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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 REFERENCES Anokye-Danso, F., Trivedi, C.M., Juhr, D., Gupta, M., Cui, Z., Tian, Y., Zhang, Y., Yang, W., Gruber, P.J., Epstein, J.A., and Morrisey, E.E. (2011). Highly efficient miRNA-mediated reprogramming of mouse and human somatic cells to pluripotency. Cell Stem Cell 8, 376–388. 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