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MiR-34 MiRNAs Provide a Barrier for Somatic Cell Reprogramming (2011)

MiR-34 MiRNAs Provide a Barrier for Somatic Cell Reprogramming (2011)

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Published by James Clement
Somatic reprogramming induced by dened transcription factors is a low-efficiency process that is enhanced by p53 deficiency. So far, p21 is the only p53 target shown to contribute to p53 repression of iPSC (induced pluripotent stem cell) generation, indicating that additional p53 targets may regulate this process. Here, we demonstrate that miR-34 microRNAs (miRNAs), particularly miR-34a, exhibit p53-dependent induction during reprogramming. Mir34a deficiency in mice significantly increased reprogramming deficiency and kinetics, with miR-34a and p21 cooperatively regulating somatic reprogramming downstream of p53. Unlike p53 deficiency, which enhances reprogramming at the expense of iPSC pluripotency, genetic ablation of Mir34a promoted iPSC generation without compromising self-renewal or differentiation. Suppression of reprogramming by miR-34a was due, at least in part, to repression of pluripotency genes, including Nanog, Sox2 and Mycn (also known as N-Myc ). This post-transcriptional gene repression by miR-34a also regulated iPSC differentiation kinetics. miR-34b and c similarly repressed reprogramming; and all three miR-34 miRNAs acted cooperatively in this process. Taken together, our findings identified miR-34 miRNAs as p53 targets that play an essential role in restraining somatic reprogramming.
Somatic reprogramming induced by dened transcription factors is a low-efficiency process that is enhanced by p53 deficiency. So far, p21 is the only p53 target shown to contribute to p53 repression of iPSC (induced pluripotent stem cell) generation, indicating that additional p53 targets may regulate this process. Here, we demonstrate that miR-34 microRNAs (miRNAs), particularly miR-34a, exhibit p53-dependent induction during reprogramming. Mir34a deficiency in mice significantly increased reprogramming deficiency and kinetics, with miR-34a and p21 cooperatively regulating somatic reprogramming downstream of p53. Unlike p53 deficiency, which enhances reprogramming at the expense of iPSC pluripotency, genetic ablation of Mir34a promoted iPSC generation without compromising self-renewal or differentiation. Suppression of reprogramming by miR-34a was due, at least in part, to repression of pluripotency genes, including Nanog, Sox2 and Mycn (also known as N-Myc ). This post-transcriptional gene repression by miR-34a also regulated iPSC differentiation kinetics. miR-34b and c similarly repressed reprogramming; and all three miR-34 miRNAs acted cooperatively in this process. Taken together, our findings identified miR-34 miRNAs as p53 targets that play an essential role in restraining somatic reprogramming.

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Published by: James Clement on Oct 27, 2011
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LETTERS
miR-34 miRNAs provide a barrier for somatic cellreprogramming
Yong Jin Choi
1,7
, Chao-Po Lin
1,7
, Jaclyn J. Ho
1
, Xingyue He
2
, Nobuhiro Okada
1
, Pengcheng Bu
1
, Yingchao Zhong
1
,Sang Yong Kim
3
, Margaux J. Bennett
1
, Caifu Chen
4
, Arzu Ozturk 
5
, Geoffrey G. Hicks
5
, Greg J. Hannon
6
and Lin He
1,8
Somatic reprogramming induced by defined transcriptionfactors is a low-efficiency process that is enhanced by
p53 
deficiency
1–5
. So far, p21 is the only p53 target shown tocontribute to p53 repression of iPSC (induced pluripotent stemcell) generation
1,3
, indicating that additional p53 targets mayregulate this process. Here, we demonstrate that miR-34microRNAs (miRNAs), particularly miR-34a, exhibitp53-dependent induction during reprogramming.
Mir34a 
deficiency in mice significantly increased reprogrammingefficiency and kinetics, with miR-34a and p21 cooperativelyregulating somatic reprogramming downstream of p53. Unlike
p53 
deficiency, which enhances reprogramming at the expenseof iPSC pluripotency, genetic ablation of
Mir34a 
promotediPSC generation without compromising self-renewal ordifferentiation. Suppression of reprogramming by miR-34a wasdue, at least in part, to repression of pluripotency genes,including
Nanog 
,
Sox2 
and
Mycn 
(also known as
N-Myc 
). Thispost-transcriptional gene repression by miR-34a also regulatediPSC differentiation kinetics. miR-34b and c similarlyrepressed reprogramming; and all three miR-34 miRNAs actedcooperatively in this process. Taken together, our findingsidentified miR-34 miRNAs as p53 targets that play anessential role in restraining somatic reprogramming.
Differentiated somatic cells can be induced to generate pluripotentstem cells that functionally resemble embryonic stem cells (ESCs;
ref.
6
).Thisreprogrammingprocessisrootedintheremarkablecellular
plasticity retained during differentiation. The process can be triggeredby exogenous expression of a set of defined ESC-specific transcription
factors, Pou5f1 (also known as Oct4), Sox2, Klf4 and c-Myc (refs
6
9
),
which constitute the core regulatory circuits controlling pluripotency 
and self-renewal. Enforced expression of these reprogramming factors
1
Division of Cellular and Developmental Biology, Molecular and Cell Biology Department, University of California at Berkeley, Berkeley, California 94705, USA.
2
40Landsdowne Street, Cambridge, Massachusetts 02139, USA.
3
Transgenetic facility, 500 Sunnyside Blvd., Woodbury, New York 11797-2924, USA.
4
Genomic AssaysR&D, Life Technologies Corporation, 850 Lincoln Centre Dr., Foster City, California 94404, USA.
5
Manitoba Institute of Cell Biology, 675 McDermot Ave. Rm.ON5029, Winnipeg, MB R3E 0V9, Canada.
6
Cold Spring Harbor Laboratory, Watson School of Biological Sciences, 1 Bungtown Road, Cold Spring Harbor, New York11724, USA.
7
These authors contributed equally to this work.
8
Correspondence should be addressed to L.H. (e-mail:lhe@berkeley.edu)Received 10 May 2011; accepted 22 September 2011; published online 23 October 2011; DOI: 10.1038/ncb2366
generates iPSCs with low efficiency and slow kinetics, suggesting the
existenceofcellularandmolecularbarrierstotheprocess
10
.
Recent studies have revealed considerable mechanistic overlap
between somatic cell reprogramming and malignant transformation
11
.
Cellular mechanisms that enhance reprogramming, including cell
proliferation and survival, evasion of DNA-damage response, and cell
immortalization,havelongbeenknowntopromotetumorigenesis
3–5,12
.
Several oncogenes and tumour suppressors also serve as essentialregulators for reprogramming
6,10,11
. Notably, the inactivation of p53,
one of the most important tumour suppressors, significantly enhances
iPSC generation
1–5,13
. As a tumour suppressor, p53’s transcriptionalregulation converges onto multiple target genes that collectively mediate its downstream effects, including cell-cycle arrest, cellular
senescence, apoptosis, DNA-damage response and genomic stability 
14
.
Similarly, p53’s role in repressing reprogramming is probably alsomediated through multiple targets. To date, the cell-cycle regulatorp21 is the only p53 target with a demonstrated role in repressingreprogramming. However,
p21
deficiency only partially phenocopies
that of 
p53
(refs
1
,
3
,
15
), suggesting that p53 represses iPSC generationthroughas-yetunidentifiedmechanism(s)andtarget(s).
Previous studies have identified the miR-34 microRNAs (miRNAs)
as
bona fide
p53 transcriptional targets, whose overexpressiontriggers cell-cycle arrest or apoptosis in a cell-type- and context-dependent manner
16–18
. miRNAs, a large family of small non-codingRNAs, primarily repress gene expression post-transcriptionally, by pairing with partially complementary messenger RNA targets
19,20
.In response to p53 activation, induced
Mir34
miRNAs can mediatep53 downstream effects by repressing specific target genes, includingcyclin D1, cyclin E2,
Cdk4
,
Cdk6 
,
Bcl2
and
c-Met 
(ref.
21
).Although p53 is primarily characterized for its role in transcriptionalactivation, it acts as a global gene regulator that both activatesand represses gene expression
14
. Direct transcriptional repression,
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1
 
LETTERS
togetherwithindirectpost-transcriptionalrepressionthroughmiRNAs
such as miR-34, constitute two major mechanisms for p53-
mediated gene repression.
The miR-34 miRNAs belong to an evolutionarily conserved family,
with three mammalian homologues, miR-34a, b and c, localized to two
distinct genomic loci,
Mir34a
and
Mir34b/c 
(
a; ref.
16
). All three
 Mir34
miRNAs were significantly induced when mouse embryonicfibroblast (MEF) reprogramming was triggered with Sox2, Oct4 and
Klf4 in the presence or absence of c-Myc (
a, data not shown). In
both cases,
Mir34
induction, similarly to that of 
p21
, was dependent ontheactivation of intact p53 (
a). Wetherefore investigatedwhether
miR-34 miRNAs are components of the reprogramming regulatory circuit downstream of p53. Among all miR-34 miRNAs, miR-34a
exhibited the highest induction level during reprogramming, whereas
miR-34b was the lowest. We therefore focused initially on the role of 
 Mir34a
in iPSC induction.
We generated
Mir34a
knockout mice using C57BL/6 ESCs(
a),
confirmed the germline transmission of the targeted allele (
b)and verified the genetic ablation of 
Mir34a
by expression studies(
c).
Mir 
34
a
/
mice were born at the expected Mendelianratio, without obvious developmental or pathological abnormalitiesup to 12 months. However, when we induced the
Mir 
34
a
/
MEFs for reprogramming, we observed a significant increase in thereprogramming efficiency (
b,d and Supplementary Fig. S1a).
Three-factor-infected
Mir 
34
a
/
MEFs exhibited a
4
.
5-fold increaseinalkalinephosphatase-positivecolonieswithtypicaliPSCmorphology 
(
b), whereas four-factor-infected
Mir 
34
a
/
MEFs yielded a
4-fold increase (Supplementary Fig. S1a). Furthermore, when weplated infected MEFs into 96-well plates at a density of one cellper well,
Mir34a
deficiency caused a similar increase in alkaline
phosphatase-positivecolonieswithtypicaliPSCmorphology(
c).
Using MEFs carrying an
Oct4
– 
Gfp
knockin reporter allele
22
, we
confirmed the effect of miR-34a in promoting reprogramming, scoringfully reprogrammed iPSCs on the basis of endogenous
Oct4
expression
indicated by green fluorescent protein (GFP). Consistently, a greaterthan fourfold increase in reprogramming efficiency was observed in
 Mir 
34
a
/
;
Oct4
Gfp
/
+
MEFsafterthree-orfour-factortransduction
(
d). A significant increase in reprogramed
Oct4
– 
Gfp
-positivecolonies could also be achieved using a locked nucleic acid (LNA)
inhibitor against miR-34a (Supplementary Fig. S1b). Notably,
Mir34a
deficiency both enhanced the overall efficiency of iPSC generation and
led to more rapid reprogramming kinetics. Small iPSC-like colonies
firstappeared7dayspost-infectioninfour-factor-transducedwild-typeMEFs,butasearlyaspost-infectionday5in
 Mir 
34
a
/
MEFs.As miR-34b and c share the miR-34a seed sequence and are similarly 
induced during reprogramming(
a), they probably also regulate
iPSCgeneration.Wegenerated
 Mir34b/c 
knockoutmice(
a
c).As
with miR-34a deficiency,
Mir34b/c 
knockout alone promoted somatic
reprogramming, although to a lesser degree (
e). Interestingly,MEFs deficient for all three miR-34 miRNAs exhibited an even
greater increase in iPSC generation(
e), suggesting a cooperative
effect among these genes, although the exact molecular and cellular
mechanisms underlying this cooperation still remained unclear. As the
 Mir 
34
a
/
;
Mir 
34
b
/
/
MEFs did not completely phenocopy 
p
53
/
MEFs, extra mechanisms may act downstream of p53 to mediate the
suppression of reprogramming.
Previous studies have identified p21 as an important mediatorof p53 suppression of reprogramming
1,12
.
Mir34
and
p21
bothexhibited
p53
-dependent induction during reprogramming (
a).
 p21
inductionalsowasobservedin
 Mir 
34
a
/
MEFs(
a).Whereas
MEFs deficient for
Mir34a
alone or
p21
alone showed comparable
increases in iPSC generation, MEFs deficient for both
Mir34a
and
p21
exhibited a cooperative increase that recapitulated a significant fraction
of the p53 effect (
c). Given the functional similarities amongthe three miR-34 miRNAs, the cooperative effects among p21 and allmiR-34 miRNAs could be even greater. Thus, the miR-34 miRNAs,together with p21, constitute important downstream effectors of p53
to mediate the repression of reprogramming.
p21 represses reprogramming efficiency primarily through itsrepression of cell proliferation
12
. Although miR-34a and p21 repressreprogramming efficiency similarly (
c), their effects on cellproliferation are quite different. The increased cell proliferation in
 p
 21
/
MEFs was highly significant (
b), yet within the time
frame of our reprogramming experiments
Mir 
34
a
/
MEFs exhibited
little increase up to passage 6. We cannot completely exclude thepossibility that a mild increase in
Mir 
34
a
/
MEF proliferationcontributed to the increased reprogramming. Yet, unlike p21, whoseeffects on reprogramming are largely attributed to its inhibitionof cell proliferation
12
, miR-34a is likely to act mostly through aseparate mechanism independent of cell proliferation. These twodistinct mechanisms may underlie the cooperative regulation of 
reprogramming by miR-34a and p21.
 Mir 
34
a
/
iPSCs resemble wild-type iPSCs and ESCs, exhibitingESC-like morphology (
a and Supplementary Fig. S1c) and
expressing key molecular markers for pluripotency. High levels of Oct4,Nanog and SSEA1 were detected (
b and Supplementary Fig. S1d).
 Mir 
34
a
/
iPSCs injected into immunocompromised nude mice yielded differentiated teratomas, containing terminally differentiatedcell types from all three germ layers(
c,d and Supplementary 
Fig. S1e). Furthermore, three independent lines of four-factor-induced
 Mir 
34
a
/
iPSCs all yielded healthy adult chimaeric mice with a high
percentage of iPSC contribution (Fig. 4e and Supplementary Fig. S1g,Table S1
). Taken together,
Mir34a
deficiency enhances the efficiency of iPSCgenerationwithoutcompromisingself-renewalorpluripotency.
Although
p53
deficiency induced reprogramming more efficiently than
Mir34a
deficiency, the
p
53
/
iPSCs exhibited compromisedself-renewal and differentiation capacity 
1,4
. As also reported inref.
1
, we have observed that four-factor-induced
p
53
/
iPSCslost ESC-like morphology after five to six passages in culture,and failed to generate highly differentiated teratomas. In contrast,four-factor-induced
Mir 
34
a
/
iPSCs remained stable beyondpassage 26, with no significant differences in self-renewal when
comparedwithwild-typeiPSCs(SupplementaryFig.S1f).Furthermore,
generation of healthy adult chimaeras from
p
53
/
iPSCs wasdifficult. The percentage of 
p
53
/
iPSC contribution was low,and the majority of such chimaeras succumbed to tumorigenesisbefore 7 weeks
1
. In contrast,
Mir 
34
a
/
iPSCs exhibited functionalpluripotency: all three four-factor-induced
Mir 
34
a
/
iPSC linestested gave rise to healthy adult chimaeras with a high percentageof iPSC contribution (
e and Supplementary S1g,
Table S1
),which remained tumour free for 6 months (to the time of 
manuscript preparation).
2NATURE CELL BIOLOGY
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LETTERS
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34a
+/+
34b/c
+/+
   F  o   l   d   u  p  r  e  g  u   l  a   t   i  o  n
34a
+/–
34b/c
+/–
34a
 –/–
34b/c
 –/–
 
: loxPEP
Δ
 Δ ΔΔ Δ
Δ
 X5
probe
   5
                  ′
   p  r  o   b  e   5
                  ′
   p  r  o   b  e   3
                  ′
   p  r  o   b  e   3
                  ′
   p  r  o   b  e
5
probe3
probe3
probe
Mir34a
EE
LacZ
P
Neo
EPKozaksequence: FRT E : EcoRI P : PsiI X : XhoI XEM : MfeI S : SpeI: loxPMMMSSSMMMSSS
PGK-Neo
Mir34bMir34c
11.8 kb10.7 kb9.0 kb6.6 kb6.4 kb5.3 kb
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miR-34a
Mir34a Mir34b/cMir34a
miR-34bmiR-34c
Mir34a Mir34b/cMir34b/cMir34a
WT
Mir34aMir34b/c
WT
Mir34b/c
flNeo
Mir34b/c
00.250.500.751.001.25
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34b/c
+/+
34b/c
+/–
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 –/–
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00.250.500.751.001.25
abc
FRT
Figure 1
Generation of
Mir34a 
and
Mir34b/c 
knockout MEFs. (
a
) Diagramsof endogenous
Mir34a 
and
Mir34b/c 
gene structure (WT) and the knockoutconstruct (
). Using recombineering, we engineered the
Mir34a 
targetingvector with a
6-kilobase (kb) homologous arm on both 5
and 3
ends, flanking a Kozak sequence, a lacZ complementary DNA and anFRT–Neo–FRT cassette. The
Mir34b/c 
targeting vector (
Neo 
) containsa
6-kb homologous arm at each end, with the
Mir34b/c 
gene and aNeo selection cassette flanked by
loxP 
sites. (
b
) Validating the germlinetransmission of the
Mir34a 
and
Mir34b/c 
targeted allele using Southernanalysis. Putative wild-type,
Mir34a 
+
/
and
Mir34a 
/
animals derivedfrom three independently targeted ESC lines were analysed by Southernblotting using probes either 5
or 3
to the homologous arms (left). Similarvalidation was carried out for wild-type,
Mir34b 
/
 
/
+
and
Mir34b 
/
+
/
animals (right). (
c
) Confirming loss of miR-34 expression in
Mir34a 
and
Mir34b/c 
knockout MEFs. Littermate-controlled wild-type,
Mir34a 
+
/
and
Mir34a 
/
MEFswereanalysedbyreal-timePCRtoquantifytheexpressionof
Mir34a 
. Whereas wild-type MEFs showed robust miR-34a inductionon culture stress, no miR-34a expression was detected in
Mir34a 
/
MEFs. The miR-34a level in
Mir34a 
+
/
MEFs was approximately halfthat of wild-type MEFs. Similar validation was carried out for
Mir34b 
/
/
MEFs. Error bar, s.d.,
n
=
3. Uncropped images of blots are shown inSupplementary Fig. S6.
Consistent with these differences, we observed that four-factor-induced
p
53
/
iPSCs, but not
Mir 
34
a
/
or wild-type iPSCs,failed to silence retroviral transgenes, thus exhibiting lower levelsof the corresponding endogenous genes (Supplementary Fig. S3a
c).The exogenous expression of reprogramming factors, particularly 
c-Myc 
(Supplementary Fig. S3e), may contribute to the impairedself-renewal and differentiation (Supplementary Fig. S3d), andpromote tumorigenesis
1
. In contrast, pluripotency was establishedeasily in
Mir 
34
a
/
iPSCs by the endogenous transcription factorcircuitry, leaving the exogenous transgenes dispensable for the
maintenance of pluripotency.
Similar to
Mir 
34
a
/
iPSCs,
Mir 
34
a
/
;
Mir 
34
b
/
/
iPSCs also
exhibited robust self-renewal and differentiation capacity, with typical
ESC morphology, key pluripotency markers and the ability to generate
differentiated teratomas (Supplementary Fig. S2a
c). Their ability to
generate chimaeras remains to be determined.The increased reprogramming efficiency and compromised pluripo-
tency of 
p
53
/
MEFs may result from aberrant apoptosis and DNAdamage response
4
. However,
Mir34a
deficiency failed to protect cellsfrom apoptosis or DNA-damage response during reprogramming
(Supplementary Fig. S4a
c). Thus, the enhanced reprogramming effi-
ciency observed in
Mir 
34
a
/
MEFs may reflect a mechanism largely independent of apoptosis and DNA damage response. These findings
contrast with the ability of 
Mir34a
overexpression to trigger apoptosis
in specific tumour cells
17,18
. These
Mir34a
effects on apoptosis are
probably cell-type dependent, differing between primary fibroblasts
16
and specific cancer cell lines
17,18
. Cells deficient for
Mir34a
may exhibit
apoptotic defects under conditions other than reprogramming. It isalso possible that the lack of apoptotic protection in reprogramming
 Mir 
34
a
/
MEFsreflectsthefunctionalredundancyofmiR-34bandc.
Enhanced reprogramming in
p53
-deficient cells has also been at-
tributed to cell immortalization and increased cell proliferation
1–5,12,15
.Whereas cell immortalization greatly promotes iPSC generation from
 p
53
/
MEFs (ref.
5
), increased cell proliferation is a key mechanism
driving stochastic reprogramming of 
p53
KD
B cells
12
. Although
Mir34
overexpression induced growth arrest and cellular senescence in pri-
mary fibroblasts
16
, no defects in senescence response were observed in
 Mir 
34
a
/
MEFs during reprogramming (Supplementary Fig. S4d and
data not shown). As described above, we did not observe a significant
MEF proliferation difference caused by 
Mir34a
deletion within thetime frame of our reprogramming experiment. Presumably, miR-34aregulates reprogramming efficiency largely through a mechanism
independentofcellproliferationandcellsenescence.To better define the molecular mechanism of miR-34a in reprogram-
ming, we initiated a search for its targets, specifically by examining
genes that promotes the iPSC generation for possible miR-34a-bindingsites.RNA22identifiedanumberofsuchgeneswithpredictedmiR-34a
sites
23
(Supplementary Fig. S5d). Of all the genes tested,
Nanog 
,
Sox2
and
Mycn
(
 N-Myc 
) emerged as top candidates(
a and Supplemen-taryFig.S5a).Allthreeexhibited
 Mir34a
-dependentrepression(
band Supplementary Fig. S5c), and each had potent effects in promoting
reprogramming
24,25
. ESCs over-expressing miR-34a, miR-34b or
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