Molecular Roadblocks for Cellular Reprogramming
Thomas Vierbuchen1,2and Marius Wernig1,*
1Institute for Stem Cell Biology and Regenerative Medicine, Department of Pathology, and Cancer Biology Program, Stanford University
School of Medicine, 265 Campus Drive, Stanford, CA 94305, USA
2Present Address: Department of Neurobiology, Harvard Medical School, 220 Longwood Avenue, Boston, MA 02115, USA
During development, diverse cellular identities are established and maintained in the embryo. Although
remarkably robust in vivo, cellular identities can be manipulated using experimental techniques. Lineage
reprogramming is an emerging field at the intersection of developmental and stem cell biology in which
a somatic cell is stably reprogrammed into a distinct cell type by forced expression of lineage-determining
factors. Lineage reprogramming enables the direct conversion of readily available cells from patients
(such as skin fibroblasts) into disease-relevant cell types (such as neurons and cardiomyocytes) or into
induced pluripotent stem cells. Although remarkable progress has been made in developing novel reprog-
ramming methods, the efficiency and fidelity of reprogramming need to be improved in order increase
the experimental and translational utility of reprogrammed cells. Studying the mechanisms that prevent
successful reprogramming should allow for improvements in reprogramming methods, which could have
significant implications for regenerative medicine and the study of human disease. Furthermore, lineage
reprogramming has the potential to become a powerful system for dissecting the mechanisms that underlie
cell fate establishment and terminal differentiation processes. In this review, we will discuss how transcrip-
tion factors interface with the genome and induce changes in cellular identity in the context of development
During development, cell fate is established and maintained by
complex regulatory networks of transcription factors that
promote expression of cell type-specific gene products and
repress regulators of other lineages. Once established, cellular
identity is remarkably stable despite numerous intrinsic and
extrinsic perturbations. This stability is likely the result of a
combination of multiple molecular features, including cis-
acting epigenetic modifications, such as DNA methylation,
posttranslational modifications of histone tails, nucleosome
positioning, incorporation of histone variants into nucleosomes,
and trans-acting regulatory factors such as sequence-specific
DNA-binding transcription factors, transcriptional coactivators,
noncoding RNAs, and chromatin remodeling complexes (Graf
and Enver, 2009; Ho and Crabtree, 2010; Yamanaka and Blau,
2010). Although generally stable in vivo, under certain experi-
mental conditions, cell fate can be dominantly reprogrammed
by forcing expression of transcription factors involved in the
establishment and maintenance of a distinct cellular lineage
(Figure 1). Identifying the relevant stimuli that can reprogram
one cell into another cell type of interest and understanding
how this process occurs are two key goals for the reprogram-
In this review, we will summarize the critical discoveries to
date, but only briefly discuss applications of cellular reprogram-
ming technologies for understanding human disease and
regenerative medicine (for more detailed reviews on these topics
see Graf, 2011; Gurdon, 2006; Holmberg and Perlmann, 2012;
Saha and Jaenisch, 2009; Vierbuchen and Wernig, 2011) and
instead focus on selected discoveries that have helped to iden-
ming. Furthermore, we propose that the nonphysiological direct
lineage reprogramming approaches will be useful for studying
physiological mechanisms of transcriptional reprogramming,
such as the establishment of cellular identity, as well as the
transcriptional regulatory networks that drive terminal differenti-
ation and functional maturation (Bussmann et al., 2009; Graf and
Enver, 2009; Vierbuchen and Wernig, 2011).
Brief Overview of Critical Discoveries in Epigenetic
Seminal work by Briggs, King, and Gurdon in the 1950s demon-
strated thatthe stability ofthe differentiated state isnottheresult
of irreversible genomic changes that occur during differentiation
(Briggs and King, 1952; Gurdon et al., 1958). This was demon-
strated by Somatic Cell Nuclear Transfer (SCNT), a technique in
which intact Xenopus nuclei from embryonic or adult cells are
transferred into an enucleated oocyte. Gurdon used this system
to demonstrate that nuclei from endoderm cells taken from tail-
bud stage frog embryos could successfully control the develop-
ment of new tadpoles. Later work showed that even nuclei from
terminally differentiated adult cells (e.g., blood cells, skeletal
muscle, kidney, and others) could generate Xenopus larvae
following nuclear transfer, albeit at reduced efficiency compared
to nuclei from embryonic cells (Gurdon, 2006; Pasque et al.,
2011). These results indicated that the oocyte contained power-
ful trans-acting reprogramming factors that could effectively
erase somatic epigenetic marks and return nuclei from differen-
tiated cells to a pluripotent state. However, it was not clear
whether these results were a testament to the unique molecular
Molecular Cell 47, September 28, 2012 ª2012 Elsevier Inc.
properties of oocytes or to the inherent plasticity of epigenetic
modifications acquired during development.
somatic cells could also exhibit cell fate plasticity (Taylor and
Jones, 1979). They found that treatment with 5-azacytidine, an
inhibitor of DNA methylation, caused fibroblasts to spontane-
ously differentiate into muscle and fat cells. This suggested
that DNA methylation is important for preventing expression of
genes that regulate differentiation into alternative lineages. In
the early 1980s,Blau and colleagues demonstrated thatmultinu-
cleated myotubes could dominantly reprogram nuclei from other
cell types to express muscle-specific gene products in hetero-
karyons (artificially fused cells that maintain distinct nuclei),
suggesting that reprogramming activity was not unique to the
oocyte and that the terminally differentiated state was actively
Figure 1. Experimental Systems for
Studying Nuclear Reprogramming
(A) Somatic cell nuclear transfer (SCNT). Nucleus
from a donor cell is inserted into an enucleated
oocyte. In mammals, the resulting cell can then be
cultured in vitro to derive nuclear transfer ESCs
(NT-ESCs), which can then be used to generate
cloned mice via standard blastocyst injection.
Alternatively, blastocysts can be derived from
oocytes in vitro (at low efficiency) and implanted
into pseudopregnant mice to develop. Measure-
ments of the efficiency of NT-ESC derivation,
blastocyst derivation from somatic nuclei, or the
generation of live pups can serve as a measure of
the efficiency of nuclear reprogramming.
(B) Cell fusion. Two distinct cell types are fused
together to generate chimeric cells with multiple
nuclei. In order to facilitate identification of tran-
scripts or proteins from each fusion partner, cells
from different species (e.g., human and mouse)
are often used. Fused cells can be purified using
fluorescent-activated cell sorting or by double
antibiotic selection. In heterokaryons, fused cells
maintain distinct nuclei and do not undergo cell
division (e.g., Bhutani et al., 2010). Cells can
also be selected for stable, dividing clones in
which nuclear fusion has occurred. These are
referred to as synkaryons or cell hybrids (e.g.,
Cowan et al., 2005).
(C) Transcription factor-mediated reprogramming.
Reprogramming transcription factors are intro-
or RNA transfection. Numerous cell fates can be
induced in addition to those shown in the figure
(see Figure 2 for a complete list). Strong artificial
promoters are generally used to ensure robust
expression. Inducible promoters (e.g., tetracy-
cline-inducible) can be used to shut off re-
programming factor expression to determine
whether cell fate reprogramming is stable in the
absence of exogenous of transcription factor
maintained by specific groups of trans-
acting factors (Blau et al., 1983). In 1987
Weintraub and colleagues showed that
the basic helix-loop-helix (bHLH) tran-
scription factor MyoD is sufficient to
convert fibroblasts into contracting myo-
cytes (Davis et al., 1987). However, when
retinal pigment epithelium, melanocytes, hepatocytes), activa-
tion of muscle markers was sometimes observed, but complete
reprogramming failed (Weintraub et al., 1989). This suggested
that a single transcription factor can be sufficient to initiate and
control the differentiation of a specific cell type, which provided
a possible mechanism for the control of terminal differentiation
processes during development (Weintraub, 1993). Gehring and
colleagues also provided dramatic proof of this principle by
showing that ectopic expression of the transcription factor
eyeless (Pax6 in mammals), a master regulator of eye develop-
ment, could generate functional eyes at various sites on the
body (Halder et al., 1995).
In 1996, Wilmut and colleagues successfully generated live
offspring from the nucleus of a mammalian somatic cell
Molecular Cell 47, September 28, 2012 ª2012 Elsevier Inc.
(Campbell et al., 1996). This important breakthrough reignited
interest in the field of epigenetic reprogramming. Nuclear trans-
fer was quickly accomplished in a variety of other species, but
the pairing of mouse genetics and nuclear transfer technology
proved to be especially fruitful, leading to a variety of insights
into the biology of pluripotency and the epigenetic control of
cell type specification (Hochedlinger and Jaenisch, 2006;
Wakayama et al., 1998). For example, mice were generated by
SCNT from the nuclei of adult lymphocytes and olfactory
neurons using a modified two-step nuclear transfer procedure
that involved first creating NT-ESCs (embryonic stem cells)
followed by injection into tetraploid blastocysts (Figure 1) (Eggan
et al., 2004; Hochedlinger and Jaenisch, 2002).
Encouraged by work in the SCNT field, as well as the demon-
stration that fusion of embryonic stem cells and fibroblasts could
activate pluripotency markers in somatic nuclei (Cowan et al.,
2005; Tada et al., 2001), Yamanaka and colleagues hypothe-
sized that reprogramming factors could be identified by their
specific expression in pluripotent cell types (Mitsui et al., 2003;
Takahashi and Yamanaka, 2006). Surprisingly, the combined
expression of 24 ESC-specific genes in mouse fibroblasts
yielded colonies of cells with pluripotent properties. After
systematic elimination, the four transcription factors Oct4,
Sox2, Klf4, and c-myc (OSKM) were shown to be sufficient for
this process, and further studies proved that these ‘‘induced
pluripotent stem (iPS) cells’’ were molecularly and functionally
equivalent to ESCs, including their capacity to contribute to
the germline (Maherali et al., 2007; Okita et al., 2007; Wernig
et al., 2007).
Again, a small group of transcription factors was able to reca-
pitulate complex developmental processes, similar to the MyoD
experiments mentioned above. However, an unresolved issue
was whether reprogramming to pluripotency was fundamentally
different than reprogramming to other somatic cell types. The
pluripotent state has been conceptualized as the ‘‘ground state’’
of cellular identity, and thus the pluripotent state could represent
a default response to the erasure of somatically acquired
epigenetic marks (Silva and Smith, 2008). However, reprogram-
ming from one somatic cell state to another would theoretically
require a highly specific erasure of the epigenetic marks of one
lineage, followed by the establishment of a new set of epigenetic
features characteristic of the new cell state. It is hard to conceive
how a transcription factor could directly control such a process,
as it seems unlikely that, for example, promoter and enhancer
elements of neuron-specific genes would be accessible for
transcription factor binding in fibroblasts or hepatocytes. For
these reasons it was assumed that lineage reprogramming
was possible between closely related cell types, such as fibro-
blasts-myocytes, lymphocytes-macrophages, or astrocytes-
neurons, because they are likely to share some epigenetic
features as a result of their recent descent from a common
progenitor cell and would thus provide a chromatin landscape
that was permissive for reprogramming factor binding and
activity (Graf and Enver, 2009; Vierbuchen and Wernig, 2011;
Zhou and Melton, 2008). For example, Graf and colleagues
had provided convincing evidence for direct conversion of
mature B cells into macrophages (Xie et al., 2004), Slack and
colleagues demonstrated acquisition of hepatic properties in
pancreatic cells (Shen et al., 2000), Gotz and colleagues showed
induction of neuronal traits in glial cells (Berninger et al., 2007;
Heins et al., 2002), and Melton and colleagues provided
evidence for the conversion of exocrine to endocrine pancreatic
cells following in vivo delivery of three transcription factors
(Zhou et al., 2008). However, like previous studies with MyoD,
the transcription factors used by Melton and colleagues were
insufficient to reprogram cells representing a different germ layer
such as embryonic fibroblasts (in vitro) or skeletal muscle cells
(in vivo) to endocrine pancreatic cells, suggesting again that
reprogramming between distantly related somatic cells might
not be possible.
In an attempt to clarify this issue, we decided to test whether
fibroblasts (representing a mesodermal lineage) could be re-
programmed into neurons (representing an ectodermal lineage).
transcription factors Brn2, Ascl1, and Myt1l (BAM) was sufficient
to convert fibroblasts to fully functional neuronal cells that we
termed induced neuronal (iN) cells (Vierbuchen et al., 2010). In
the following year, other groups used a similar approach to
show that fibroblasts can be reprogrammed into functional
cardiomyocyte-like and hepatocyte-like cells (Huang et al.,
2011; Ieda et al., 2010; Sekiya and Suzuki, 2011) (Figure 2).
These data confirmed that reprogramming between distantly
related somatic lineages is possible without passing through
the pluripotent state and suggested that reprogramming fibro-
blasts into pluripotent stem cells might represent an active
conversion process rather than a return to the default cellular
state following erasure of chromatin marks associated with
Reprogramming by Cell Fusion
As cells differentiate during development, they acquire lineage-
specific patterns of epigenetic modifications that reinforce cell
fate decisions and promote the faithful transmission of cellular
identity during cell division (Ho and Crabtree, 2010). Thus, the
pre-existingchromatin stateofacellrepresents apotentialroad-
block to lineage reprogramming. Experiments performed in
a variety of reprogramming systems suggest that lineage
conversion tends to be more difficult the more distantly related
the two cell types are. For example, Gurdon and colleagues
showed that the efficiency of deriving swimming tadpoles
decreases dramatically when nuclei from more differentiated
cells are used as donors for nuclear transfer (Gurdon, 2006;
Pasque etal.,2011). Similarly, nucleifrommouse ESCs generate
NT-ESCs following nuclear transfer at much higher efficiency
than more differentiated cells, and generation of live pups from
terminally differentiated cell types (e.g., olfactory neurons and
T cells) by NT is extremely inefficient without passage through
a NT-ESC state (Hochedlinger and Jaenisch, 2006; Yamanaka
and Blau, 2010). However, iPS cells have been derived from
most tissue types tested, albeit with variable efficiency, and in
some cases specific experimental modifications were required
for success (Hochedlinger and Plath, 2009).
Cell fusion experiments also indicated that cellular identity
affected the transcriptional response to ectopic trans-acting
factors. For example, although myotube heterokaryons could
activate muscle-specific genes in nuclei of cells derived from
Molecular Cell 47, September 28, 2012 ª2012 Elsevier Inc.
ectodermal lineages exhibited slower kinetics of transcriptional
activation, which suggested that the lineage-specific patterns
ramming factors found in myotubes (Blau et al., 1985). Building
on the experiments of Taylor and Jones (discussed above),
Blau and colleagues determined that pretreatment of fusion
partners with 5-azacytidine could elicit expression of muscle-
specific genes from previously nonresponsive HeLa cell nuclei
following fusion with myotubes, which suggested that pre-
existing DNA methylation in fibroblast nuclei is one barrier that
prevents ectopic induction of muscle genes during reprogram-
ming (Chiu and Blau, 1985).
More recent studies have established heterokaryons between
somatic cells and pluripotent stem cells. In this system, nuclei
from somatic cells rapidly activate genes associated with pluri-
et al., 2010; Pereira et al., 2010; Piccolo et al., 2011). However,
due to the nature of heterokaryon formation, it is difficult to
analyze the extent and stability of fusion-mediated reprogram-
ming. Heterokaryons have also been used to investigate the
requirement of specific genes for reprogramming activity in the
pluripotent fusion partner. For example, transcriptional activa-
tion of some pluripotency genes required AID-mediated DNA
demethylation (Bhutani et al., 2010), and Oct4 (but not Sox2)
and polycomb complex activity were also required for complete
activation of pluripotency genes in human lymphocytes (Pereira
et al., 2008; 2010). Recently, Lahn and colleagues generated
stable, dividing hybrid cell lines from fused rat fibroblasts and
mouse ESCs, but were unable to detect AID expression in this
system, perhaps as a result of the different methods used
(Foshay et al., 2012). Further studies are certainly needed to
clarify the details of reprogramming kinetics in hybrid cell lines
and heterokaryons and to what extent the ratio of nuclei in fusion
products and the species of donor nuclei affects reprogramming
activity (e.g., Palermo et al., 2009).
In myotube-fibroblast cell fusion, it has been proposed that
some myogenic genes are activated more slowly in fibroblast
nuclei (Gaetz et al., 2012). This suggests that some genes can
be readily activated by trans-acting factors while others might
be kept silent by specific cis-acting epigenetic modifications
(referred to as ‘‘occluded’’ genes) (Lahn, 2011). Surprisingly,
when bacterial artificial chromosomes (BAC) containing these
‘‘occluded’’ genes (e.g., Myf5) with their associated regulatory
Figure 2. Transcription Factor-Mediated Conversion of Fibroblasts into Diverse Cellular Lineages
Summary of thediverse celltypes generated directlyfrom mouse and human fibroblastsby lineagereprogramming.Factors listed in parentheses are required for
Caiazzo et al., 2011; Davis et al., 1987; Feng et al., 2008; Huang et al., 2011; Ieda et al., 2010; Kajimura et al., 2009; Lujan et al., 2012; Pang et al., 2011; Pfisterer
et al., 2011; Qiang et al., 2011; Sekiya and Suzuki, 2011; Son et al., 2011; Szabo et al., 2010; Takahashi and Yamanaka, 2006; Yoo et al., 2011.
Molecular Cell 47, September 28, 2012 ª2012 Elsevier Inc.
regions are transfected into mouse fibroblasts, they tend to be
transcriptionally active. However, in fibroblasts from Myf5/6
BAC-transgenic mice, the Myf5-BAC reporter was not activated.
This suggests that cell type-specific cis-acting epigenetic marks
acquired during development prevent the expression of
occluded genes in heterokaryons and that fibroblasts do not
initiate epigenetic silencing of these loci de novo. No specific
DNA methylation status, histone modification pattern, or binding
of chromatin factors was found in promoters of ‘‘occluded’’
genes, raising the possibility that enhancer elements may be
more informative or that other, as yet unidentified marks could
provide an explanation for the differential gene activation
(Lahn, 2011). Unbiased genome-wide analysis of histone modifi-
cations may provide further insight into the chromatin dynamics
of those two classes of loci.
Given its muscle-inducing activity in fibroblasts, MyoD is
certainly a strong candidate for a trans-acting factor responsible
for reprogramming in fibroblast-myotube heterokaryons. It is
clear, though, that there are other factors involved in hetero-
karyon reprogramming, For example, MyoD was not sufficient
to activate muscle genes in cells derived from different germ
layers, such as hepatocytes, whereas cell fusion was (Miller
et al., 1988). Furthermore, heterokaryon formation between
MyoD-expressing hepatocytes and fibroblasts could also acti-
vate muscle-specific genes in the hepatocyte nucleus, showing
that fibroblasts contain additionally required trans-acting factors
(Scha ¨fer et al., 1990).
It is important to point out that in cell fusion and nuclear
implantation experiments, cells are exposed to much lower
(i.e., closer to physiological) levels of the reprogramming factors,
which are limited to genes expressed in the fusion partners
(Figure 1). Although iPS cell reprogramming factors are all ex-
pressed in ESCs, the transcription factors used for lineage
conversion in some other tissues are not always expressed in
the target cell type. For example, the key iN cell reprogramming
factor Ascl1 is not expressed in mature, differentiated neurons,
and therefore fibroblast-neuron heterokaryons might fail to acti-
vate neuronal genes in fibroblast nuclei (although to our knowl-
edge this experiment has not yet been performed). Thus, it is
important to consider that in this context a failure to reprogram
is not necessarily due to insurmountable barriers encoded in
chromatin, when it can also be explained by the absence of
essential reprogramming factors or cofactors (as was demon-
strated in MyoD-expressing hepatocytes fused to fibroblasts).
Reprogramming by Forced Expression of Transcription
As described earlier, it is now well established that transcription
factors can induce distantly related cell fates (Figure 2) (Vierbu-
chen and Wernig, 2011). We believe these experimental systems
provide an excellent opportunity to investigate the mechanisms
of reprogramming. Compared to cell fusion methods, reprog-
ramming with transcription factors is a much simpler experi-
mental system because the reprogramming factors are more
clearly defined. Currently, insights into direct reprogramming
between distantly related cell types have been gained exclu-
sively by studying the mechanism of reprogramming cells to plu-
ripotency using the OSKM factors. These studies have benefited
from rigorous functional assays and the fact that the transcrip-
tional networks that control pluripotency are relatively well
understood. However,theutility ofthissystem forunderstanding
the mechanisms controlling reprogramming is limited by the low
efficiency of reprogramming, the prolonged period required to
required for the acquisition of pluripotency, and the multitude of
cell divisions that are required (Hanna et al., 2010). For these
reasons, direct conversion of fibroblasts to neurons and other
distantly related cell types provides a perfect complement to
theiPScell systemfor understanding
processes. For example, iN cell reprogramming using mouse
embryonic fibroblasts can reach efficiencies of 20% and gener-
2010).Whenmoreclosely related neonatal astrocytes were used
as donor cells, neuronal induction required fewer factors and
was more efficient and rapid (Heinrich et al., 2010). Also,
MyoD-mediated induction of the muscle fate was reported to
be up to 50% efficient, and dramatic morphological changes
(e.g., formation of multinucleate cells) occurred within 3 days
after induction (Davis et al., 1987). Similarly, in vitro conversion
of a pre-B cell line into macrophages by forced expression of
C/EBPa can induce phenotypic characteristics of macrophages
as early as 10 hr after induction with nearly 100% efficiency
(Bussmann et al., 2009). The complementary nature of these
tools should provide insights into the transcriptional and epige-
netic changes that occur during reprogramming and can begin
to elucidate general principles of reprogramming that are shared
between diverse systems. In summary, both the epigenetic state
of the donor cells and trans-acting factors regulate the activation
of previously silent genes in the context of lineage reprogram-
ming. However, it is also clear that specific experimental manip-
ulations, such as treatment with histone deacetylase inhibitors
(HDAC inhibitors), 5-azacytidine, or other inhibitors of epigenetic
programming in various reprogramming protocols (Xu et al.,
Targeting Reprogramming Transcription Factors to the
For reprogramming to occur, it is probably necessary that tran-
scription factors be able to access a majority of their relevant
binding sites (i.e., the sites that they bind to in order to regulate
gene expression during normal differentiation processes from
tissue-specific stem cells). However, due to the preponderance
of potential binding sites in the genome for most transcription
factors, binding is thought to be constrained to a specific subset
ofpotential sitesbyboththe celltype-specific chromatincontext
in which the transcription factor is normally expressed and/or by
a requirement for cell type-specific cofactors that bind to the
adjacent regulatory elements and promote stable interaction
with DNA. For example, the bHLH transcription factor Scl/Tal,
which recognizes the generic E-Box motif CANNTG, binds to
a different set of sites in the different hematopoietic cell types
in which it is expressed (Palii et al., 2011; Wilson et al., 2010).
Similarly, certain bHLH transcription factors also control differ-
entiation more than one type of cell (e.g., Ngn3, Ascl2) during
development, which could also involve binding to different
Molecular Cell 47, September 28, 2012 ª2012 Elsevier Inc.
regulatory regions in the genome and/or interaction with cell
type-specific cofactors (Bertrand et al., 2002). With respect to
use of transcription factors in reprogramming, these observa-
tions suggest a potential paradox: how is specificity of DNA
binding achieved in the absence of some or all context-specific
cues? As we will later discuss in greater detail, one potential
explanation is that the reason multiple factors are required for
reprogramming between distantly related cell types in order to
provide the cooperative interactions necessary to confer binding
specificity or stability lacking in the cell type to be reprog-
rammed. A second possibility is that the reprogramming factors
can actas‘‘pioneer’’transcription factors,meaning thattheycan
recognize their target sites irrespective of the pre-existing chro-
matin state (Zaret and Carroll, 2011). These scenarios are not
mutually exclusive and could provide a conceptual starting point
to study how successful reprogramming is accomplished and
why under many conditions reprogramming fails (see Figure 3
for a summary). It also raises important questions about how
transcription factors regulate cellular differentiation during
development. For example, how does cell lineage affect the
activity of lineage-specific transcription factors (Tapscott,
2005)? More specifically, how do transcription factors initiate
cell type-specific differentiation programs when initially acti-
vated in a stem/progenitor cell type?
Cis-Acting Repression of Reprogramming by Chromatin
It is generally believed that cells sequester unneeded genes into
densely packed heterochromatin, which is thought to inhibit
binding of transcription factors, thus preventing activation of
transcription at that locus (Beisel and Paro, 2011). The combina-
tion of classic methods to study accessibility, such as DNase
sensitivity, with high-throughput sequencing have made it
possible to map accessible regions genome-wide, which has
begun to provide insight into the relationship between transcrip-
tion factor binding and DNA accessibility on a global scale (Bell
et al., 2011). Careful studies of the PHO5 promoter in yeast
have demonstrated the functional consequences of DNA acces-
sibility on transcriptional activation. Nucleosomes positioned in
the PHO5 promoter region limit Pho4 binding to a single acces-
sible site in the promoter, which allows Pho4 to recruit chromatin
remodeling proteins that remove or shift these nucleosomes
and make additional Pho4 binding sites in the promoter
accessible (Almer and Ho ¨rz, 1986; Bell et al., 2011). Similarly,
on a genome-wide scale, cell type-specific patterns of DNA
accessibility appear to be important for limiting binding of the
glucocorticoid receptor (GR) to a subset of its response
elements (John et al., 2011). These data provide compelling
evidence that nucleosome positioning plays an important role
Figure 3. Models of Transcription Factor
Binding during the Initiation of
(A) Permissive enhancer model (Taberlay et al.,
2011). Genes that have promoters that exhibit
marks of polycomb-mediated epigenetic silencing
(i.e., H3K27me3) can have enhancer elements that
exist in a permissive state (i.e., H3K4me1-
enriched) and allow for reprogramming factor
binding and subsequent chromatin remodeling at
(B) Pioneer factor model. Reprogramming factors
with pioneer activity can bind nucleosomal DNA
and can thus access cis-regulatory elements that
exist in a chromatin state that is thought to
preclude binding of most transcription factors.
Pioneer factor binding can displace nucleosomes
and recruit chromatin modifying proteins or addi-
tional transcription factors, leading to activation of
previously ‘‘nonpermissive’’ genes.
(C) Spontaneous accessibility. Reprogramming
transcription factors bind to cis-regulatory regions
during transient unwrapping of nucleosome-
bound DNA that would normally be sterically
occluded. High levels of reprogramming factor
expression could help to increase the likelihood of
this occurring at a high enough fraction of impor-
tant cis-regulatory regions that it is relevant for
(D) Accessibility during cell division. Reprogram-
occluded cis-regulatory regions during cell divi-
sion. Copying the genome requires pre-existing
nucleosomes to be temporarily displaced and the
insertion of initially unmodified histones into newly
copied DNA strands. These processes are likely to
interrupt the stable epigenetic silencing of these
loci and could thus allow reprogramming factors
(especially when expressed at high levels) to bind
to these temporarily available sites and cause
chromatin remodeling, leading to stable tran-
Molecular Cell 47, September 28, 2012 ª2012 Elsevier Inc.
in restricting transcription factor binding to specific sites. How-
ever, during reprogramming, transcription factors need to gain
access to (presumably) occluded cis-regulatory regions. Thus,
the success of reprogramming suggests that these cis-acting
roadblocks can be overcome by forced expression of transcrip-
tion factors (Figure 3).
To What Extent Are Transcription Factor Binding Sites
In the ‘‘immediate access’’ scenario, transcription factor access
to the critical cis-regulatory elements of a gene is rapid and
unimpeded by cis-acting chromatin modifications. This could
happen in one of two ways. First, target regions could be in
a heterochromatic, ‘‘repressive’’ state (e.g., trimethylated his-
tone H3 lysine 27 (H3K27me3-enriched), bound by polycomb
complexes, nucleosome-enriched). Alternatively, the region of
interest could be in a pre-existing ‘‘permissive’’ chromatin state
(e.g., H3K4me1-enriched, nucleosome-depleted) that allows
unimpeded binding of reprogramming factors. The first scenario
would suggest that reprogramming can only be accomplished
by pioneer transcription factors (Zaret and Carroll, 2011).
Pioneer transcription factors are thought to be the first to bind
to lineage-specific regulatory elements during organogenesis,
which can help to displace nucleosomes (either through active
or passive mechanisms) and create a permissive binding envi-
ronment for other regulatory factors that do not have pioneer
activity. This model is supported by the fact that many transcrip-
pioneer factors during development and have been shown to be
capable of binding to nucleosomal DNA in vitro (e.g., GATA and
FOXA family transcription factors; see Figure 1) (Cirillo et al.,
2002). The idea that many sites are in fact permissive for binding
is intriguing because it would be a simple molecular explanation
for why closely related cell types are more amenable to lineage
conversion. Jones and colleagues recently provided experi-
mental support for this idea (Taberlay et al., 2011). Using a
well-characterized MyoD enhancer and promoter as an experi-
mental starting point, they examined how the pre-existing chro-
matin configuration of these autoregulatory elements affects
MyoD binding in various cell lines that do not express MyoD.
They found that a minimal MyoD enhancer element could exist
in a ‘‘permissive’’ state for MyoD binding (nucleosome-depleted,
H3K4me1-enriched, flanked by H2AZ-containing nucleosomes,
not bound by polycomb complex components or enriched for
H3K27me3) even when the promoter region exhibited a ‘‘repres-
sive’’ chromatin state (see Figure 3). This ‘‘multivalent’’ epige-
netic state of the MyoD locus allowed for MyoD binding to the
enhancer element in the first 24 hr after ectopic MyoD expres-
sion, which caused nucleosome displacement at the promoter
region and the acquisition of H3K4me3 enrichment by 48 hr,
but was insufficient to induce transcription from the MyoD locus.
Conversely, in a colorectal cancer cell line in which both the
MyoD promoter and proximal enhancer were nucleosome
bound, ectopic MyoD binding was not observed. Thus, the pres-
ence of enhancer elements in a permissive state might be able to
predict whether a gene can be activated by reprogramming
factors. Using computational approaches and publicly available
ChIP-seq data, the authors found that this type of permissive
enhancer is present at a substantial fraction of polycomb-
repressed genes across a wide range of human cell types,
providing a potential explanation for global epigenetic remodel-
ing induced by ectopic transcription factors. Of note, using
ChIP-seq, MyoD was recently shown to bind predominantly to
accessible sites in fibroblasts (Fong et al., 2012).
Interestingly, the MyoD enhancer also contains an Oct4
binding site. When Oct4 is expressed in cell lines with a permis-
sive MyoD enhancer, Oct4 binds almost immediately to the
enhancer and promotes formation of a bivalent chromatin state
at the MyoD promoter, which is similar to the chromatin state
of the MyoD promoter in ESCs. This suggests that diverse re-
programming factors can gain immediate access to critical
regulatory elements at individual genes through these permis-
sive enhancer elements and catalyze reprogramming of the
It will likely be informative to examine how these permissive
enhancers are established and maintained during normal
differentiation processes and to determine to what extent their
locations vary between different cell types. Recent work has
identified a chromatin signature for ‘‘active’’ enhancer elements,
which are distributed across the genome in highly cell type-
specific patterns (Creyghton et al., 2010; Rada-Iglesias et al.,
2011). The identification of permissive enhancers genome-
wide could help to provide a rational method for determining
observations could also provide a mechanistic explanation for
the ‘‘occluded’’ genes observed in heterokaryon experiments
(Lahn, 2011). For example, a transcriptionally silent gene would
be occluded if its enhancers are not permissive for DNA binding.
However, ChIP-seq studies of MyoD binding in fibroblasts and
myotubes showed no evidence of dramatic differences in
MyoD occupancy between these two cell types, consistent
with the fact that MyoD can efficiently reprogram fibroblasts to
functional muscle cells (Cao et al., 2010). The immediate access
model predicts that reprogramming factor binding at critical
target genes should be detectable shortly after introduction
into the cells. Most studies of reprogramming have focused on
later time points, so surprisingly little is known about global
changes in transcription and chromatin modifications during
the earliest phases of reprogramming. Work by Tapscott and
colleagues detailing the kinetics of gene activation following
MyoD expression in fibroblasts indicated that MyoD targets
exhibit gene-specific kinetics of activation (Bergstrom et al.,
2002). For example, a subset of MyoD targets can be activated
within 6 hr of MyoD induction, and in some cases these genes
can be activated in the absence of protein synthesis, suggesting
that they are activated directly by MyoD (potentially with help
from fibroblast trans-acting factors). At genes that exhibit slower
kinetics of activation, MyoD binding is not detected at early time
points, but appears to increase concomitantly with transcrip-
tional activation, suggesting that MyoD either does not have
access to these regions at early time points or that it can access
the DNA but is missing cofactors that might stabilize its interac-
tion with these sites. It is also possible that binding to enhancers
of late-stage genes precedes binding to the promoter regions
assessed for MyoD binding in this paper, as suggested by Jones
and colleagues (discussed above) (Taberlay et al., 2011).
Molecular Cell 47, September 28, 2012 ª2012 Elsevier Inc.
Repeating these experiments and measuring genome-wide
MyoD binding at similarly early time points could begin to clarify
this issue. These data also demonstrate the idea that feed-
forward activation of gene expression by MyoD helps to pattern
the temporal activation of its target genes even when it is
expressed ectopically in fibroblasts (Tapscott, 2005). Because
the regulatory regions of each of its target genes have specific
requirements for activation, correct temporal activation of
MyoD downstream genes are thought to be controlled by
different requirements for cooperating factors (e.g., bFGF
signaling, p38 and MAPK signaling, and MEF2, Pbx/Meis, and
Six family transcription factors), some of which are downstream
targets of MyoD themselves (Aziz et al., 2010).
Graf and colleagues observed even more rapid kinetics of
reprogramming during in vitro lineage conversion between B
cells and macrophages by forced expression of the transcription
factor C/EBPa (Bussmann et al., 2009). In these studies, induc-
tion of C/EBPa in a highly homogeneous B cell line leads to
dramatic gene expression changes within 3 hr. Further analysis
of C/EBPa binding and the pre-existing chromatin state at the
macrophage-specific gene regulatory elements in B cells might
clarify the influence of chromatin on reprogramming factor
binding and transcriptional activation in this reprogramming
The idea of immediate access is also consistent with the
finding that short-term expression of iPS cell reprogramming
factors predominantly induces the activation of genes whose
promoters are marked by H3K4me3 in fibroblasts (Koche
et al., 2011). Surprisingly, out of five histone modifications
examined, only the H3K4me2 mark appeared to be dynamic at
early stages of reprogramming, but these changes were not
associated with transcriptional activation. Instead, H3K4me2
enrichment was found largely at putative enhancer elements
(defined in this case as nonpromoter elements with H3K4me2
enrichment), which showed a shift away from fibroblast-specific
enhancers to ESC-specific enhancers. In a related study,
Plath and colleagues examined promoter binding of the iPS
cell reprogramming factors in both partially reprogrammed and
fully reprogrammed mouse iPS cells (Sridharan et al., 2009).
During reprogramming, stable lines of partially reprogrammed
cells can be established that share some morphological, func-
tional, and molecular characteristics with ESCs, but have failed
to activate the complete program of pluripotency (Meissner
et al., 2007). It is also known that in clonal populations of partially
reprogrammed cells, complete reprogramming can occur
spontaneously at a low frequency, suggesting that stochastic
events are required for the transition to a fully pluripotent
state (Meissner et al., 2007). Interestingly, the OSKM factor
binding sites in partially reprogrammed cell lines were sub-
stantially different than those in ESCs. The authors proposed
that differential binding might be explained by the absence of
Nanog (and potentially other factors) in the partially reprog-
rammed cells, as the Nanog DNA binding motif was enriched
in sites bound in ESCs, but not in partially reprogrammed
cells. These data are consistent with reprogramming occurring
as a step-wise process, in which access to certain DNA
regulatory regions is dependent on specific cofactors that
are induced during the process of reprogramming and argue
against a pioneering mechanism for the iPS cell reprogramming
Access to Critical Regulatory Elements
by Nonphysiological Mechanisms
In another model, a transcription factor gains access to impor-
tant regulatory regions by means of a nonphysiological mecha-
nism. For example, high levels of reprogramming factor
expression, which are typical of lineage reprogramming experi-
ments, might allow for limited and transient access to important
binding sites during normal nucleosome turnover or stochastic
unwinding of nucleosomal DNA, which causes the nucleosome
to be displaced and allows for the subsequent recruitment of
chromatin modifying complexes to stably remodel the chromatin
ming to pluripotency have suggested that stochastic events,
presumably related to the cell cycle, are likely to be rate limiting
during certain phases of reprogramming (Hanna et al., 2009). For
example, the increased efficiency of reprogramming in p53-
deficient cells is cell cycle dependent, and targeted chromatin
remodeling seen during early phases of reprogramming seems
to be enhanced in cells that have undergone multiple rounds of
cell division (Koche et al., 2011). How might cell division promote
reprogramming? During cell division, nucleosomes must be
partitioned to newly synthesized DNA, and it is not known to
what extent histone modifications are replaced when newly
synthesized histones are incorporated into nucleosomes (Probst
et al., 2009). Presumably, new and recycled histone subunits are
distributed randomly among the two double helices. Thus, cell
division could provide a window for transcription factor access
to otherwise occluded cis-regulatory regions, which could
prevent the subsequent re-establishment of repressive marks
or positioned nucleosomes at these loci.
Although cell cycle obviously plays an important role in iPS
cell reprogramming, many somatic lineage reprogramming
experiments do not require cell division, such as iN cell reprog-
conversion (Di Tullio and Graf, 2012; Heinrich et al., 2010; Marro
et al., 2011; Vierbuchen et al., 2010). In these cases, transient
transcription factor binding could also be mediated by
stochastic, localized alterations of histone marks due to random
activity of chromatin modifying enzymes, referred to as chro-
matin ‘‘breathing.’’ In agreement with this idea is the observation
that pharmacological HDAC inhibition has been shown to
promote reprogramming in a variety of systems (Huangfu et al.,
2008; Xu et al., 2008). HDAC activity might be important for
reinforcing specific chromatin states at gene regulatory
elements, which when disrupted could potentially lead to global
changes in the patterns of occluded genes. It is also possible
that HDAC inhibition could cause transcriptional activation of
tors necessary for reprogramming.
Repression of Reprogramming Activity by Cell
Type-Specific Trans-Acting Factors
Heterokaryon experiments provided evidence that cellular
identity is established and actively maintained by trans-acting
factors (Yamanaka and Blau, 2010). Furthermore, fusion of
Molecular Cell 47, September 28, 2012 ª2012 Elsevier Inc.
keratinocytes and muscle cells demonstrated that gene
activation in heterokaryons is in fact bidirectional, and the ratio
of regulatory factors between the two cell types (heterokaryons
often have more than two nuclei) determines which fate is
dominant (Palermo et al., 2009). During transcription factor-
mediated reprogramming, high expression levels of reprogram-
ming factors (often from strong artificial promoters) are likely to
help facilitate cell fate conversion. However, in some cases
high expression alone is not sufficient for reprogramming factors
to override the transcriptional program of the host cell. For
example, mature B lymphocytes could only be reprogrammed
to pluripotency by the OSKM factors with the concomitant
knockdown of the B cell transcription factor Pax5 or overexpres-
sion of C/EBPa, suggesting that pre-existing lineage-specific
transcriptional programs are an impediment to reprogramming
may be more general repressors of cell fate changes. The REST/
NRSF complex, which blocks expression of neuronal genes in
nonneural cell types, may be one such example (Chong et al.,
1995). For example, recent work has shown that conditional
ablation of REST in fibroblasts causes an upregulation of some
neuronal genes, but does not appear to cause overt neuronal
conversion (Aoki et al., 2012).
Cell type-specific microRNAs are a second mechanism em-
ployed by various cell types to prevent translation of lineage-
inappropriate transcripts (Hornstein and Shomron, 2006).
Accordingly, the brain-specific microRNAs miR-9 and miR-
124 could promote the generation of iN cells from human fibro-
blasts when combined with the transcription factors ASCL1,
NEUROD2, and MYT1L (Yoo et al., 2011). These miRNAs have
been previously shown to downregulate Baf53a, REST,
Co-REST, and PTBP1, which are all thought to actively prevent
neuronal fate acquisition (Yoo et al., 2009). Even more surprising
was the claim that iPS cell reprogramming could be achieved by
miRNAs in combination with HDAC inhibition (Anokye-Danso
et al., 2011; Miyoshi et al., 2011). It remains unclear how these
miRNAs, which are not thought to be capable of directly
activating gene expression, can induce pluripotency genes.
One explanation may be the inhibition of potential repressors
of pluripotency-associated genes analogous to REST for the
neuronal lineage. Another possibility is that miRNAs are indeed
able to upregulate gene products (Machlin et al., 2011).
Another potential mechanism for interference with transcrip-
tional activity is the competition of exogenous and endogenous
transcription factors for shared generic cofactors. This has been
proposed as a potential mechanism helping to regulate neuronal
versus glial cell fate choice in cultured neural stem/progenitor
cells (Sun et al., 2001). In this system, forced expression of the
bHLH transcription factor Ngn1 efficiently induces neurogenesis
and also blocks acquisition of astrocyte fate. The induction of
neurogenesis requires Ngn1 transcriptional transactivation
whereas inhibition of astrocyte fate is DNA binding independent.
Instead of directly suppressing astrocyte genes, Ngn1 acts to
sequester CBP-Smad1-activating complexes from genes that
promote astrocytic fate, such as STAT transcription factors.
Furthermore, lineage-specific bHLH factors (such as Ascl1
and MyoD) require heterodimerization with widely expressed
E-proteins (Tcf3) for DNA binding (Bertrand et al., 2002). Thus,
high levels of ectopic bHLH factor expression might limit the
pool of E-proteins available to endogenous bHLH transcription
factors, which could lead to downregulation of some elements
of the host transcriptional program, and thus facilitating changes
in cellular identity.
Fidelity of Lineage Conversion: Epigenetic Memory
‘‘Epigenetic memory’’ refers to remnants of transcriptional pro-
perties or chromatin features typical of the starting cell type
after reprogramming. The persistence of epigenetic memory is
a critical issue in the reprogramming field because it has the
potential to modify the behavior of reprogrammed cells, which
could have large consequences for in vitro disease modeling
studies and any future clinical applications of reprogrammed
cells. Gurdon and colleagues demonstrated that there is epige-
netic memory in cloned Xenopus embryos derived from embry-
onic nuclei that had initiated expression of lineage-specific
regulators. For example, 81% of embryos derived from neuro-
ectodermal nuclei (Sox2+) inappropriately expressed Sox2
in endoderm (Ng and Gurdon, 2005). This suggests that an
epigenetic mark of transcriptional activity persisted through
the reprogramming process, leading to lineage-inappropriate
Sox2 expression. Similarly, epigenetic memory of MyoD ex-
pression in nuclear transfer embryos derived from muscle
cell nuclei could persist for 24 cell divisions during reprogram-
ming (in the absence of MyoD transcription) and correlated
with K4-trimethylated H3.3 retention at the MyoD promoter
(Ng and Gurdon, 2008).
There is also evidence that iPS cells exhibit specific transcrip-
tional and epigenetic signatures associated with their cell type of
origin (Kim et al., 2010, 2011; Polo et al., 2010). This epigenetic
continued passage in vitro, or differentiation followed by subse-
quent reprogramming. In contrast, early-passage ESCs derived
from nuclear transfer exhibited less evidence for epigenetic
memory, perhaps because of differences in the kinetics of
DNA demethylation that occurs during reprogramming in these
two systems (Kim et al., 2010). Similarly, iN cells derived from
hepatocytes were shown to efficiently downregulate the hepato-
cyte-specific transcriptional program, but small remnants of
hepatic gene expression were detectable on the single-cell level
(Marro et al., 2011). Further work will be required to determine
the extent to which epigenetic memory affects the functional
properties of reprogrammed cells. It will also be interesting to
determine the molecular basis of epigenetic memory, whether
different reprogramming methods lead to more or less epige-
netic memory, and whether certain loci are more resistant to
complete reprogramming than others, as has been seen during
reprogramming to pluripotency under certain experimental
conditions (Carey et al., 2011; Stadtfeld et al., 2012).
Cellular reprogramming research has been energized by its
potential asa critical tool for the next generation of medical diag-
nostics and cell-based regenerative therapies, as well as the
study of human embryonic development and organogenesis. It
is now possible to generate a large variety of cell types in vitro
from induced pluripotent stem cells derived from human
Molecular Cell 47, September 28, 2012 ª2012 Elsevier Inc.
patients. This provides unprecedented access to rare popula-
tions of genetically matched cells such as motor neurons or
cardiomyocytes that are nearly impossible to obtain from live
patients (Saha and Jaenisch, 2009). These cell types can then
be rigorously characterized and compared to matching cell
types from healthy individuals, with the hope that specific
manifestations of the disease process are recapitulated in vitro
(Han et al., 2011; Ming et al., 2011). The recent progress in direct
lineage reprogramming also suggests that readily available
human fibroblasts can be directly converted into cells resem-
bling several types of neurons found in the central nervous
system (Caiazzo et al., 2011; Pang et al., 2011; Pfisterer et al.,
2011; Yoo et al., 2011). These human iN cells have even shown
some potential to demonstrate specific in vitro manifestations
of disease (Qiang et al., 2011) (see Figure 1). The combination
of these approaches should provide a powerful toolkit to study
human disease processes and the developmental biology of
human tissues in a culture dish, which could have transformative
consequences for regenerative medicine.
Our work is generously supported by the National Institutes of Health (NIH
grants RC4 NS073015-01, R01MH092931, AG010770-18A1), the California
Institute for Regenerative Medicine (CIRM grants DR1-01454, RT2-02061),
the Department of Defense (PR100175P1), the Ellison Medical Foundation,
the Stinehart-Reed Foundation, and the Baxter Foundation. M.W. is a New
York Stem Cell Foundation-Robertson Investigator and T.V. is a California
Institute for Regenerative Medicine predoctoral fellow (TG2-01259). We would
also like to thank Karen Jann for generating all of the figure illustrations.
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