The expression of myogenic microRNAs indirectly
requires protein arginine methyltransferase
(Prmt)5 but directly requires Prmt4
Chandrashekara Mallappa1, Yu-Jie Hu1, Priscilla Shamulailatpam2, Sookil Tae2, Saı ¨d Sif2
and Anthony N. Imbalzano1,*
1Department of Cell Biology, University of Massachusetts Medical School, 55 Lake Avenue North, Worcester,
MA 01655 and2Department of Molecular and Cellular Biochemistry, The Ohio State University College of
Medicine, 1645 Neil Avenue, Columbus, OH 43210, USA
Received August 3, 2010; Revised September 20, 2010; Accepted September 21, 2010
of muscle development and differentiation. To
better understand the roles of chromatin-modifying
myogenic microRNA expression, we have func-
tionally analyzed two different protein arginine
methyltransferases, Prmt5 and Prmt4, both of
which have previously been implicated in the regu-
lation of myogenic mRNA expression. Both Prmts
are required for myogenic microRNA induction
during differentiation. Prmt5 is indirectly required
due to the necessity of Prmt5 for expression of the
expression of myogenin eliminates Prmt5 depend-
ency. By contrast, Prmt4 binds to the upstream
regulatory regions of myogenic microRNAs and is
required for dimethylation of the Prmt4 substrate,
Deletion of Prmt4 does not alter MyoD binding at
prevents the binding of both myogenin and the
Brg1 ATPase that catalyzes SWI/SNF-dependent
chromatin remodeling, resulting in an inhibition of
post-transcriptionally regulate gene expression and that
are required for organismal development (1–3). They
function by binding to complementary sequences, gener-
ally in the 30untranslated regions of mRNAs, leading to
messenger RNA (mRNA) degradation or inhibition of
translation (4). Considerable effort has been expended to
understand the specific targets and functions of individual
miRNAs; however, the mechanisms by which miRNA ex-
pression is regulated have not been as well studied.
Given the requirement for miRNAs and the miRNA
processing enzymes in most aspects of development and
cell differentiation, we have focused our efforts on under-
standing the induction of developmentally regulated
miRNAs. Cardiac and skeletal muscle development is
marked by the induction and function of several
miRNA molecules. miR-1 inhibits muscle growth and
promotes differentiation by inhibiting the expression of
a Hand family transcriptional activator that promotes
cell growth and proliferation and by targeting histone
deacetylase 4, which is an inhibitor of muscle differenti-
ation (5,6). miR-206 also promotes myogenic differenti-
ation by targeting the DNA polymerase-a, resulting in
DNA synthesis inhibition (7). By contrast, miR-133
promotes growth and inhibits differentiation by targeting
the serum response factor, which is a key activator of
myogenesis (5). Other miRNAs have been implicated in
cardiac and skeletal muscle disease (8–12).
Studies examining the regulation of myogenic miRNAs
in skeletal muscle have indicated a role for known
myogenic transcriptional regulatory proteins. Several
E-boxes are present in the regions upstream of myogenic
miRNA genes and have been shown by chromatin
immunoprecipitation (ChIP) to be occupied by MyoD
and myogenin (13–15). Other studies demonstrated that
the MyoD binding sites and myocyte enhancer factor 2
(Mef2) sites present in myogenic miRNA regulatory se-
quences mediate miRNA expression (6,16). In addition,
the Twist transcriptional regulator was identified as a
regulator of a myogenic miRNAs in Drosophila (17).
More recently, the expression of several myogenic
miRNAs was shown to be dependent upon the chromatin
*To whom correspondence should be addressed. Tel: +1 508 856 1029; Fax: +1 508 856 5612; Email: email@example.com
Published online 14 October 2010Nucleic Acids Research, 2011, Vol. 39, No. 41243–1255
? The Author(s) 2010. Published by Oxford University Press.
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/
by-nc/2.5), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
remodeling function of the Brg1 ATPase that is the cata-
lytic subunit of some SWI/SNF chromatin remodeling
enzymes (13). More detailed analysis of how myogenic
miRNAs expression is regulated is lacking.
Post-translational modifications of histones can alter
nucleosomes, cause conformational changes in chro-
matin structure, lead to recruitment of proteins or
protein complexes and contribute to the regulation of
transcription. The histone-modifying enzymes, together
with ATP-dependent chromatin remodeling enzymes
and transcription factors, cooperate to modulate RNA
polymerase II function. Histone modifiers comprise
a large family of enzymes that add or remove a
variety of moieties from histone proteins. These modifica-
tions include acetylation, deacetylation, methylation,
demethylation, phosphorylation, ubiquitylation, sumoy-
lation, poly(ADP-ribosylation) and biotinylation (18–24).
The requirement for individual enzymes and the interplay
and functional relationships between different enzymes
is an area of intense interest.
Among the histone-modifying enzymes are the protein
arginine methyltransferases (Prmts), which asymmetrically
(type I) or symmetrically (type II) dimethylate arginine
residues in substrate proteins (25). Nine Prmts have been
identified in mammals (26). Prmts have roles in multiple
cellular processes such as regulation of cell signaling,
cytokine production, differentiation, transcription, RNA
Investigation of cellular differentiation, in particular the
differentiation of skeletal muscle, has led to identification
of roles for two Prmts in myogenesis. Prmt5 is a type II
Prmt that dimethylates histone 3 at arginine 8 (H3R8) and
histone H4 at arginine 3 (27,28). The induction of
myogenin requires Prmt5, and H3R8 dimethylation at
the myogenin promoter is dependent upon Prmt5 (21).
methyltransferase 1 (Carm1), and hereafter referred to as
Carm1/Prmt4, asymmetrically dimethylates H3R17 and
H3R26 (29,30). Carm1/Prmt4 binds to genes expressed
at late times of skeletal muscle differentiation and is
required for the expression of these genes (20,31).
Interestingly, both Prmt5 and Carm1/Prmt4 can associate
with SWI/SNF chromatin remodeling enzymes (28,32,33),
suggesting functional links between these different classes
of enzymes. Indeed deficiency of either Prmt5 or Carm1/
Prmt4 leads not only to an absence of arginine
dimethylated histones at target promoters but, in both
cases, also results in an inhibition of SWI/SNF enzyme
binding at target genes. Consequently, the target genes
do not undergo chromatin remodeling and are not tran-
scriptionally induced (20,21).
In the current study, we asked whether Prmt5 and
Carm1/Prmt4 are involved in the induction of myogenic
miRNA expression during differentiation. The results
indicate that while both Prmts are required for myogenic
miRNA expression, the mechanism by which each enzyme
functions is distinct. Prmt5 is indirectly needed for
myogenic miRNA expression via its requirement for
myogenin expression. By contrast, Carm1/Prmt4 binds
to myogenic miRNA regulatory sequences, modifies
histones in these regions, and is required for the binding
of both the Brg1 ATPase of SWI/SNF chromatin re-
modeling enzymes and myogenin. The results demonstrate
a role for multiple Prmts in the induction of myogenic
miRNAs during differentiation and reinforce the idea
remodeling enzymes can cooperate during gene activation.
MATERIALS AND METHODS
Cell culture, retrovirus generation and myogenic
NIH 3T3 cells were derived by passaging cells from a
mouse embryo (strain NIH/Swiss) using a classical 3T3
protocol, resulting in an immortalized mouse embryo
fibroblast (MEF) line (34). These were purchased from
ATCC. Prmt5 antisense cells are NIH 3T3 fibroblasts
that constitutively express a Prmt5 antisense vector.
Clone 12 (C12) was previously described (28). Clone 1
(C1) is another independently derived antisense clone
that has not previously been published but that phenotyp-
ically is identical to previously published Prmt5 antisense
clonal cell lines (21,28). The wild-type and Carm1/Prmt4
null immortalized MEFs were generated by passaging cells
from a mouse embryo (mixed strain TC-1/Black Swiss)
using a classical 3T3 protocol (35). All cell lines were
maintained in Dulbecco’s modiEed Eagle’s medium
(DMEM) supplemented with 10% calf serum, penicillin
and streptomycin. Prmt5 antisense cells were cultured in
the presence of 2.5ug/ml of puromycin. The pBABE-
MyoD retroviral construct (21) was transfected into
BOSC23 cells (36) for generation of retroviral particles,
infection and differentiation as described (21,37). To
induce myogenic differentiation with myogenin and
Mef2D1b, a blasticidin resistant pBABE-myogenin con-
struct and neomycin-resistant pBABE-Mef2D1b construct
were used to generate retrovirus. Cells were infected with
the myogenin encoding virus, subjected to drug selection,
then infected with the Mef2D1b retrovirus, and again sub-
jected to drug selection to ensure efficient double infection
prior to differentiation. Samples for RNA and ChIP were
collected at different time points of differentiation.
RNA isolation, reverse transcription-polymerase chain
reaction and northern blots
Total RNA was isolated from cells using TRIzol
(Invitrogen) reagent according to the manufacturer’s
instructions. Five-hundred nanograms total RNA was
used for reverse transcription reactions to generate
cDNA using superscript III (Invitrogen) reverse tran-
Quantitative polymerase chain reaction (PCR) was per-
formed with SYBR green master mix (ABI) according to
the manufacturer’s protocol using MyoD, Mef2D and
myogenin primers described (39,40). Primers for Acta1
and primary miRNA transcripts were published (13).
Primers for SRF were 50- atgccccatcccttaaaatccctttgg-30
StepOne Plus System. Values for each gene or miRNA
were normalized to levels of EF1alpha mRNA. Primers
1244Nucleic Acids Research, 2011,Vol.39, No. 4
for EF1alpha were described (39). Northern blots were
miR-133a, miR-29a and U6 (13). The probe sequence
for miR-143 was 50-gagctacagtgcttcatctca-30.
Western blots to detect Prmt5, MyoD and Carm1/Prmt4
were performed on whole cell extracts as described (41)
using rabbit polyclonal antisera against Prmt5 (Santa
Cruz sc-22132), MyoD (Santa Cruz sc-32758) and Prmt4
ChIP assays were carried out as previously described (21).
Analysis of immunoprecipitated DNA was performed
by quantitative real-time PCR using SYBR green master
mix (ABI) on an ABI StepOne Plus RT-PCR system.
Primers used for ChIP assays were described (13).
step included rabbit polyclonal antisera or partially
purified antibodies against Prmt5 (32), Carm1/Prmt4
07-214), MyoD (Santa Cruz sc-304), myogenin (Santa
Cruz sc-576) and Brg1 (41). IgG (Millipore 12-370) was
used as a control.
Kinetics of miRNA expression
miRNA-1 and miRNA-133a are skeletal and cardiac
muscle-specific miRNAs. Expression of these miRNAs is
induced during myogenesis (5,6). To further understand
the kinetics of induction of these miRNAs during skeletal
muscle differentiation, we utilized a well-defined culture
model for myogenesis(42).
were differentiated along the skeletal muscle lineage
by introducing a retrovirus encoding MyoD, allowing
the cells to become confluent and inducing differentiation
via exposure to a low serum media. Primary transcripts
were induced by 12h post-differentiation in the MyoD-
differentiated cells, and the expression levels continued
to increasewithtime post-differentiation.
vector infected cells did not express these miRNAs
NIH 3T3 fibroblasts
Prmt5 is indirectly required for the expression of miR-1
Prmt5 and Carm1/Prmt4 play important roles in the regu-
lation of myogenic gene expression and differentiation.
Although Prmt5 is required for the expression of the
myogenin gene, which is expressed at early times
post-differentiation, it is not directly required for activa-
tion of myogenic late genes (20,21). Conversely, Carm1/
Prmt4 is required for the expression of late genes but is
dispensable for the expression of myogenin during
myogenic differentiation (20). To investigate whether
Prmt5 plays a role in the regulation of myogenic
miRNA expression, we differentiated NIH 3T3 and
Prmt5 antisense (AS) cell line clones C12 and C1 following
the introduction of retrovirus encoding MyoD or an
empty vector control. We confirmed the knockdown of
Prmt5 by western blot in both the C1 and C12 Prmt5
AS lines and the equivalent expression of MyoD in the
cells infected with the MyoD retrovirus (Figure 2A). The
expression of miR-1 and miR-133a primary transcripts at
24h post-differentiation was completely inhibited in both
of the Prmt5 AS cell lines, indicating that Prmt5 is
required for the induction of myogenic miRNA expression
To investigate whether the regulation of miRNA
expression by Prmt5 was direct, we performed ChIP for
Prmt5 and dimethylated histone 3 arginine 8 (diMeH3R8),
a Prmt5-mediated histone modification (28), at miRNA
regulatory regions in MyoD-differentiated cells. As previ-
ously reported (21), we found that Prmt5 and diMeH3R8
localized to the myogenin promoter in differentiated cells,
but we could not detect the binding of Prmt5 or
diMeH3R8 on any of the previously characterized
myogenic regulatory regions upstream of miR-1 or
miR-133a (data not shown). This suggests that the
requirement for Prmt5 during myogenic miRNA induc-
tion is indirect. As Prmt5 is directly required for
myogenin expression during differentiation (21), we
hypothesized that the failure
miRNAs was due to the lack of myogenin expression
when Prmt5 levels were reduced. To address this possi-
bility, we took advantage of our previous findings that
these fibroblasts could be differentiated into myotubes in
the absence of MyoD by simultaneously expressing
muscle-specific isoform of Mef2D that cooperates with
myogenin to induce differentiation but that is lacking in
these fibroblasts (39,43,44). If the lack of myogenic
miRNA expression in Prmt5 AS cells is due to the lack
of myogenin, then one would expect that introducing
restore the ability of the differentiating cells to express
the myogenic miRNAs.
NIH 3T3 and C1 Prmt5 AS cells were infected with
retroviruses encoding myogenin and Mef2D1b in a
sequential manner and differentiated. We could detect
the expression of myogenin and Mef2D1b in both the
NIH 3T3 and the C1 cells from the onset of differentiation
throughout the differentiation time course (Figure 3A and
B), demonstrating that each of the cell lines at each of the
myogenin and Mef2D1b. We then tested the expression
observed that all four were robustly expressed in a
(Figure 3C–F). These results demonstrate that loss of
miRNA expression in Prmt5 AS cell lines could be com-
indicating that the Prmt5 requirement for myogenic
miRNA expression is indirect via the induction of
myogenin. In this experiment, we also noted that
miRNA expression was induced earlier when cells were
differentiated by myogenin and Mef2D1b than when
to induce myogenic
Nucleic Acids Research,2011, Vol.39, No. 41245
they were differentiated by MyoD (compare Figures 1 and
3C–F). This observation argues that myogenic miRNAs
may be targets for activation by myogenin because the
onset of myogenic microRNA expression correlated with
the introduction of myogenin. This observation is also
consistent witha previous
myogenin can bind to E boxes upstream of these
myogenic miRNA sequences (14).
report indicating that
Figure 1. Kinetics of myogenic miRNA expression during myogensis. (A–D) qPCR analyses of miR-1-1, miR-1-2, miR-133a-1 and miR-133a-2 primary
transcripts in MyoD-differentiated NIH 3T3 cells. Relative expression was analyzed at the indicated times post-differentiation. The expression level
Figure 2. Myogenic miRNA expression is compromised in Prmt5 antisense cell lines. (A) Immunoblot showing the Prmt5 and MyoD protein levels
in vector- and MyoD-infected NIH 3T3 and Prmt5 antisense cell lines C1 and C12 at the onset of differentiation. The same blot was stripped and
probed with PI3 Kinase (PI3K) antibody as a loading control. (B) qPCR analyses of primary transcripts of miR-1 and miR-133a upon
MyoD-mediated differentiation of NIH 3T3 cells and the Prmt5 antisense cell lines, C1 and C12, along with the empty vector (EV) control.
Expression levels were monitored 24h post-differentiation. The expression of each miRNA in NIH 3T3 cells infected and differentiated with the
empty vector retrovirus was normalized to 1. Results are the average of three independent experiments±standard deviation.
1246 Nucleic Acids Research, 2011,Vol.39, No. 4
Carm1/Prmt4 is required for myogenic miRNA expression
and interacts with miRNA regulatory sequences
Carm1/Prmt4 is an arginine methyltransferase that methy-
lates arginines 17 and 26 on histone H3 (29,30). Carm1/
Prmt4 is required for the expression of late myogenic
genes but is dispensable for the expression of myogenin,
which is expressed at early times post-differentiation (20).
To determine whether Carm1/Prmt4 contributes to
myogenic miRNA expression, we utilized cell lines that
were derived from wild type and Carm1/Prmt4-deficient
mouse embryos via a 3T3 passaging protocol (35). An
immunoblot with Carm1/Prmt4 antibody confirmed the
absence of Carm1/Prmt4 protein in the knockout (KO)
fibroblasts (Figure 4A). We then tested whether Carm1/
Prmt4 was required for the proper expression of myogenic
miRNA genes. We differentiated wild type and Carm1/
Figure 3. Expression of myogenin and Mef2D1b complements the loss of myogenic miRNA expression in a Prmt5 AS cell line. (A and B) Relative
expression of myogenin and Mef2D1b upon differentiation in NIH 3T3 and in the Prmt5 AS cell line, C1, infected with retrovirus encoding
myogenin and Mef2D1b. (C–F) qPCR analyses of primary transcripts of miR-1 and miR-133a in NIH 3T3 and C1 cells expressing myogenin
and Mef2D1b at various times post-differentiation. The data represent the average of three independent experiments±standard deviation.
Expression at Time 0 in the empty vector (EV) control is normalized to 1. h, hours.
Nucleic Acids Research,2011, Vol.39, No. 41247
Prmt4 KO fibroblasts by expressing MyoD or the empty
vector as a control. Cells infected with the MyoD
encoding retrovirus expressed similar levels of MyoD
(Figure 4B). Differentiated Carm1/Prmt4 KO fibroblasts
expressed normal levels of myogenin but failed to express
late marker gene Acta1, in agreement with previous results
[(20); Figure 4C and D]. When tested for the expression
of primary transcripts of miR-1 and miR-133a, no or
minimal miRNA induction was observed in Carm1/
Prmt4 KO cells at all times post-differentiation, whereas
normal induction of miRNA expression was observed in
the wild-type fibroblasts upon myogenic differentiation
Figure 4. Carm1/Prmt4 is required for the induction of myogenic miRNA expression during myogenesis. (A) Immunoblot demonstrating the absence of
Carm1/Prmt4 in the knockout (KO) cells. A cross-reacting band (asterisk) demonstrates equal loading between gel lanes. (B–D) Relative expression of
MyoD, myogenin and Acta1 mRNAs in the wild-type (WT) and Carm1/Prmt4 KO cells differentiated along the myogenic pathway by expressing MyoD
or the empty vector (EV) control. (E–H) Relative expression of primary transcripts of miR-1-1, miR-1-2, miR-133a-1 and miR-133a-2 at various times
post-differentiation in the WT and Carm1/Prmt4 knockout (KO) cells. (I and J) Relative expression of Mef2D and SRF mRNAs at various times
post-differentiation in the WT and Carm1/Prmt4 knockout (KO) cells. The data represent the average of three independent experiments±standard
deviation. The expression at time 0 in empty vector infected cells was normalized to 1. h, hours.
1248 Nucleic Acids Research, 2011,Vol.39, No. 4
(Figure 4E–H). Analysis of other myogenic regulatory
protein such as Mef2D and SRF showed that the absence
of Prmt4/Carm1 did not alter the expression of these genes
(Figure 4I and J). These results show that Carm1/Prmt4
is required for miR-1 and miR-133a expression during
To determine whether Carm1/Prmt4 is required for the
accumulation of mature microRNAs, we performed
northern blots. Figure 5 shows differentiation-dependent
accumulation of the mature forms of miR-1 and miR-133
that is severely reduced in the absence of Carm1/Prmt4.
By contrast, the widely expressed microRNAs miR-29a
and miR-143 were unaffected by differentiation or
by the absence of Carm1/Prmt4. These data indicate
that the accumulation of mature myogenic microRNAs,
like the accumulation of myogenic microRNA primary
transcripts, is dependent on Carm1/Prmt4.
We next addressed whether Carm1/Prmt4 binds to the
upstream regulatory regions of miRNA genes and whether
the Carm1/Prmt4 substrate, dimethylated H3R17, is also
present at these sequences. A schematic diagram illustrates
the positions of E boxes upstream of the miR-1-1,
sequences (Figure 6A). Open rectangles indicate E box
containing regions that we previously showed were
bound by both MyoD and the Brg1 ATPase that is the
enzymatic subunit of the SWI/SNF ATP-dependent chro-
matin remodeling enzyme. Solid rectangles indicate E box
containing sequences that we previously showed were not
bound by either MyoD or Brg1 [Figure 6A; (13)]. ChIP
experiments for Carm1/Prmt4 and diMeH3R17 at each of
these putative miRNA regulatory regions in MyoD-
differentiated wild-type and Carm1/Prmt4 KO fibroblasts
showed that Carm1/Prmt4 binds to the proximal three E
box containing regions of the miR-1-1 upstream region
but not to the two more distal E-boxes (Figure 6B–F).
Carm1/Prmt4 also interacted with the proximal E box
containing sequences of the miR-1-2, miR-133a-1 and
miR-133a-2 upstream regions. However, no binding was
observed at the distal E box containing sequences
upstream of miR-133a-2 (Figure 7A–D). Carm1/Prmt4
binding at these regions correlated precisely with the
(Figures 6B–F and 7A–D). Binding of both Carm1/
Prmt4 and diMeH3R17 was first observed at 12h
post-differentiation, consistent with the onset of miRNA
expression (Figure 1) and was maintained through the
48-h post-differentiation time point. These results demon-
strate that Carm1/Prmt4 and diMeH3R17 are present in
the upstream regulatory regions of myogenic miRNAs,
miR-1 and miR-133a, that dimethylation of H3R17 at
these sequences required Carm1/Prmt4, and that the
occurred at the time of myogenic miRNA gene induction.
In combination with studies from our previous work (13),
we have demonstrated that each of the sites bound by
Carm1/Prmt4 and diMeH3R17 also are bound by
MyoD and by Brg1.
Carm1/Prmt4 is required for the binding of myogenin and
Brg1 at miRNA regulatory regions
Brg1-based SWI/SNF chromatin remodeling enzyme
function is required for the expression of miR-1 and
miR-133a primary transcripts during myogenesis (13). In
addition, MyoD has been shown to interact with upstream
differentiated C2C12 myoblasts (14) as well as in MyoD-
differentiated fibroblasts and in skeletal muscle tissue (13).
The myogenin factor also binds to some of these sequences
in differentiated C2C12 myoblasts (14). To address the
relationship between Carm1/Prmt4, the dimethylation of
H3R17, and the binding of Brg1, MyoD and myogenin,
we carried out ChIP assays on wild-type and Carm1/
Prmt4 KO cells that were differentiated by MyoD or the
empty vector. Consistent with our previous report (13),
MyoD was found bound to the proximal but not distal
regulatory regions of miR-1 and miR-133a in wild-type
cells both at the onset of and during differentiation
(Figures 8 and 9). Binding of MyoD at these sites was
not affected in the Carm1/Prmt4 KO cells (Figures 8
and 9), indicating that MyoD could access these E boxes
in the absence of Prmt4 and in the absence of
dimethylated H3R17. By contrast, myogenin binding
was not observed at any region upstream of the
microRNAs at the onset of differentiation. However,
myogenin did bind to the same sequences bound by
MyoD, Carm1/Prmt4 and diMeH3R17 at 12h post-
differentiation and beyond in MyoD-differentiated cells
(Figures 8 and 9). Intriguingly, myogenin binding was
completely inhibited in the absence of Carm1/Prmt4
Figure 5. Carm1/Prmt4 is required for the accumulation of mature
myogenic miRNAs during myogenesis. Duplicate Northern blots were
prepared using RNA isolated from the indicated samples at the
indicated times. Levels of the myogenic microRNAs, miR-1 and
miR-133a, and the widely expressed microRNAs, miR-29a and
miR-143, were determined. U6 snRNA was probed as a loading
control. Blot 1 was sequentially probed for miR-1, miR-29a and U6.
Blot 2 was sequentially probed for miR-133a, miR-143 and U6. The
differences in the two U6 blots were negligible; only the U6 image from
Blot 1 is shown.
Nucleic Acids Research,2011, Vol.39, No. 4 1249
(Figures 8 and 9). This was not due to a change in
myogenin expression in the Carm1/Prmt4-deficient cells
(Figure 4C), suggesting instead that the local chromatin
environment is insufficient to permit myogenin binding to
the miRNA regulatory regions of miR-1 and miR-133a in
the absence of Carm1/Prmt4.
Additional ChIP experiments revealed binding of Brg1
to the proximal regulatory regions but not to the distal
E-boxes of miR-1-1 in the wild-type MyoD-differentiated
cells (Figure 8A–E). Brg1 was also found to interact with
regulatory regions upstream of miR-1-2 and miR-133a-1
and to the proximal but not distal regions of miR-133a-2
upon myogenic differentiation.
was observed at any of these sites in the Carm1/Prmt4
KO cells at any time (Figure 9A–D). These results
demonstrate that Carm1/Prmt4 is required for the
binding of myogenin and Brg1 and suggest that the
requirement for Carm1/Prmt4 for the proper expression
of myogenic miRNAs during myogenic differentiation is
based on its ability to facilitate Brg1 and myogenin
binding to the miRNA regulatory sequences.
An increasing number of miRNAs, both muscle-specific
and ubiquitously or widely expressed, have been shown to
affect muscle cell proliferation, development and differen-
tiation (45–48). In addition, there is a growing appreci-
ation that specific miRNAs are involved in the onset of
disease states affecting all three types of muscle (8,10).
miRNAs, therefore, play an important and essential role
in the regulation of muscle formation and function, and
defining the principles controlling the expression of these
Figure 6. Prmt4/Carm1 binds and dimethylates H3R17 at regulatory regions upstream of myogenic miR-1-1 miRNA. (A) A schematic diagram
showing the location of consensus E-boxes, the cis elements that mediate the interaction of myogenic regulatory factors, upstream of miR-1 and
miR-133a genes. This schematic diagram is a modified version of the diagram published in Figure 6 of ref. (13), that was amended with permission
from the American Society for Microbiology. (B–F) ChIP experiments demonstrating the interaction of Carm1/Prmt4 and the presence of
dimethylated (diMe) H3R17 at the three proximal E-boxes but not at the two distal E-boxes of miR-1-1 in WT but not in the Carm1/Prmt4
KO cells at various times during myogenic differentiation. No binding of IgG was found at any of the E-boxes at any time point. The binding at time
0 in the empty vector (EV) control in WT and Carm1/Prmt4 KO cells was normalized to 1. Data represent the average of three independent
experiments ± standard deviation. h, hours.
1250Nucleic Acids Research, 2011,Vol.39, No. 4
miRNAs is critical to better understanding muscle biology
and muscle disease.
We have focused on the transcriptional regulation of
expression of several miRNAs that are induced during
muscle differentiation and are largely muscle specific in
their expression. The miRNAs are critical regulators of
myoblast proliferation and differentiation (5). Previous
studies have implicated MyoD, myogenin, Mef2 and the
respective binding sites for these factors in myogenic
miRNA expression (5,14,16). Thus, the data support the
idea that myogenic, DNA-binding transcription factors
that control myogenic mRNA expression also control
myogenic miRNA expression.
Considerable effort has been made to understand how
epigenetic regulators influence myogenic mRNA expres-
sion. These studies have revealed roles for numerous
histone-modifying enzymes, specific histone modifications
and ATP-dependent chromatin remodeling enzymes (49–
51). Whether such chromatin modifiers also contribute
myogenic miRNA expression has only recently begun to
be investigated. Recently, we used morpholinos to reduce
Brg1 levels in developing zebrafish and observed that
?40% had altered tail development with morphologically
altered somite structure. In addition, these embryos had
altered sarcomeric actin organization in the tissue.
Analysis of myogenic miRNA expression in these altered
tissues showed that there was a significant decrease in
expression relative to the controls (13). This phenotype
was remarkably similar to that observed by others when
in the inductionof
the microRNA processing enzyme, Dicer, was mutated or
when the levels of myogenic miRNAs miR-1 and miR-133
were reduced by morpholino injection (52). Together, the
two studies suggest that Brg1 and miRNAs are part of the
same regulatory pathway. Subsequent studies in Brg1-
deficient myoblasts, in primary skeletal muscle tissue and
in a cell culture model for skeletal muscle differentiation
indicated that Brg1 is not only required for skeletal muscle
mRNA expression and differentiation, as previously
myogenic miRNA expression (13). To date, no other chro-
matin modifiers or remodeling enzymes have been shown
to contribute to myogenic miRNA regulation.
Given the recently demonstrated roles for the arginine
methyltransferases Prmt5 and Carm1/Prmt4 in myogenic
mRNA expression, we asked whether these enzymes are
involved in myogenic miRNA regulation. We previously
showed that Prmt5 is directly required for expression of
the myogenin gene (21), but despite binding to regulatory
sequences controlling myogenic genes expressed at late
times of myogenesis in cultured cells, it was not required
for the expression of these genes. Carm1/Prmt4, in
contrast, bound to late gene regulatory sequences and
was required for expression during differentiation in
culture (20). Our studies of Prmt5 and Carm1/Prmt4
function during myogenic miRNA expression revealed
that therequirements for
transferases were similar to their functions in regulating
myogenic late gene mRNA expression. Thus, there is con-
servation between Prmt5 and Carm1/Prmt4 function in
Figure 7. Carm1/Prmt4 binding and the presence of dimethylated (diMe)H3R17 at miR-1-2, miR-133a-1 and miR-133a-2 regulatory regions. ChIP
experiments show the presence of Carm1, and diMeH3R17 at E-box containing sequences upstream of miR-1-2 (A) and miR-133a-1 (B) and
proximal E-boxes of miR-133a-2 (C) but not the distal E-boxes of miR-133a-2 (D) at various times following MyoD mediated differentiation in
the WT but not the Carm1/Prmt4 KO cells. Values for empty vector (EV) infected cells at time 0 of differentiation were normalized to 1. Data
represent the average of three independent experiments ± standard deviation. h, hours.
Nucleic Acids Research,2011, Vol.39, No. 41251
the regulation of myogenic miRNAs and in the regulation
of a subset of myogenic mRNAs. It is important to note,
however, that despite the similarity to the regulation of
myogenic late gene mRNAs, this does not indicate that
the myogenic miRNAs should be considered ‘late’ express-
ing genes. In vivo analysis of the expression of transgene
constructs controlled by regulatory sequences of several
myogenic miRNAs indicates that they can be detected in
somites at E9.5 and at later stages of development (6,16).
The ChIP experiments performed in the Carm1/
insights into how Carm1/Prmt4 functions at myogenic
miRNA regulatory sequences. First, incorporation of
the Carm1/Prmt4 substrate, dimethylated H3R17, at
these sequencesis absolutely
presence of Carm1/Prmt4 (Figures 6 and 7). Second,
the subset of E box containing sequences that showed
Carm1/Prmt4 binding correlated exactly with those se-
quences previously found to bind both MyoD and the
Brg1 SWI/SNF ATPase (13). This suggests that factors
regulating myogenic miRNA expression are functioning
at the same cis-acting sequences. Third, cells lacking
Carm1/Prmt4 not only were deficient for the H3R17
modification, but also failed to target the Brg1 ATPase
of SWI/SNF chromatin remodeling enzymes to the regu-
latory sequences (Figures 8 and 9). We have previously
entirely dependent upon Brg1 (13). The absence of
Brg1 indicates that these sequences cannot undergo chro-
matin remodeling and are therefore not accessible for
active gene expression. The mechanism(s) by which
binding remains to be determined, but we can speculate
that Carm1/Prmt4 mediated histone modifications likely
contribute to targeting the ATP-dependent chromatin
The last significant conclusion from the ChIP experi-
ments with Carm1/Prmt4 deficient cells is the novel obser-
vation that MyoD binding to these sequences was
(Figures 8 and 9). The data presented here and in our
binding was abolished
Figure 8. The myogenic regulatory factors MyoD and myogenin and the chromatin remodeling enzyme Brg1 interact with the proximal E-boxes
containing sequences upstream of miR-1-1. ChIP experiments demonstrated the binding of MyoD, myogenin and Brg1 at regions containing the
proximal E-boxes (A–C) but not to the distal E-boxes (D and E) of miR-1-1 at the indicated times in MyoD differentiated WT and Carm1/Prmt4
KO cells. Values at time 0 in the empty vector (EV) control were normalized to 1. The experiment was repeated three times, and the data represent
the average of three experiments ± standard deviation. h, hours.
1252Nucleic Acids Research, 2011,Vol.39, No. 4
prior study (13) indicate that MyoD binding to these se-
quences is independent of Brg1 function and the presence
of Carm1/Prmt4. Thus, MyoD binding does not require
these particular enzymes or the chromatin structural
required for MyoD binding at these sequences is not
known. By contrast, myogenin binding is completely
similarities between the MyoD and myogenin proteins,
it has long been established that MyoD is intrinsically
better at functioning in a chromatin environment than is
myogenin (55,56). How the structural differences between
MyoD and myogenin relate to the Carm1/Prmt4 depend-
ence of binding at miRNAs regulatory sequences is an
presence of MyoD at these sequences in both wild-type
and Carm1/Prmt4-deficient cells suggests that MyoD
binding is not sufficient for myogenic miRNA expression.
Instead, the binding of myogenin correlates with tran-
scriptional competence, suggesting that it is myogenin
that is facilitating transcriptional activation. Previous
cooperativity between Carm1/Prmt4 and both myogenin
and Mef2 has been reported (20,31), supporting the idea
that Carm1/Prmt4 acts as a co-activator for myogenin and
Mef2 to promote myogenic miRNA expression.
The functional interrelationships between two distinct
types of Prmts and the Brg1 chromatin-remodeling
enzyme during differentiation-mediated induction of
myogenic miRNAs suggest that regulation of these
myogenic miRNAs will be as complex as the regulation
of myogenic mRNAs. It is therefore likely that histone
acetyltransferases, lysine methyltransferases and other
histone-modifying enzymes will also be involved in
myogenic miRNA expression. Because histone modifica-
tions are dynamically regulated, there also will likely be
roles for deacetylating and demethylating enzymes. How
the activities of multiple
integrated with the functions of SWI/SNF enzymes and
gene specific transcription factors to regulate myogenic
miRNA expression will be an interesting topic for future
Finally, the similarity in the regulation of the miR-1
because miR-1 and miR-133 have opposing functions;
promotes myoblast proliferation (5). We showed that
miR-1-1, miR-1-2, miR-133a-1 and miR-133a-2 are
equivalent in MyoD-differentiated fibroblasts and that
the four myogenic microRNAs are also induced with
similar kineticsin myogenin/Mef2D1b-differentiated
fibroblasts (Figures 1 and 3). This is consistent with the
work of others who have seen differentiation-dependent
increases in miR-1 and miR-133 expression in C2C12 cells
(5,7,14,57) and with data from transgenic animal studies
indicatingthat the intragenic
two miR-1 and miR-133 clusters both drive reporter
gene expression in the heart and somites with similar
kinetics (6,16). The reason that the induction and
regulation of two microRNAs that promote opposite
However, others have speculated that differential regula-
tion of the miRNAs at steps downstream of the
Figure 9. Interaction of MyoD, myogenin and Brg1 at upstream regulatory regions of miR-1-2, miR-133a-1 and miR-133a-2. ChIP experiments
demonstrate the interaction of MyoD, myogenin and Brg1 at sequences containing the E-box regulatory elements upstream of miR-1-2 (A),
miR-133a-1 (B) and the proximal (C) but not the distal E-boxes (D) of miR-133a-2 at the indicated times following MyoD-mediated differentiation
of WT and Carm1/Prmt4 KO cells. The values from the empty vector (EV) control at time 0 in both WT and Carm1/Prmt4 KO cells were
normalized to 1. The results are the average of three independent experiments±standard deviation. h, hours.
Nucleic Acids Research,2011, Vol.39, No. 4 1253
transcription of these microRNAs might promote their
differences in function, as might the obvious differences
in the functions of the proteins encoded by their target
mRNAs (16,58,59). In addition, despite the similarities
in regulation by chromatin-modifying and remodeling
remains possible that subtle, yet uncharacterized, differ-
ences in regulatory mechanisms or in the timing of
microRNA expression could promote differences in
miR-1 and miR-133 function.
and previously(13), it
We thank Silvana Konda for retrovirus preparation, Dr.
Mark Bedford for providing the Carm1/Prmt4 deficient
cell line and its wild-type counterpart, and Qiong Wu
for reviewing the manuscript.
National Institutes of Health (grants GM56244 to A.N.I.
and CA116093 to S.S.). A.N.I. is a member of the
University of Massachusetts Medical School Diabetes
Endocrine Research Center supported by National
Institutes of Health grant DK32520. Funding for open
access charge:National Institutes
(GM56244 to A.N.I.).
Conflict of interest statement. None declared.
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