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Cell Stem Cell
Article
Klf4 Organizes Long-Range Chromosomal Interactions
with the Oct4 Locus in Reprogramming and Pluripotency
Zong Wei,1,2 Fan Gao,1,2,3,4 Sewoon Kim,1,2 Hongzhen Yang,1,2 Jungmook Lyu,5 Woojin An,2 Kai Wang,3,4
and Wange Lu1,2,*
1Eli
and Edythe Broad Center for Regenerative Medicine and Stem Cell Research
of Biochemistry and Molecular Biology
3Zilkha Neurogenetic Institute
4Department of Preventive Medicine and Psychiatry
University of Southern California, Los Angeles, CA 90033, USA
5Catholic Institute for Visual Science, Department of Ophthalmology and Visual Science, College of Medicine,
The Catholic University of Korea, 137-040, Seoul, Korea
*Correspondence: [email protected]
http://dx.doi.org/10.1016/j.stem.2013.05.010
2Department
SUMMARY
Epigenetic mechanisms underlying somatic reprogramming have been extensively studied, but little
is known about the nuclear architecture of pluripotent stem cells (PSCs). Using circular chromosome conformation capture with high-throughput
sequencing (4C-seq) and fluorescence in situ hybridization (FISH), we identified chromosomal regions
that colocalize frequently with the Oct4 locus in
PSCs. These PSC-specific long-range interactions
are established prior to transcriptional activation of
endogenous Oct4 during reprogramming to induced
PSCs and are facilitated by Klf4-mediated recruitment of cohesin. Depletion of Klf4 leads to unloading
of cohesin at the Oct4 enhancer and disrupts longrange interactions prior to loss of Oct4 transcription
and subsequent PSC differentiation, suggesting a
causative role for Klf4 in facilitating long-range interactions independent of its transcriptional activity.
Taken together, our results delineate the basic
nuclear organization at the Oct4 locus in PSCs and
suggest a functional role for Klf4-mediated higherorder chromatin structure in maintaining and
inducing pluripotency.
INTRODUCTION
The discovery of direct reprogramming by overexpression of
the transcription factors Oct4, Sox2, cMyc, and Klf4 has
transformed stem cell research and inspired immense interest
in the application of induced pluripotent stem cells (iPSCs) to
therapeutic purposes (Takahashi and Yamanaka, 2006; Yu
et al., 2007). Understanding the molecular mechanisms underlying direct reprogramming is critical to generate high quality
iPSCs (Hanna et al., 2010; Orkin and Hochedlinger, 2011).
Changes in various epigenetic modifications and histone modifiers observed at early reprogramming stages support the hypothesis that reprogramming requires conversion from a
36 Cell Stem Cell 13, 36–47, July 3, 2013 ª2013 Elsevier Inc.
somatic to a pluripotent chromatin state (Doege et al., 2012;
Koche et al., 2011; Mansour et al., 2012; Onder et al., 2012).
Nonetheless, the dynamics of that higher-order chromatin structure remain mostly unclear.
Pioneering experiments in nuclear transfer revealed that nuclear enlargement and chromatin decondensation precede alterations in gene expression (Gurdon and Melton, 2008). The
distinct nuclear architecture of PSCs compared to somatic cells
(Jaenisch and Young, 2008; Meshorer et al., 2006) suggests that
somatic cells undergo enormous changes in higher-order chromatin structure during reprogramming. Furthermore, since these
higher-order structures are implicated in genome-wide transcriptional activity (Chakalova and Fraser, 2010), changes in
nuclear architecture occurring during reprogramming may
significantly drive transformations in both the epigenome and
the transcriptome required to establish induced pluripotency.
The recent development of chromosome conformation capture (3C)-based methods has enabled detection of long-range
interactions and discovery of novel interactions locally and
genome-wide (Dekker et al., 2002; Dixon et al., 2012; Fullwood
et al., 2009; Lieberman-Aiden et al., 2009; Simonis et al., 2006;
Zhao et al., 2006). Long-range interactions that recruit distant
enhancers in cis and in trans have various biological functions
(Williams et al., 2010). In PSCs long-range intrachromosomal interactions have been described for the Nanog and Gata4 loci,
which are important for self-renewal and differentiation, respectively (Levasseur et al., 2008; Tiwari et al., 2008). Nevertheless,
how these dynamic changes in chromatin organization are regulated and their relevance to activation of pluripotency circuitry
remain topics of intense investigation.
In the present study, we focused on the pluripotency master
gene Oct4 (also known as Pou5f1), an essential player in the pluripotency circuitry (Kim et al., 2009) known to be indispensible
during late stages of reprogramming (Anokye-Danso et al.,
2011; Heng et al., 2010; Kim et al., 2009; Plath and Lowry,
2011). To trace dynamic changes occurring at the Oct4 locus
in single cells over the course of reprogramming, we applied
circular chromosome conformation capture (4C-seq) and fluorescence in situ hybridization (FISH) to identify a group of chromosomal regions in cis and in trans that colocalize with Oct4 in
PSCs specifically. Analysis of these colocalizations during reprogramming revealed that long-range interactions at the Oct4
Cell Stem Cell
Long-Range Interactions at the Oct4 Locus
Figure 1. Identification of Intrachromosomal and Interchromosomal Interactions at Endogenous Oct4 Loci Using the 4C-Seq Approach
(A) Summary of sequencing results. Each tag represents a read that can be mapped to the Oct4 bait primer sequence as well as another region, both of which
contain intact HindIII restriction sites. Total mapped tags are separated into proximal tags (<1 MB from the Oct4 enhancer), distal intrachromosomal tags, and
interchromosomal tags.
(B) Schematic plot showing intrachromosomal interacting regions from two biological replicates (BR1 and BR2). Red and blue boxes represent interacting regions
defined in BR1 and BR2, respectively. Gene density of chromosome 17 is indicated by gray scale.
(C) Circos plots showing interactions between the Oct4 locus and interacting partners with each line representing an interaction. Chromosomes are plotted
across the circle. Two biological replicates are shown separately.
See also Figure S1 and Table S1.
locus emerge specifically in a subset of cells prior to activation of
endogenous Oct4. Oct4 loci participating in interactions were
correlated with sites of ongoing transcription in PSCs. Furthermore, we demonstrated that either depletion or overexpression
of Klf4 altered these long-range interactions independent of
Oct4 transcription and PSC differentiation, suggesting that Klf4
regulates long-range interactions independent of its role as a
transcription factor. Finally, we identified the interaction between
Klf4 and cohesin, which colocalizes with Klf4 and mediates DNA
looping at the Oct4 enhancer. We discovered that Klf4 is
required for loading of cohesin at the Oct4 enhancer. Taken
together, the results of our study define Klf4-mediated longrange interactions involving the Oct4 enhancer and serve as evidence that these interactions play critical roles in activating Oct4
during reprogramming.
RESULTS
Identifying Putative Long-Range Interactions of the
Oct4 Distal Enhancer
To identify potential long-range interactions involving the Oct4
locus, we performed circular chromosome conformation capture
followed by high throughput sequencing (4C-seq) in embryonic
stem cells (ESCs) (Figure S1A available online). We focused on
the Oct4 distal enhancer (DE), which is located 2 kb upstream
of the transcription start site and is required for Oct4 activation in
ESCs (Pan et al., 2002). Previous ChIP-seq data revealed binding
of Oct4, Sox2, Klf4, and several other transcription factors to this
region (Chen et al., 2008). Thus we applied the 4C-seq assay in
ESCs using the DE region as bait (Figure S1B and S1C). Similar
to findings reported in previous studies (Noordermeer et al.,
2011b; Splinter et al., 2011), we observed a large percentage
(64%–68%) of 4C-seq captured sequences located within
1 MB of the bait region (Figure 1A), with approximately 85%
located in cis (Figure 1A). Using pipelines similar to published
4C-seq protocols (van de Werken et al., 2012), we identified a
large number of putative distal interacting regions in cis and in
trans in two biological replicates (Figures 1B and 1C, Table
S1). As expected, Oct4-interacting regions are mostly gene
rich (Figure 1B), similar to what was previously revealed using
HiC in various cell types (Dixon et al., 2012; Lieberman-Aiden
et al., 2009). Most intrachromosomal and interchromosomal interactions defined in the two replicates were overlapping (Figure 1B, Figures S1D and S1E). By combining our 4C-seq and
published mRNA-seq results (Brookes et al., 2012), we found
that the genes located in the positive Oct4-interacting regions
are expressed at a higher level than genes located outside in
ESCs (Figure S1F). Similarly, a higher than average percentage
of genes were found to be positive for active gene markers
such as RNA polymerase II (RNAPII)-S5P, RNAPII-S2P, and
H3K4Me3, while the percentage of Suz12-positive genes remained unchanged (Figure S1G). When we classified all the
genes into three categories—active, bivalent, and inactive—
based on the published genome-wide profiling data (Brookes
et al., 2012), 71% of the genes in 4C-seq defined regions are
classified as ‘‘active,’’ while only 45% of genes in the whole
genome are considered ‘‘active’’ (Figure S1H). Interestingly,
4C-seq defined regions are also early in replication timing (Figure S1I) (Hiratani et al., 2010), suggesting a link between chromatin structure and replication timing (Ryba et al., 2010).
Cell Stem Cell 13, 36–47, July 3, 2013 ª2013 Elsevier Inc. 37
Cell Stem Cell
Long-Range Interactions at the Oct4 Locus
Figure 2. Long-Range Interactions between Oct4 and Various Loci Reveal a
Unique Higher-Order Chromatin Structure
at the Oct4 Locus in PSCs
(A) DNA FISH of Oct4 and various loci in ESCs.
(B) Summary of percentage of interchromosomal
colocalizations at the Oct4 locus and candidate
loci in ESCs. Probes located in putative interacting
regions have significantly greater colocalization
frequency compared to control probes located
outside interacting regions. Black bars indicate
probes located in Oct4 interacting regions. White
bars indicate control probes. Probe locations are
shown in Figure S1E and Table S2. (***p < 0.001.)
(C) Boxplots showing the distribution of interprobe
distances. The bottom and top of the box correspond to the 25th percentile and the 75th percentile, respectively, and the internal band represents
the median. The plot whiskers correspond to the
lowest and highest data points within a 1.5 interquartile range of the lower and upper quantiles.
(D) Images showing colocalization of the Oct4
locus and its interacting partners 143F14, 474J5,
and 280H9 in ESCs, iPSCs, pre-iPSCs, and NSCs.
(E–G) Colocalization percentages of Oct4-143F14
(D), Oct4-474J5 (E), and Oct4-280H9 (F) colocalization in ESCs, iPSCs, pre-iPSCs, and NSCs. n
indicates the total number of nuclei analyzed in
two biological samples. (*p < 0.05, **p < 0.01,
***p < 0.001.)
(H) Images showing Oct4-143F14 colocalization in
iPSC-derived NSCs and native NSCs isolated
from E14.5 mouse forebrains.
(I) Quantification of analysis shown in (G). iPSCderived NSCs and native NSCs showed no
significant difference in colocalization frequency
between Oct4 and 143F14.
(J) Nuclear size as measured by DAPI staining in
NSCs, iPSCs, ESCs, and pre-iPSCs. Nuclei are
larger in iPSCs and ESCs relative to NSCs and preiPSCs, indicating that the increase of colocalization
frequency seen in PSCs is not due to a smaller nuclear size. Error bars represent SEM. (***p < 0.0001.)
See also Figure S2 and Table S2.
Identification of Long-Range Interactions at the SingleCell Level in PSCs and Differentiated Cells
To verify that interactions occur in vivo, we selected six loci
discovered by 4C-seq and six control loci not covered by 4Cseq and performed DNA FISH on ESCs using bacterial artificial
chromosome (BAC)-derived probes containing these loci
together with a BAC probe containing the endogenous Oct4 locus (Figures 2A and 2B; see Table S2 and Figure S1E for names
and locations of BAC probes). Compared to the control group
(Figure 2B, white bars), 4C-seq positive regions (black bars)
show significantly higher colocalization frequencies (Figure 2B),
as well as differences in interprobe distance distribution (Fig38 Cell Stem Cell 13, 36–47, July 3, 2013 ª2013 Elsevier Inc.
ure 2C). These studies confirm significant
increase of colocalization frequency between Oct4 loci and several putative interacting partners at the single-cell level.
To investigate whether long-range interactions at the Oct4 locus are specific
to PSCs, we compared colocalization frequency in iPSCs,
ESCs, pre-iPSCs (or partial iPSCs), and iPSC-derived neural
stem cells (NSCs). Several long-range interactions were specific
to iPSCs and ESCs only and were not present in iPSC-derived
NSCs (Figures 2C–2F) or primary cultured NSCs (Figures 2G
and 2H), indicating that interactions are unique to pluripotent
cells. Interestingly, colocalization frequency in pre-iPSCs was
lower than that seen in PSCs. pre-iPSCs are considered to be
cells ‘‘trapped’’ at an intermediate stage of reprogramming (Mikkelsen et al., 2008; Theunissen et al., 2011). That the frequency of
long-range interactions increases between pre-iPSCs and
iPSCs suggests that the Oct4 locus in the former has not yet
Cell Stem Cell
Long-Range Interactions at the Oct4 Locus
Figure 3. PSC-Specific Long-Range Interactions at the Oct4 Locus Are Established Early in Reprogramming
(A–D) Representative images (A and B) and quantification (C and D) showing colocalization of Oct4 with interacting partners 474J5 (A and C) and 143F14 (B and
D). Enrichment is seen only in the SSEA1+ cell population. Scale bar, 5 mm. Error bars represent standard deviation with n = 3 biological replicates in (C) and (D).
(Mean ± SEM, *p < 0.05, **p < 0.01.)
(E) DNA FISH images showing colocalization over the course of AZA treatment of pre-iPSCs. Insets show an enlarged single nucleus. Scale bar, 50 mm.
(F) Summary showing statistical analysis of colocalization frequency during reprogramming.
See also Figure S3.
developed a higher-order chromatin structure characteristic of
PSCs and supports previous reports that Oct4-GFP-negative
pre-iPSCs do not express other pluripotency genes (Ichida
et al., 2009; Mikkelsen et al., 2008). To exclude the possibility
that the higher colocalization efficiency seen in PSCs was a
consequence of these cells having a smaller nuclei, we
measured the nuclear staining area using 40 ,6-diamidino-2-phenylindole (DAPI) in the four cell types evaluated. PSCs, in fact,
exhibited relatively larger nuclei compared to NSCs and preiPSCs (Figure 2I).
Dynamics of Long-Range Interactions during
Reprogramming
Next we investigated how cells establish PSC-specific, longrange interactions during reprogramming. Previously we established a doxycycline-inducible reprogramming system harboring
an Oct4-GFP reporter to study reprogramming mechanisms
(Wei et al., 2009). Using that system, upon induction iPSCderived NSCs can be reprogrammed back into second-generation iPSCs within 2 weeks (Wei et al., 2009), during which time
cells show a gradual increase in the percentage of cells positive
for the pluripotency surface marker SSEA1 (Figures S2A and
S2B). GFP expression begins after day 13, indicating that endogenous Oct4 is activated after approximately 2 weeks. For this
study we used quantitative PCR (qPCR) to confirm that endogenous Oct4 expression remained low at day 8 in both SSEA1+ and
SSEA1 population (Figure S2C).
To determine when NSCs begin to form long-range interactions favoring pluripotency, we collected samples at days 1, 5,
8, and 13 after induction of reprogramming and applied
immuno-FISH to examine colocalization frequencies between
the Oct4 locus and loci recognizing two other probes located
in trans, 143F14, and 474J5, at these stages. In contrast to
endogenous Oct4 expression, which remained low at day 8 (Figure S2C), colocalization levels at the Oct4 locus began to rise as
early as day 5 (Figures 3A–3D), indicating that long-range interchromosomal interactions are forming at early reprogramming
stages and, more importantly, are detectable prior to activation
of endogenous Oct4. Moreover, colocalization frequency was
greater in SSEA1+ cells (Figures 3C and 3D). Since mature iPSCs
arise from the SSEA1+ population, this observation indicates
that long-range interactions occur predominantly in progenitors
of mature iPSCs.
To determine whether colocalization dynamics observed
during the transition from pre-iPSCs, which were isolated from
colonies without iPSC morphology during reprogramming and
remain stable unless treated with various inhibitors, to iPSCs
occurs in a manner similar to that in normal reprogramming
from somatic cells, we examined colocalization during reprogramming from pre-iPSCs to mature iPSCs (Figures 3E
and 3F). Similar to what was described in a previous study (Mikkelsen et al., 2008), after treatment with the DNMT1 inhibitor
AZA, all pre-iPSCs required approximately 5 days to be reprogrammed into mature iPSCs, as shown by GFP expression (Figure 3E). However, the extent of interchromosomal colocalization
at day 1 after addition of AZA rose to a level similar to that of
PSCs (Figure 3F). Colocalization was also observed in GFPnegative cells (Figure 3E), indicating that initiation of long-range
interchromosomal interactions precedes activation of endogenous Oct4.
Cell Stem Cell 13, 36–47, July 3, 2013 ª2013 Elsevier Inc. 39
Cell Stem Cell
Long-Range Interactions at the Oct4 Locus
Figure 4. Long-Range Interactions Are Correlated with Endogenous Oct4 Transcription in PSCs
(A–C) Images of triple-labeled RNA/DNA FISH for Oct4 RNA (green), Oct4 DNA (yellow), and DNA probes 143F14 (red) (A), 474J5 (red) (B), or 280H9 (red) (C) in
ESCs. Side panels show enlarged images of colocalized signals (top to bottom: yellow, red, green, yellow and green, red and yellow, triple labeled).
(D–F) Percentage of Oct4 loci positive for RNA in triple-labeled RNA/DNA FISH. The percentage of Oct4 RNA-positive 143F14-interacting Oct4 loci (D), 474J5interacting Oct4 loci (E), or 280H9-interacting Oct4 loci (F) is significantly greater than the percentage of noninteracting Oct4 alleles in the same cell. n indicates
the total number of nuclei analyzed in two biological samples. (**p < 0.01.)
PSC-Specific Long-Range Interactions Are Associated
with Local Transcription at the Oct4 Locus
The function of higher-order chromatin structures varies from
organizing transcription factories to maintaining condensed
heterochromatin (Chakalova and Fraser, 2010; Probst and
Almouzni, 2011). Since a key event during reprogramming is to
‘‘jump-start’’ the endogenous pluripotency circuitry (Jaenisch
and Young, 2008; Plath and Lowry, 2011), represented primarily
by expression of endogenous Oct4 and Nanog, we reasoned
that formation of a higher-order chromatin structure at the
Oct4 locus in PSCs governs Oct4 transcription. To test this hypothesis, we performed both RNA/DNA FISH and triple-label immuno-DNA FISH in ESCs. For RNA/DNA FISH, a probe targeting
the first intron of Oct4 served as a marker of Oct4 transcription.
Using this probe we detected Oct4 primary transcripts in 40%–
50% of ESCs (Figures S3A–S3E), a proportion similar to that re40 Cell Stem Cell 13, 36–47, July 3, 2013 ª2013 Elsevier Inc.
ported previously for pluripotency gene Zfp42 (also known as
Rex1) expression in ESCs (Hiratani et al., 2010). This ‘‘snapshot’’
indicates that only a subset of cells actively transcribe Oct4 at a
given time. When we combined RNA FISH for Oct4 with DNA
FISH for Oct4, 143F14, 474J5, and 280H9, 66%–80% of
143F14-, 474J5-, and 280H9-interacting Oct4 alleles were positive for Oct4 RNA FISH (Figures 4D and 4E). This is in sharp
contrast with the other Oct4 allele in the same cell that does
not colocalize with 143F14, 474J5, or 280H9. In that case only
40% of the noninteracting Oct4 loci were positive for Oct4
RNA FISH. Considering that Oct4 likely has multiple long-range
interacting partners, the 40% of transcriptionally active Oct4 alleles seen in controls may represent loci engaged in other longrange interactions. We also performed triple-label immuno-DNA
FISH using an antibody recognizing RNAPII-S5P and observed
staining at most Oct4 loci above the median intensity seen in
Cell Stem Cell
Long-Range Interactions at the Oct4 Locus
A
C
D
E
F
G
Figure 5. Klf4 Is Closely Associated with
Long-Range Interactions at the Oct4 Locus
and Occupies These Loci Specifically in
the SSEA1+ Subpopulation during Reprogramming
B
H
the same nucleus. Notably, Oct4 loci participating in long-range
interactions are preferentially located within RNAPII-S5P-dense
regions, compared with ‘‘noninteracting’’ Oct4 loci (Figures S3F–
S3I). Overall, these results show that the Oct4 loci participating in
long-range interactions are more likely to be in an active transcriptional state, suggesting the direct association between
long-range interactions and local transcription.
Klf4 Associates with Specific Pluripotent Long-Range
Interactions at Oct4 Loci
The DE region of Oct4 used as our 4C assay bait contains Oct4,
Sox2, and Klf4 binding sites (Chen et al., 2008). Interestingly,
Oct4-interacting regions defined by 4C-seq are enriched in
Klf4 binding sites, and to a lesser extent by Oct4, Sox2, and
Nanog sites (Figure 5A). Previous studies demonstrated that
Oct4, Sox2, and Klf4 cooperatively activate Oct4 and other pluripotency genes (Sridharan et al., 2009; Wei et al., 2009). Among
these factors, Klf4 is of particular interest, as another Sp/Klf
family member, EKLF/Klf1, is a well characterized regulator of
long-range interactions at b-globin loci (Drissen et al., 2004).
Specifically, Schoenfelder et al. (2010) demonstrated Klf1’s
role in mediating interactions between coregulated genes in
erythroid cells. Moreover, Klf1 can replace Klf4 in reprogram-
(A) Enrichment in pluripotency factor binding sites
in Oct4-interacting regions. Klf4, Oct4, Sox2,
Nanog, and CTCF binding sites are all enriched.
(B) Immunostaining indicating that Klf4 protein
(green) forms punctate structures that overlay with
RNAPII-S5P (red) inside the nucleus.
(C) Immuno-FISH showing colocalization of Klf4
protein (green) and genomic loci, including the
control locus 102A1 (red) and 143F14, 474J5,
280H9, and Oct4 (red). Side panels show enlarged
images of colocalization (top to bottom: red,
green, double labeled).
(D) Graph showing percentages of various loci
colocalized (black) or not colocalized (white) with
Klf4 protein. The red line shows the ‘‘background’’
colocalization percentage.
(E) Triple-labeled immuno-FISH for Klf4 protein
(green) and colocalized Oct4-143F14 or Oct4474J5 (yellow and red, as indicated). Side panels
show enlarged colocalization signals (top to bottom: red, yellow, red and yellow, green, triple
labeled).
(F) Graph showing interacting pairs (143F14-Oct4,
474J5-Oct4, and 280H9-Oct4) colocalized (black)
or not colocalized (white) with Klf4 foci.
(G) Schematic illustration of the experimental
procedure in (G).
(H) Ratio of Klf4 enrichment in SSEA1+ to SSEA1
cells at the Oct4, 143F14, and 474J5 loci at reprogramming days 5 (white bar) and 10 (black bar).
(Mean ± SEM, *p < 0.05, **p < 0.01.)
ming, although it is a less efficient inducer than Klf4 (Nakagawa
et al., 2008), suggesting partial redundancy. Klf1 is reportedly
located in discrete sites overlapping with RNAPII-S5P foci,
which function as transcription factories (Schoenfelder et al.,
2010). Interestingly, Klf4 immunostaining revealed that Klf4 foci
also overlapped with RNAPII-S5P foci (Figure 5B). To determine
whether Oct4 and its interacting loci are located inside Klf4 foci,
we performed immuno-FISH to simultaneously detect Klf4
protein with Oct4, 143F14, 474J5, and 280H9, all of which
contain multiple Klf4 binding sites (Chen et al., 2008). All of these
loci were found to be preferentially associated with Klf4 foci (Figures 5C and 5D). More importantly, when the Oct4 locus colocalized with partner 143F14, 474J5, and 280H9 loci, the frequency
of association with Klf4 foci was further increased (Figures 5E
and 5F). These results strongly suggest that interactions between the Oct4 locus and interacting partners occur inside
Klf4- and RNAPII-S5P-enriched foci.
Oct4 Enhancer and Oct4-Interacting Loci Are Occupied
by Klf4 Preferentially in SSEA1+ Cells during
Reprogramming
Klf4 is ectopically expressed in all cells during reprogramming,
whereas Oct4-locus long-range interactions appear to occur
Cell Stem Cell 13, 36–47, July 3, 2013 ª2013 Elsevier Inc. 41
Cell Stem Cell
Long-Range Interactions at the Oct4 Locus
A
B
F
C
D
G
J
K
E
H
L
I
M
N
Figure 6. Depletion or Overexpression of Klf4 Alters Oct4-Associated Interchromosomal Interactions
(A and B) qPCR of expression of Klf4 (A) and Oct4 (B) following Klf4 depletion by shRNA. (Mean ± SEM.)
(C–E) Frequencies of colocalization of Oct4-143F14 (C), Oct4-474J5 (D), and Oct4-280H9 (E) in control and Klf4 knockdown samples.
(F–H) Distribution of interprobe distances shows changes after Klf4 depletion. White boxes represent control and gray boxes represent Klf4 knockdown samples.
The bottom and top of the box correspond to the 25th percentile and the 75th percentile, respectively, and the internal band represents the median. The plot
whiskers correspond to the lowest and highest data points within a 1.5 interquartile range of the lower and upper quantiles. *p < 0.05.
(I) The percentage of RNA-positive Oct4 alleles that show no significant decrease 48 hr after induction of Klf4 shRNA knockdown. (Mean ± SEM.)
(J–L) Colocalization frequencies of Oct4-143F14 (J), Oct4-474J5 (K), and Oct4-280H9 (L) at transcriptionally active (RNA-positive) and inactive (RNA-negative)
Oct4 alleles.
(M and N) Colocalization frequencies of Oct4-143F14 (M) and Oct4-474J5 (N) in control and Klf4-overexpressing cells.
In (C)–(N), n indicates the total number of nuclei analyzed in two biological samples. *p < 0.05, **p < 0.01. See also Figures S4 and S5 and Table S3.
only in SSEA1+ cells. To examine a role for Klf4 in organizing
Oct4-locus long-range interactions during reprogramming, we
asked whether Klf4’s occupancy of these genes differs between
SSEA1+ and SSEA1 cells. Cells at different reprogramming
stages, from NSCs to iPSCs, were sorted by flow cytometry
into SSEA1+ and SSEA1 populations (Figure 5G). A control region (positive for Klf4 binding but not related to pluripotency or
Oct4 long-range interactions) showed a ratio of binding intensity
of 1 for SSEA1+ versus SSEA1 cells, while enrichment of Klf4
at Oct4, 143F14, 474J5, and 280H9 was much higher in SSEA1+
than that in SSEA1 cells (Figure 5H). These differences were
seen as early as day 5 after initiation of reprogramming and
increased by day 10. This finding suggests that Klf4 expression
is induced immediately after reprogramming initiation in all cells,
but its occupancy of the Oct4 locus with interacting partners
(143F14 and 474J5) occurs only in SSEA1+ cells. Differential
Klf4 recruitment supports the hypothesis that Klf4 accessibility
42 Cell Stem Cell 13, 36–47, July 3, 2013 ª2013 Elsevier Inc.
to its target determines its function as a long-range interaction
mediator only in a subset of cells, despite its universal expression during reprogramming.
Depletion or Overexpression of Klf4 Has Direct Effects
on Long-Range Interaction Frequency
To determine whether Klf4 is required for long-range interactions, we generated stable ESC lines harboring inducible Klf4
shRNA. shRNA induction by doxycycline treatment for 2 days
promoted a decrease of Klf4 mRNA and protein (Figure 6A and
Figure S4A), while cells in which depletion was prolonged for
more than one passage underwent differentiation (Figure S4B).
On knockdown day 2, Oct4 mRNA and protein levels were maintained (Figure 6B and Figure S4B), but Oct4 interchromosomal
interactions began to decrease significantly (Figures 6C–6E).
The distribution of interprobe distance is also changed after
Klf4 depletion (Figures 6F–6H). On differentiation days 1 and 3,
Cell Stem Cell
Long-Range Interactions at the Oct4 Locus
Figure 7. Klf4 Interacts with Cohesin and
Recruits Cohesin to the Oct4 Distal
Enhancer
A
C
B
D
(A) Smc1 (component of cohesin) can be detected
by western blotting after immunoprecipitation of
ESCs expressing flag-tagged Klf4 with flag antibody. However, Med12, which is a component of
Mediator complex, cannot be detected by western
blotting using the same condition.
(B) Schematic presentation of Oct4 locus. The distal
enhancer (DE) is occupied by Klf4, Smc1, Med12,
Oct4, Sox2, and several other transcription factors.
(C and D) ChIP-qPCR results of Smc1a (C) and
Med12 (D) occupancy at Oct4 DE in doxycycline
inducible Klf4 knockdown cell lines. The binding of
both Smc1a and Med12 are significantly reduced
after 48 hr knockdown of Klf4. (Mean ± SEM, *p <
0.05, **p < 0.01.)
(E) ChIP-qPCR results of Oct4 occupancy at Oct4
intron region after Klf4 depletion in the condition
similar to (C) and (D). The binding of Oct4 is not
affected after knockdown. (Mean ± SEM.)
E
of-function analysis indicate that Klf4
is essential for organization of specific
long-range interchromosomal interactions at the Oct4 locus.
Klf4-depleted cells maintained normal morphology but did not
start to differentiate until the second passage (5 days after Klf4
shRNA induction) (Figure S4C), indicating that the dissociation
is unlikely the result of cell differentiation.
It is possible that the dissociation of long-range interactions
seen after Klf4 knockdown results from loss of transcription at
the Oct4 locus. However, the total Oct4 mRNA level did not
significantly decrease in the first 2 days of knockdown (Figure 6B). Furthermore, RNA FISH labeling of nascent Oct4 RNA
did not indicate significant decreases in the percentage of
Oct4 RNA-positive alleles at day 2 (Figure 6I), indicating that
immediately after loss of Klf4, transcription of Oct4 remains at
the same level. Additionally, inhibition of RNAPII transcription
by a-amanitin did not alter colocalization frequency (Figure S4D),
in agreement with previous studies (Palstra et al., 2008; Takebayashi et al., 2012). Most importantly, at Klf4 knockdown day
2 when we separated Oct4 alleles into transcriptionally active
versus transcriptionally inactive groups based on RNA FISH,
reduced colocalization was seen in both subgroups (Figures
6J–6L), supporting the idea that Klf4 depletion promotes dissociation of Oct4-interchromosomal interactions independent of
its transcriptional activity (see Discussion).
Finally, we examined potential changes in colocalization frequencies in Klf4 gain-of-function studies. Klf4-overexpressing
ESCs can self-renew independent of LIF signaling (Zhang
et al., 2010). When we induced Klf4 overexpression in ESCs (Figure S5), we observed a significant increase in colocalization
frequencies (Figures 6M and 6N). Overall, both loss- and gain-
Klf4 Recruits Cohesin to the Oct4
Enhancer Region
To further explore the molecular mechanisms of Klf4’s role in mediating longrange interactions, we focused on the potential interacting
partners of Klf4 in PSCs. Previous ChIP-seq data have demonstrated that Klf4 colocalizes with cohesin complex (Nitzsche
et al., 2011), which has been shown to be a critical player in
DNA looping (Kagey et al., 2010; Phillips and Corces, 2009).
Thus we applied immunoprecipitation assay to detect the potential interactions between Klf4 and looping factors. Due to the lack
of high quality Klf4 antibody, we performed the assay with
our inducible Klf4-flag ESCs cell lines. Low-concentration
(50 ng/ml) doxycycline was used to induce ectopic Klf4 expression and total Klf4 level was similar to endogenous Klf4 (data
not shown). After immunoprecipitation with flag antibody, we
were able to detect cohesin component Smc1 (Figure 7A), which
indicates that Klf4 can interact with cohesin complex in PSCs.
Interestingly, we did not find an interaction between Klf4 and
Mediator complex protein Med12. This indicates that although
Klf4, cohesin, and Mediator co-occupy the Oct4 DE (Figure 7B),
Klf4 specifically interacts with cohesin, but not with Mediator.
Cohesin and Mediator have been implicated in mediating the
DNA looping between enhancer and promoter (Kagey et al.,
2010). As Klf4 interacts and colocalizes with cohesin at the
Oct4 enhancer, we examined whether the interaction is critical
for loading of cohesin at the Oct4 enhancer. We performed
ChIP-qPCR in Klf4 inducible knockdown lines with Smc1a and
Med12 antibody. Immediately after Klf4 depletion, we observed
a significant decrease of Smc1a binding (Figure 7C), indicating
that Klf4 is essential for cohesin binding at the Oct4 enhancer,
likely through direct interaction. Interestingly, Med12 occupancy
Cell Stem Cell 13, 36–47, July 3, 2013 ª2013 Elsevier Inc. 43
Cell Stem Cell
Long-Range Interactions at the Oct4 Locus
is also decreased after Klf4 depletion (Figure 7D). This is consistent with the model that cohesin stabilizes the looping structure for the mediator to bridge the enhancer with the promoter
(Cuylen and Haering, 2010). Meanwhile, immediate knockdown
of Klf4 seems to have little effect on Oct4’s binding to its own
enhancer (Figure 7E). Together, our results show that Klf4
specifically recruits looping factor cohesin to the Oct4 DE. This
coordination likely contributes to Klf4’s role in mediating longrange interactions in PSCs.
DISCUSSION
Although the epigenetic landscape in reprogramming in terms of
genome-wide DNA methylation and histone modification has
been systematically studied in cells undergoing reprogramming
(Mikkelsen et al., 2008), the dynamic changes in nuclear architecture occurring in the process remain largely unexplored. As
seen after nuclear transfer (Gurdon and Melton, 2008), changes
in higher-order chromatin structure are likely highly correlated
with adoption of a pluripotent chromatin signature. Our finding
that long-range interactions are associated with activation and
transcription of endogenous Oct4 and establishment of pluripotency supports this idea.
Over the course of reprogramming, reprogramming factors
are ubiquitously expressed, but only a small fraction of cells
become iPSCs, indicating that these factors alone are not sufficient to overcome dedifferentiation ‘‘barriers’’ and that additional
stochastic events are required to activate pluripotency. We have
shown that a transition to a higher-order chromatin structure,
represented here by long-range interactions at the endogenous
Oct4 locus, occurs only in a subpopulation of cells and is highly
correlated with Oct4 transcription. The contribution of longrange interactions to local gene expression, especially in the
case of trans activation, has been reported in a recent study of
b-globin activation (Noordermeer et al., 2011a). Instead of occurring universally, trans activation only occurs in so-called
‘‘jackpot’’ cells enriched for interchromosomal interactions
(Noordermeer et al., 2011a). Similarly, the establishment of a
PSC-specific higher-order chromatin structure may constitute
one of the stochastic steps to pluripotency and serve to overcome a reprogramming barrier.
Recently developed technologies have enabled the discovery
of numerous novel long-range interactions (Fullwood et al.,
2009; Lieberman-Aiden et al., 2009). How these interactions
are regulated remains unclear. Multiple transcription factors
reportedly regulate higher-order chromatin structure: for
example, DNA looping at the b-globin locus primarily requires
the lineage-specific transcription factors EKLF (Klf1), GATA-1,
Fog-1, and Ldb1 (Drissen et al., 2004; Song et al., 2007; Vakoc
et al., 2005). However, work reported by others (Palstra et al.,
2008; Takebayashi et al., 2012) and presented here (Figure S5D
and S5E) shows that inhibition of transcription does not promote
dissociation of long-range interactions but rather suggests that
transcription factors may have a ‘‘tethering’’ function in addition
to a transactivating function. This hypothesis is supported by
our finding that either depletion or overexpression of Klf4 alters
long-range interaction frequency at the Oct4 locus, independent
of Oct4 transcription. Similarly, a recent, elegant study by Deng
et al. (2012) illustrates a tethering function for the transcription
44 Cell Stem Cell 13, 36–47, July 3, 2013 ª2013 Elsevier Inc.
factor Ldb1. In erythroid development, Ldb1 interacts with the
DNA-binding protein GATA-1 to organize DNA looping between
the locus control region (LCR) and the b-globin promoter to activate b-globin transcription. In a GATA-1 null cells, overexpression of Ldb1 fused to an artificial zinc finger, which binds to
the promoter and LCR without GATA-1, promotes long-range
interactions and recruits RNAPII-S5P, promoting b-globin
expression. Interestingly, GATA-1/EKLF interactions require
zinc finger domains of both proteins (Merika and Orkin, 1995).
Similarly, we previously reported that interaction of Klf4 with
Oct4/Sox2 requires zinc fingers at the Klf4 C terminus (Wei
et al., 2009). Whether Klf4 zinc fingers serve as ‘‘docking sites’’
to organize interactions and enhance a pluripotency-associated
higher-order chromatin structure is a question for future
investigation.
The finding that Klf4 depletion first results in dissociation of
long-range interactions and then promotes inactivation of Oct4
transcription seems contradictory to the conventional model of
Klf4’s function as a transactivator. Several factors may account
for this discrepancy. First, Oct4 is regulated by a large number of
transcription factors. Among them, Klf4 protein has a rather short
(4 hr) half-life (Gamper et al., 2012); other factors regulating Oct4
may be more stable and temporarily support Oct4 transcription.
Although stable depletion of Klf4 causes differentiation, it has
also been shown that in transient transfection only knocking
down Klf2/4/5 together can cause differentiation (Jiang et al.,
2008). Thus, Klf2 and Klf5 redundancy may also contribute to
this delay. Also, our finding that cohesin’s binding at Oct4
enhancer is determined by Klf4’s occupancy at the same regions
suggest a model in which Klf4 recruits cohesin to the Oct4
enhancer and therefore is critical for cohesin’s stabilization function between enhancers and promoters (Kagey et al., 2010).
Moreover, studies in other systems suggest that Klf4 collaborates with p300/CBP and/or HDACs to activate downstream
genes including Oct4 (Evans et al., 2007; Kidder and Palmer,
2012; Noti et al., 2005). Following Klf4 depletion, dissociation
of HAT or HDAC complexes from the Oct4 enhancer may also
need to occur prior to transcriptional silencing. Lastly, although
long-range interactions alone can activate gene expression,
that effect is reportedly minimal (15%) compared to normal
activation levels (Deng et al., 2012). Thus, Klf4’s regulation of
Oct4 transcription is partly through directly controlling longrange interactions at the Oct4 enhancer.
The ring-shaped cohesin complex has been originally demonstrated to mediate sister chromatid cohesion (Remeseiro and
Losada, 2013). However, recent evidences suggest that during
interphase, cohesin can regulate organization of transcription
(Remeseiro and Losada, 2013), although it is largely unclear
whether it works in a similar way to that in mitosis. This is of
particular interest for the biology of long-range interaction, as cohesin has been shown to work together with Mediator to coordinate the looping between distal regulatory elements and gene
promoters. Our discovery of Klf4’s role in recruiting cohesin
and Mediator complex to the Oct4 enhancer brings Klf4 to the
center of the regulatory mechanism. Since Mediator and cohesin
binding sites seem to lack a specific pattern in ESCs, Klf4 is likely
responsible for bringing specificity to the long-range interactions. Recently, it has been shown that, compared to other
stem cell factors such as Oct4 and Nanog, Klf4’s binding is
Cell Stem Cell
Long-Range Interactions at the Oct4 Locus
particularly enriched at the ‘‘super enhancers,’’ defined by a high
level of Mediator intensity (Whyte et al., 2013). This further supports the idea that Klf4’s role in mediating long-range interaction
is through cohesin and Mediator complex. In addition, a very
recent paper by Apostolou et al. (2013) identified a role for cohesin in mediating genome-wide chromatin interactions of the
Nanog locus in PSCs.
The causal relationship between long-range interactions and
gene regulation is constantly under debate. Our data start to
shed light on the functional relevance of long-range interactions
in gene regulation. However, it should be noted that the direct
evidence and detailed mechanisms remain to be discovered.
The recent advances, including using synthetic zinc finger proteins to ‘‘force’’ the formation of looping (Deng et al., 2012) and
real-time visualization of lamin-interacting domain in single cells
(Kind et al., 2013), provide powerful tools for future investigation.
In addition, to directly correlate long-range interactions with
specific regulatory elements, ideally the size of long-range interacting regions need to be at the kilobase level. To this end, integration of emerging high-resolution genome-wide techniques
such as ChIA-PET (Fullwood et al., 2009), will be crucial for
further studies.
EXPERIMENTAL PROCEDURES
4C Assay
4C was performed using a modified protocol adapted from a 3C-qPCR
protocol (Splinter et al., 2011; van de Werken et al., 2012). Briefly, 107 ESCs
were trypsinized to single cells and resuspended in 0.5 ml GMEM/10%
FBS. Then 9.5 ml of 2% paraformaldehyde/10% FBS was added and cells
were incubated for 10 min. After being quenched with glycine, cells were lysed
in 5 ml lysis buffer (10 mM Tris-HCl [pH7.5]; 10 mM NaCl; 5 mM MgCl2; 0.1 mM
EGTA; 13 protease inhibitor) for 10 min on ice and centrifuged to remove the
supernatant. Nuclei were then digested by HindIII at 37 C. After inactivation
by 1.6% SDS at 65 C for 20 min, samples were diluted in 6.125 ml of 1.153
ligation buffer and 100U T4 ligase and incubated at 16 C for 4 hr and then
25 C for 30 min. Ligated chromatin was digested by proteinase K and purified
by phenol-chloroform extraction, and DNA was ethanol precipitated. The
products were purified and further digested by DpnII and then circularized using T4 ligase. After purification, 16 parallel PCR reactions, each containing
200 ng of DNA, were performed using primers shown in Table S3. PCR products were combined and purified by Agencourt AMPure XP para-magnetic
beads and the size and distribution of purified products were verified by an
Agilent High Sensitivity DNA assay. Bar-coded DNA libraries were subject
to single-end sequencing with a 50 bp read length using the Illumina
HiSeq2000.
4C-Seq Data Analysis
Sequencing reads with the 50 end aligned to the forward inverse PCR primer
sequence were selected. The rest of the selected reads (including the HindIII
sites) were mapped to the mm9 assembly using BWA to locate ligation sites
in the genome. The mapped ligated HindIII sites were further matched to a
reduced genome including locations of all HindIII sites. For statistical analysis,
we followed the previous 4C-seq data analysis protocol (data not shown;
Splinter et al., 2011; van de Werken et al., 2012). To define nonrandom longrange intrachromosomal interactions, we used FDR 0.05 as a threshold and
compared our 4C-seq data to randomly permutated data sets. A window
size of 100 was used. To define interchromosomal interactions, we randomly
permuted the data set 100 times using FDR 0.05 as a threshold. A window size
of 500 was used.
ACCESSION NUMBERS
4C-seq data has been deposited to GEO (GSE45418).
SUPPLEMENTAL INFORMATION
Supplemental Information for this article includes Supplemental Experimental
Procedures, five figures, and three tables and can be found with this article
online at http://dx.doi.org/10.1016/j.stem.2013.05.010.
ACKNOWLEDGMENTS
We thank all members of the Lu laboratory for discussion, C. Yang for
providing the inducible shRNA construct, G. Coetzee and M. Pera for critical
comments, and L. Barsky for FACS sorting. This work was partially supported
by a grant from CIRM (RB1-01353).
Received: November 21, 2012
Revised: April 11, 2013
Accepted: May 15, 2013
Published: July 3, 2013
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