSIG-2 and FBXW7
are also targeted by the corresponding human miRNAs (Fig-
ure S4). To test this prediction, HeLa cells were cultured in the
presence of sterols, where nuclear levels of SREBPs are low
and cells were treated with individual pre-miRNAs correspond-
ing to human miR-96, miR-182, miR-183, or the combination
of all three pre-miRNAs (Figure 6A). Nuclear SREBP-1 was
There was a similar increase in SREBP-2 nuclear accumulation,
and SREBP target genes were stimulated in parallel with
changes in nuclear SREBPs (Figure S7).
To determine whether the regulation of SREBP levels by this
miRNA pathway had a significant physiologic impact on endog-
enous lipid synthesis, we measured the effects of the combina-
tion of all three pre-miRNAs on the synthetic rates for fatty acids
andCh inHeLacells. Thelowlevel ofendogenous lipid synthesis
in sterol-treated HeLa cells was significantly enhanced by the
pre-miRNA combination (Figure 6B).
In classic experiments dating to the middle of the last century,
hepatic Ch synthesis was suppressed when animals were fed
a diet supplemented with excess Ch (Gould, 1951; Langdon
and Bloch, 1953). This first demonstration of end-product
repression in a complex mammalian system in vivo predated
most of the key experiments that defined the fundamental
molecular mechanisms for nutrient sensing in bacteria (Monod
et al., 1963). Since that time, mammalian Ch metabolism has
been an experimentally rich and clinically relevant experimental
system for understanding how the classic regulatory mecha-
nisms for small-molecule sensing have evolved to maintain
homeostasis in a complex and highly integrated multicellular
eukaryotic environment (Brown and Goldstein, 2009).
Using an updated version of the original animal feeding proto-
col, we have uncovered a role for a coordinately expressed clus-
control of intracellular lipid metabolism. In an unbiased screen,
we noticed that levels of miR-96, miR-182, and miR-183 were
dramatically increased in the livers of mice fed a chow diet
Robust Induction of Lipid Biosynthesis
(A) HeLa cells were transfected with the indicated human pre-miRNAsalone or
in combination (10 nM, Ambion), as indicated and cultured in antibiotic-free
medium. After 24 hr, the dishes were switched to DMEM containing 5%
lipoprotein-deficient serum with or without sterol mixture (12 mg/ml Ch and
1mg/ml25-hydroxycholesterol)and incubatedfor24hrat37?Cand harvested.
Western blot analysis for the precursor (P) and nuclear (N) form of SREBP-1
was performed. Fatty acid synthase protein was also analyzed by immuno-
blotting (FASN), and b-actin was measured as a control.
(B) Companion dishes of HeLa cells were treated as in (A), and de novo syn-
thesis of fatty acid and Ch were measured with [14C] acetate incorporation.
Either control miRNA (Con) or a mixture of all three specific miRNAs (miR-96,
miR-182, and miR-183) were added together. The p values for differences
relative to control were *p = 0.035 for fatty acids and **p < 0.0001 for Ch.
Data are represented as mean ± SEM. See also Figure S7.
An miRNA Operon Regulates Mammalian SREBP Pathway
Cell Metabolism 18, 51–61, July 2, 2013 ª2013 Elsevier Inc. 57
supplemented with LE relative to a group fed chow supple-
mented with excess Ch. These three conserved miRNAs are
transcribed together in the same transcription unit to from a
miRNA locus on mouse chromosome 6 and the homologous re-
gion from human chromosome 7 (Chien et al., 2011; Xu et al.,
2007). Nuclear SREBP-2 levels increase dramatically by the LE
feeding protocol, and the expression of this miRNA locus was
also induced along with the known SREBP-2 transcriptional pro-
gram driving lipid accumulation. We also showed that SREBP-2
binds to the promoter for the miRNA locus, providing a mecha-
nism for the LE-dependent induction.
Additional studies demonstrated that the promoter driving the
expression of the miRNA locus encoding miR-96/182/183 is
directly activated by SREBP-2. We also show that miR-96 in-
hibits Insig-2, that miR-182 inhibits Fbxw7, and that both of
these proteins have well-described roles in limiting the accumu-
lation of nuclear SREBPs, as diagrammed in Figure 3A (Sundqv-
ist et al., 2005; Yabe et al., 2002). Insig-2 reduces the proteolytic
activation of the membrane-bound SREBP precursor, and
Fbxw7 is the E3 ubiquitin ligase that targets nuclear SREBPs
for turnover by the proteasome. In fact, the major effect of a he-
hepatic lipid accumulation (Onoyama et al., 2011), which further
emphasizes the importance of Fbxw7 in hepatic lipid accumula-
tion and the SREBP pathway. Additionally, an FBXW7 siRNA
titration experiment showed that a similar change in FBXW7
mediated by miR-182 resulted in an increase in nuclear SREBP
levels in HeLa cells.
Treatment of HeLa cells with pre-miR-183 also increased nu-
involved in regulating SREBP levels. We were initially encour-
aged when a target scan predicted that miR-183 might target
Insig-1 directly. However, we have been unable to confirm this
by direct studies with pre-miR-183 and the INSIG-1 30UTR re-
porter. Unfortunately, there were no other obvious SREBP-
pathway-associated genes within the list of putative miR-183
candidates predicted by TargetScan (data not shown) or addi-
tional prediction programs. Our results strongly suggest that
miR-183 targets akey gene thatregulates nuclear SREBP levels;
however, its identification will require the development of more
robust and accurate methods for identifying miRNA target
genes. Because miRNAs often target several genes in the
same pathway, it is also possible that future studies will identify
additional relevant target genes of miR-96 and miR-182 as well.
The miRNA regulatory pathway described here is conserved
from mice to humans, and we show that the introduction of the
corresponding pre-miRNAs into human cells increases nuclear
levels of both SREBP-1 and SREBP-2. Importantly, this is
accompanied by an increase in the rates of synthesis for fatty
acids and Ch, which are major physiological outcomes for
increased SREBP activity. It is noteworthy that the increase in
lipid synthesis occurs in cells cultured in the presence of excess
sterols, indicating that this miRNA pathway can significantly
affect lipid metabolism in the absence of other signals that in-
crease SREBP activity in responseto lowintracellular sterol con-
ditions. Even though the addition of ectopic miRNAs can drive
significant SREBP accumulation in transfected cells, this miRNA
pathway represents only a portion of the overall multifaceted
mechanism for regulating SREBP levels in response to physio-
logic cues. This is evident from the experiment where Ch was
added to the diets of mice that were pretreated with LE to induce
SREBP-2. In this experiment, SREBP-2 protein levels decline
rapidly and dramatically, whereas the levels of miR-182 decline
more slowly over time.
miR-182 has also been implicated in oncogenesis, and anti-
miRNA targeting of miR-182 decreases hepatic metastasis in a
mouse melanoma model (Huynh et al., 2011). Although compar-
ative microarray analyses showed several putative miR-182
target genes were altered by the anti-miRNA targeting in this
study, the identity of key oncogenic targets were not clearly es-
tablished. On the basis of our studies and the known role of
Fbxw7 in regulating the turnover of cyclins (Koepp et al., 2001)
and oncogenes such as c-Myc and c-Jun (Nateri et al., 2004;
Yada et al., 2004), it is possible that Fbxw7 is an important target
in this liver metastasis model as well.
The hepatic induction of miR-182 by LE treatment was signif-
ential accumulation of miR-182 was also observed when the
locus was activated during the clonal expansion of T helper cells
of miR-182 relative to the others is not clear because all three are
processed from the same initial transcript. However, individual
miRNAs are assembled into an active RISC after a multistep pro-
cessing and assembly pathway, and the mechanistic details are
not fully understood (Bartel, 2004, 2009). Thus, it is likely that the
differential accumulation and loading of specific miRNAs into the
RISC is related to differences in the efficiency of pre-miRNA pro-
cessing and differential complex assembly.
The concentration of miR-182 decreased by approximately
50% from its peak value in the LE treatment group after 1 day
of Ch supplementation, and it declined more over the course
of the experiment. Thus, hepatic miR-182 levels respond more
rapidly than miRNAs in general, which have been reported
to be quite stable and to have an average half-life of approxi-
mately 5 days (Gantier et al., 2011). miR-182 levels also change
quickly during the light-dark transition in the retina (Krol et al.,
2010). Thus, relatively rapid changes in miR-182 levels are
compatible with a significant role in more dynamic metabolic
miRNA regulation often results in modest decreases in target
protein expression. However, singular miRNAs are known to
target several proteins in a common pathway, so, even though
individual changes are modest, the overall effect on pathway
flux can be quite significant (Small and Olson, 2011). Our studies
where two separate miRNAs that are encoded from a common
RNA transcript (Chien et al., 2011; Xu et al., 2007) target different
steps in a pathway that regulates a transcription factor that con-
trolsexpression fromthecorresponding miRNApromoter. Inthis
way, our study reveals a regulatory loop whereby SREBP-2 con-
trols expression from a genetic locus that produces miRNAs that
regulate SREBP activity (Figure S7C). This mechanism is remi-
niscent of the classic ‘‘operon’’ paradigm for coordinate regula-
tion of biological processes in bacterial systems where gene
products that function together in a common pathway are coor-
dinately expressed from one primary transcript and downstream
from one promoter that is regulated by the biological process
associated with the gene products encoded by the operon
An miRNA Operon Regulates Mammalian SREBP Pathway
58 Cell Metabolism 18, 51–61, July 2, 2013 ª2013 Elsevier Inc.
(Jacob and Monod, 1961; Monod et al., 1965). In bacteria, end-
product repression of amino acid operons occurs through a
lation. It is interesting that this common feature is also shared
with the miRNA-SREBP-2 regulatory circuit we describe here
(Figure S7C). Because a prominent feature of the operon mech-
anism of genetic control is a polycistronic mRNA, it was un-
known whether this mechanism was conserved in eukaryotic
organisms where mRNAs are monocistronic and transcription
and translation occur in separate cellular compartments. How-
ever, because miRNAs are encoded in polycistronic units and
the RNAs function as the active gene products, it was formally
possible that miRNA operons might exist in eukaryotic organ-
isms. The SREBP-regulated miRNA operon described here con-
stitutes an example of a true eukaryotic operon.
Overall, this study has uncovered a unique role for miRNA-
grated with the INSIG-SCAP pathway for controlling nuclear
SREBP levels. Several other miRNAs were differentially ex-
pressed in the TLDA array profile from LE versus Ch feeding
groups. Recent studies indicate that SREBPs link lipid meta-
bolism with additional physiologic processes (Jeon and
Osborne, 2012), and it is likely that future studies will reveal
newrolesfortheseothermiRNAsand,perhaps, additional target
genes of the miR-96/182/183 locus in the integrated processes
controlled by SREBP action.
All animal experiments were performed in accordance with accepted stan-
dards of animal welfare and with permission of the Sanford-Burnham Medical
Research Institute atLake Nona InternationalAnimalCare and Use Committee
(protocol 2012-88). We obtained 6-week-old male C57BL/6 mice from the
Jackson Laboratory and maintained them on a chow diet for 1 week with a
12 hr light, 12 hr dark cycle for acclimatization.
For miRNA expression profiling, mice were separated into two groups of six
animals per group and treated as described by Seo et al. (2011). In brief, one
group was fed with normal chow supplemented with Ch (1% w/w) for 10 days,
and another group was fed with chow supplemented with a mixture of lova-
statin (100 mg lovastatin [2.5 tablet equivalents]/100 g chow, w/w; Mylan)
and ezetimibe (from Schering-Plough Pharmaceuticals; 21 mg ezetimibe [2.1
tablet equivalents]/100 g chow, w/w) for 7 days. All mice were sacrificed at 8
a.m. (at the end of the dark cycle) via CO2asphyxiation followed by cervical
dislocation. This basic feeding regimen was used in all experiments, and spe-
cific variations are described in the appropriate figure legends.
For the anti-miRNA experiment, mice were fed Ch or LE as above, and con-
trol or experimental anti-miRNA oligonucleotides were dissolved in 1 3 PBS
and intraperitoneal injected at 30 mg/kg on days 4, 5, and 6 at 8 a.m. (end of
the dark cycle). Mice were sacrificed on day 7 at the end of the dark cycle.
RNA Isolation, qRT-PCR, and miRNA Expression Profiling and
Total RNA was isolated from mouse liver and cultured cells with a mirVana
miRNA Isolation Kit (Ambion). Primer sequences used in this study are pro-
ribosomal protein L32 and human glyceraldehyde 3-phosphate dehydroge-
nase mRNA as a control and calculated by the comparative threshold cycle
method. miRNA expression profiling was carried out with the TaqMan Rodent
Array MicroRNA Card Set v2.0 (Applied Biosystems) in triplicate at the
Sanford-Burnham Genomics Core facility. Then, differential expression was
assessed with the Partek Genomics Suite (Partek). Then, expression levels
for miRNAs were quantified with a TaqMan MicroRNA Assay kit (Applied
Biosystems) with a CFX96 Real-Time PCR Detection System (Bio-Rad).
miRNA expression levels were normalized to sno202 (for mouse) and RNU48
(for human) expression.
Cell Culture and Small RNA Transfection
HeLa cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM)
supplemented with 10% heat-inactivated fetal bovine serum (FBS) and antibi-
otics in an atmosphere of 5% CO2at 37?C. HeLa cells were transfected with
10 nM pre-miRNAs (Ambion) with Lipofectamine RNAiMAX Reagent (Invitro-
gen) or FBXW7 siRNA (Dharmacon) using a Dharmafect 1 reagent. Manipula-
tions were performed according to the manufacturer’s instructions, and cells
were cultured in DMEM with 10% FBS without antibiotics. HeLa cells were
switched to DMEM containing 5% lipoprotein-deficient serum (LPDS,
Sigma-Aldrich) and sterols (12 mg/ml Ch, 1 mg/ml 25-hydroxycholesterol)
24 hr after transfection. Cells were harvested 24 hr later. Where indicated,
plasmids encoding the full-length human SREBP-2 with three copies of the
FLAG epitope (a gift from J. Rutter) or human FBXW7 with a GST tag (Sundqv-
ist et al., 2005) were included in the transfection.
Chromatin preparations for ChIP assays with mouse livers were performed as
previously described (Bennett et al., 2008; Seo et al., 2009). For gene-specific
ChIP, qPCR analysis of SREBP-2 binding to specific gene promoters was per-
parallel, and enrichment was measured by SYBR green incorporation with the
use of a CFX-96 Real-Time PCR Detection System. Analyses were performed
by the standard curve method, and values were normalized relative to a
nontarget control region from the ribosomal L32 gene. The qPCR oligonucle-
otide pairs for the mouse promoters are provided in Figure S7.
De Novo Lipid Biosynthesis Assay
HeLa cells were transfected with pre-miRNAs as described above. Cells were
switched to DMEM containing 5% LPDS with or without sterols for 24 hr. Cells
were incubated in DMEM containing 5% LPDS 24 hr later, with or without ste-
rols, plus 0.5 mM sodium [14C]-acetate for the indicated times up to 3 hr. The
cells were harvested by scraping into 0.5 ml 0.1 N NaOH followed by 0.5 ml
of the lysates as previously described (Horton et al., 1999) and spotted onto
plastic-backed silica gel thin-layer chromatography (TLC) plates (Macherey-
were excised and transferred to scintillation vials containing 10 ml Ultima Gold
XR scintillation fluid (PerkinElmer) for radioactive counting of [14C] and [3H].
The rate of incorporation for the 3 hr time course was linear under all assay
conditions, indicating that the endogenous acetate pool was unaffected by
thesterol manipulation(datanotshown).Alldata foreachsample were normal-
ized to starting protein concentration and extraction efficiency with the internal
[3H]-chloroform and [3H]-oleic acid standards. Data were reported as [14C]-
acetate incorporation per unit mass of protein (nmol/mg protein).
The data are presented as mean ± SEM or mean ± SD, as detailed in the figure
legends. Differences between the means of the individual groups were
assessed by one-way ANOVA with a Dunnet’s multiple comparison test and
a Student’s t test. Differences were considered significant at p < 0.05. The sta-
tistical software package Prism 5.0 (GraphPad) was used for these analyses.
Supplemental Information contains Supplemental Experimental Procedures,
four figures, and two tables can be found with this article online at http://dx.
This work was supported, in part, by a grant from the NHLBI/NIH (HL48044).
Y.-A.M. was supported by HL020948-36. We thank R. Debose-Boyd for
An miRNA Operon Regulates Mammalian SREBP Pathway
Cell Metabolism 18, 51–61, July 2, 2013 ª2013 Elsevier Inc. 59
supplying the HMG CoA reductase and Insig-2 antibodies, J. Rutter for the
FLAG-tagged SREBP-2 vector, and J. Ericsson for the GST-tagged FBXW7
Received: November 26, 2012
Revised: March 13, 2013
Accepted: June 12, 2013
Published: July 2, 2013
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