Super-Enhancers at the Nanog Locus Differentially Regulate ...

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Super-Enhancers at the Nanog Locus Differentially Regulate Neighboring Pluripotency-Associated Genes Graphical Abstract

Authors Steven Blinka, Michael H. Reimer, Jr., Kirthi Pulakanti, Sridhar Rao

Correspondence [email protected]

In Brief Super-enhancers are tissue specific cisregulatory elements that are transcribed and regulate genes critical to cell identity. Blinka et al. show that three neighboring super-enhancers differentially regulate the core pluripotency transcription factor Nanog, and eRNAs produced at one distal enhancer specifically activate a different neighboring gene by stabilizing chromatin looping.

Highlights d

d

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Genomic editing reveals functional differences in superenhancer regulation of Nanog 45 enhancer regulates both nearest neighbor genes, Nanog and Dppa3 45 eRNAs specifically regulate Dppa3 by stabilizing chromatin looping

Blinka et al., 2016, Cell Reports 17, 19–28 September 27, 2016 ª 2016 The Author(s). http://dx.doi.org/10.1016/j.celrep.2016.09.002

Cell Reports

Report Super-Enhancers at the Nanog Locus Differentially Regulate Neighboring Pluripotency-Associated Genes Steven Blinka,1,2 Michael H. Reimer, Jr.,1,2 Kirthi Pulakanti,2 and Sridhar Rao1,2,3,4,* 1Department

of Cell Biology, Neurobiology, and Anatomy, Medical College of Wisconsin, Milwaukee, WI 53226, USA Research Institute, BloodCenter of Wisconsin, Milwaukee, WI 53226, USA 3Department of Pediatrics, Medical College of Wisconsin, Milwaukee, WI 53226, USA 4Lead Contact *Correspondence: [email protected] http://dx.doi.org/10.1016/j.celrep.2016.09.002 2Blood

SUMMARY

Super-enhancers are tissue-specific cis-regulatory elements that drive expression of genes associated with cell identity and malignancy. A cardinal feature of super-enhancers is that they are transcribed to produce enhancer-derived RNAs (eRNAs). It remains unclear whether super-enhancers robustly activate genes in situ and whether their functions are attributable to eRNAs or the DNA element. CRISPR/Cas9 was used to systematically delete three discrete super-enhancers at the Nanog locus in embryonic stem cells, revealing functional differences in Nanog transcriptional regulation. One distal super-enhancer 45 kb upstream of Nanog (45 enhancer) regulates both nearest neighbor genes, Nanog and Dppa3. Interestingly, eRNAs produced at the 45 enhancer specifically regulate Dppa3 expression by stabilizing looping of the 45 enhancer and Dppa3. Our work illustrates that genomic editing is required to determine enhancer function and points to a method to selectively target a subset of super-enhancer-regulated genes by depleting eRNAs. INTRODUCTION Embryonic stem cells (ESCs) are distinguished by two unique properties: the ability to undergo unlimited self-renewal while maintaining pluripotency and the capacity to differentiate into all embryonic germ layers. Transcription factors (TFs) form the core machinery that maintain pluripotency by binding ESC specific cis-regulatory elements (CREs) that regulate transcription independent of distance and orientation (Chen et al., 2008). While there have been great advances in identifying the genomic location of ESC-specific enhancers based on epigenetic signatures, much remains unknown about their exact mechanism and contribution to cell identity. Murine ESCs are an ideal model to study epigenetic regulation of cell identity, as the TF and chromatin interaction networks (e.g., chromatin immunoprecipitation sequencing [ChIP-seq] and high-throughput chromosome conformation capture [Hi-C]) controlling pluripotency have been well characterized, resulting in identification of highly active cell-type-specific CREs,

termed super-enhancers or stretch enhancers, which associate with genes critical to cell identity (Whyte et al., 2013). In addition, super-enhancers play a critical role in driving disease processes such as cancer, and targeting them with small molecule inhibitors is an active area of research (Loveń et al., 2013). Murine ESCs have 231 super-enhancers, which are large (10 kb) CREs occupied by an extremely high density of the pluripotency-critical TFs Nanog, Oct4, and Sox2 (NOS) and coactivators such as Mediator (Whyte et al., 2013). Further, super-enhancers are defined by very high levels of the active enhancer epigenetic mark histone 3 Lysine 27 Acetylation (H3K27Ac), are bound by the initiating form of RNA polymerase II (serine 5 phosphorylated [Ser5P] RNAPII), and are bidirectionally transcribed to produce unspliced long non-coding RNAs (lncRNAs) termed enhancer RNAs (eRNAs; Pulakanti et al., 2013; Hnisz et al., 2013). eRNAs are a mark of highly active enhancers (Ørom et al., 2010; Kim et al., 2010; De Santa et al., 2010; Pulakanti et al., 2013) and have diverse roles in regulating transcription, including stabilizing enhancer-promoter looping by tethering cohesin and Mediator complexes at interacting CREs (Li et al., 2013; Lai et al., 2013; Hsieh et al., 2014; Feng et al., 2006; Figure S1A). How discrete super-enhancers in close proximity work together to regulate genes and the relative contribution of the DNA element and its transcribed product (eRNA) to gene expression are currently unknown. The 160-kb extended Nanog locus on murine chromosome 6 contains four genes (Apobec1, Gdf3, Dppa3, and Nanog) that have critical roles early in embryonic development and are NOS regulated in ESCs. Nanog is a homeobox transcription factor and is required for pluripotency and self-renewal in ESCs (Chambers et al., 2003; Mitsui et al., 2003). Enhancer-promoter interactions at the extended Nanog locus have been studied, revealing a complex network of putative CREs that interact and may co-regulate genes (Levasseur et al., 2008; Apostolou et al., 2013; de Wit et al., 2013). Interestingly, the Nanog locus contains three super-enhancers, but the relative contribution of each CRE to Nanog expression has not been studied in situ. We have previously demonstrated one distal superenhancer 45 kb upstream of Nanog is transcribed and produces ESC-specific eRNAs (Pulakanti et al., 2013). Thus, the extended Nanog locus is an ideal genomic region to study the function of multiple super-enhancers and the regulatory capacity of the eRNAs they produce. Here, we demonstrate striking differences in super-enhancer transcriptional regulation of Nanog. In addition, we show that a

Cell Reports 17, 19–28, September 27, 2016 ª 2016 The Author(s). 19 This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

A Chr6:mm9

H3K4me1 H3K27Ac Nanog Oct4 Med1 Smc1a

122,620kb

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(0-120) (0-250) (0-60) (0-230) (0-150) (0-20)

Apobec1

-45 SE

Dppa3

Gdf3

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+60 SE

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45 kb

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Slc2a3

5 kb

Fold Change Relative to Nanog Promoter Only

B

10

WT ESCs Fibroblasts

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8

**

7 6 5

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**

4 3 2 1 0

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*

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-45 Plus -45 Minus -5 Plus -5 Minus +60 Plus +60 Minus Enhancer Enhancer Enhancer Enhancer Enhancer Enhancer Nanog Promoter

Luciferase

Enhancer

Figure 1. Nanog Neighbors Three Putative Super-Enhancers (A) Integrated Genome Viewer (IGV) image of genome-wide chromatin immunoprecipitation (ChIP-seq) datasets in wild-type (WT) ESCs at the extended Nanog locus. Super-enhancers are indicated below in red. (B) Luciferase reporter plasmids containing the Nanog promoter and a fragment of the 45, 5, or +60 enhancer in both orientations were transfected into WT ESCs or a fibroblast cell line (NIH/3T3). All samples had a fold change (y axis) and statistical analysis calculated relative to a plasmid containing Nanog promoter only, which was set to one (dashed line). n = 3. Error bars indicate the SEM between experimental replicates. *p < 0.05, **p < 0.01 using a one-sample Student’s t test.

distal super-enhancer differentially regulates multiple neighboring genes and that eRNAs produced at this super-enhancer selectively activate expression of one neighboring gene. Thus, eRNAs are critical to enhancer function. This study provides insight into the mechanism of transcribed enhancers in a cluster of co-regulated genes, specifically parsing out genes that cluster with a highly active transcribed enhancer versus a subset of genes the enhancer regulates. RESULTS Super-Enhancers Cluster with Nanog Whyte et al., (2013) identified 231 tissue-specific super-enhancers in murine ESCs, which represent 10-fold increase in Dppa3 expression, consistent with a previous report that Nanog represses Dppa3 (Sharov et al., 2008; Figure S4A). Dppa3 depletion led to minimal changes in Nanog expression and other pluripotency genes such as Sall4 (Figure S4B), demonstrating that Dppa3 does not regulate pluripotency. We identified an interaction of the 45 enhancer with the Dppa3 promoter in ESCs using 3C, which was not present in fibroblasts (Figure 4A). Finally, we show that the 45 enhancer activates Dppa3 in reporter assays in ESCs, but not in fibroblasts (Figure 4B). Thus, we determined that the 45 enhancer regulates both nearest neighbor genes within a highly active region of co-regulated genes in ESCs. Further, the modest increase in Dppa3 expression in 5 enhancer monoallelic ESCs demonstrates that the 5 enhancer regulates Nanog, but not Dppa3 (Figures 3B and 3C). Interestingly, despite studies showing clustering of Apobec1 and Gdf3 with Nanog (Levasseur et al., 2008; Apostolou et al., 2013), our genome editing results reveal that none of the super-enhancers regulate these genes (Figure 3B). This suggests linear DNA proximity of the super-enhancer is of functional importance within this compact cluster of co-regulated CREs. eRNAs Produced at the 45 Enhancer Specifically Regulate Dppa3 In just a few years since their discovery, eRNAs have been shown to have diverse functions in transcriptional regulation, including roles in enhancer-promoter looping (Lai et al., 2013; Li et al., 2013; Hsieh et al., 2014). We previously identified ESC-specific eRNAs produced at the 45 enhancer (Pulakanti et al., 2013). The 45 enhancer serves as an interesting opportunity to study eRNA function, as this CRE regulates two neighboring genes, Nanog and Dppa3. To identify activating properties of 45 eRNAs at these two genes, we targeted eRNAs for depletion with modified antisense oligonucleotides (ASOs). eRNAs are restricted to the nucleus, and therefore ASOs, which act by a nuclear-specific RNase H mechanism, have been used to efficiently deplete them. ASOs were designed to target peak levels of eRNAs as determined by global run-on sequencing (GRO-seq; Kaikkonen et al., 2013; Li et al., 2013; Figure S2D), which detects nascent RNA transcription and thus is a sensitive technique to detect eRNAs. Our level of eRNA depletion is comparable to other studies, as ASOs reproducibly depleted eRNAs by >60% relative to a non-targeting ASO (Kaikkonen et al., 2013; Li et al., 2013; Figure 5A). Maintaining >50% depletion is challenging,

A

Cas9 gRNA

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Nanog mRNA Fold Change Relative to WT ESCs

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0.4 0.2 0 -5 Enhancer +/-

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Apobec1 mRNA

Gdf3 mRNA

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1.6 Fold Change Relative to WT ESCs

Fold Change Relative to WT ESCs

1.4 1.2 1 0.8 0.6 0.4 0.2 0 -5 Enhancer +/-

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1.2 1 0.8 0.6 0.4 0.2 0 -5 Enhancer +/-


Gene Expression Changes Following Enhancer Deletion -5 Enhancer -45 Enhancer Monoallelic Biallelic Deletion Deletion

+60 Enhancer Biallelic Deletion

Nanog Dppa3 Figure 3. Genomic Editing Reveals Functional Differences in Super-Enhancer Regulation of Nanog (A) Schematic of CRISPR-mediated super-enhancer deletion at the Nanog locus. (B) Expression of genes within the extended Nanog locus in WT ESCs. Two representative clones are shown for 45- (Neo present), 5-, and +60-enhancerdeleted clones. Error bars indicate the SEM between experimental replicates. n = 3. All samples had a fold change (y axis) and statistical analysis (one-sample Student’s t test) calculated relative to WT ESCs that were set to one (dashed line). A two-sample Student’s t test was used to compare clones. *p < 0.05, **p < 0.01 and ## p < 0.01 using a one-sample and two-sample Student’s t test, respectively. (C) Chart depicting changes in gene expression of Nanog and Dppa3 following deletion of each enhancer.

Cell Reports 17, 19–28, September 27, 2016 23

1.2 Normalized Interaction Ratio

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Primers HaeIII H3K27Ac Nanog Smc1a

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Dppa3

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Figure 4. The 45 Enhancer Regulates Dppa3 (A) A looping event is detected between the 45 enhancer and Dppa3 by 3C in WT ESCs. The x axis indicates genomic distance (kilobases) from the Dppa3 promoter (anchor), and the y axis indicates the normalized interaction ratio. The peak interaction was normalized to one for each WT ESC experimental replicate. The IGV image showing HaeIII cut sites, 3C primers, and ChIP-seq binding profiles in WT ESCs is shown below the graph. (B) A luciferase reporter plasmid containing the Dppa3 promoter and a fragment of the 45 enhancer in both orientations was transfected into WT ESCs or a fibroblast cell line (NIH/3T3). All samples had a fold change (y axis) and statistical analysis calculated relative to a plasmid containing Dppa3 promoter only, which was set to one (dashed line). n = 3. Error bars indicate the SEM between experimental replicates. *p < 0.05 and # p < 0.05 using a one-sample and two-sample Student’s t test, respectively.

24 Cell Reports 17, 19–28, September 27, 2016

as eRNAs are a short-lived molecule and restricted to focal region(s) of the nucleus. Interestingly, 24-hr depletion of eRNAs at the 45 enhancer showed a >40% decrease in Dppa3 expression, with no change in Nanog expression (Figure 5A). This is in contrast to changes following 45 enhancer deletion, which result in decreased expression of both genes (Figure 3B). This demonstrates specificity of eRNAs to regulate only one of the nearest neighbor genes and that eRNAs are partially required for the cis-activating properties of this super-enhancer. Previous studies have shown that lncRNAs interact with epigenetic regulators involved in DNA methylation (Berghoff et al., 2013), catalyzation of enhancer histone marks H3K27Ac (Wang et al., 2008), and histone 3 Lysine 4 methylation (H3K4me; Yang et al., 2014). To examine the mechanism by which eRNAs regulate Dppa3, we first tested for changes in active epigenetics marks of the enhancer. Following eRNA depletion, we observed no change in H3K4me1 or H3K27Ac levels at the 45 enhancer and a putative transcribed enhancer upstream of Oct4 (control genomic region on a different chromosome) by chromatin immunoprecipitation (Figures S5A and S5B). Highly active enhancers also exhibit DNA hypomethylation compared to typical enhancers. Following eRNA depletion, we observed no change in DNA methylation at two CpG clusters within the 45 enhancer (Pulakanti et al., 2013; Figure S5C). In sum, transient depletion of eRNAs does not result in alteration of epigenetics marks of an active enhancer, implying eRNAs are not required for their maintenance. Recent work highlights a role of eRNAs in stabilizing enhancerpromoter looping through interactions with the cohesin and Mediator complexes (Li et al., 2013; Lai et al., 2013). Cohesin is enriched at super-enhancers, which are RNAPII bound and transcribed (Hnisz et al., 2013). Using published enhancer datasets, we show that Smc1a (a component of the cohesin complex) is enriched at all ESC-transcribed enhancers relative to eRNAnegative enhancers (Pulakanti et al., 2013; Figure S5D). This suggests that eRNAs may have a generalized role to tether looping factors such as cohesin to stabilize chromatin interactions in ESCs. To determine the mechanism underlying altered Dppa3 expression, we tested whether eRNA depletion disrupts enhancer-promoter looping by 3C. Using eRNA ASO 1, which results in the most efficient depletion of eRNAs, we observed a 50% decrease in looping of the 45 enhancer at the Dppa3 promoter (Figure 5B). There was no change in interaction of the 45 enhancer with Nanog or the 5 enhancer (Figure 5B). Other interactions at the Nanog locus were also unchanged following eRNA depletion (Figure S5E). Thus, 45 eRNAs stabilize specific enhancer-promoter interactions within a cluster of pluripotency-associated genes to drive robust Dppa3 expression. Our results are in agreement with studies showing no change in active enhancer marks following disruption of enhancer-promoter looping by depleting cohesin (Seitan et al., 2013). Finally, we confirmed that deletion of the 45 enhancer does not alter other super-enhancer interactions with Nanog (Figure 5C), further substantiating that 45 eRNAs specifically regulate a single gene within the cluster of CREs and that the 5 enhancer is sufficient to maintain pluripotency (Figures 3B and 5C).

B 1.4

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1.2 1

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0.6

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0.4

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Nanog mRNA Dppa3 mRNA

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Nanog

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eRNA ASO 2

0.8

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Dppa3

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Normalized Interaction Ratio

Fold Change Relative to Non-Targeting ASO

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0.6 0.4 0.2

-45 eRNAs

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Transcript

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-45:Nanog

Chromatin Interactions

C

D 5’

-45

-5

Nanog

+60

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Nanog

eRNA Depletion

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Dp

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Nanog

Dppa3

Normalized Interaction Ratio

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Dppa3

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Gene

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Super-Enhancer

Chromatin Looping Machinery

0.2

Bidirectional RNAPII (Ser5P) Elongating RNAPII (Ser2P)

0 Nanog:-5

Nanog:+60

eRNAs

Chromatin Interactions Figure 5. 45 eRNAs Regulate Dppa3 Expression by Stabilizing Enhancer-Promoter Interactions (A) 24-hr depletion of 45 eRNAs using antisense oligonucleotides (ASOs) results in a reduction in Dppa3 expression. All samples had a fold change (y axis) and statistical analysis calculated relative to a non-targeting ASO, which was set to one (dashed line). n = 4. (B) Chromatin interactions (3C) between the 45 enhancer and neighboring cis-regulatory elements following eRNA depletion using eRNA ASO 1. Statistical analysis was calculated relative to the non-targeting ASO, which was set to one (dashed line). n = 3. (C) Chromatin interactions (3C) between Nanog and the 5 and +60 enhancers in a 45-enhancer-deleted ESC line (Neo removed). Statistical analysis was calculated relative to WT ESCs, which was set to one (dashed line). n = 3. For (A)–(C) error bars indicate SEM between experimental replicates. *p < 0.05, **p < 0.01 using a one-sample Student’s t test. (D) Two-dimensional schematic of the extended Nanog locus before and after 45 eRNA depletion. 45 eRNAs maintain interaction of Dppa3 and the 45 enhancer and high levels of Dppa3 expression.

DISCUSSION In summary, our data demonstrate that in situ genomic editing is required to unravel the functional role of enhancers, particularly when numerous co-regulated CREs cluster within a compacted chromatin space. Expression changes following CRE

ablation suggests there is a super-enhancer functional hierarchy at the Nanog locus, where the 5 enhancer is the only CRE required for robust Nanog expression and pluripotency. Further, by combining genome editing with eRNA depletion, we demonstrate that eRNAs have a functional role by specifically regulating one of the enhancer associated genes


(Figure 5D). This is in contrast to recent work by Paralkar et al., (2016), where depletion of an enhancer-transcribed long intergenic non-coding RNA (Lockd), which belongs to a class of lncRNAs distinct from eRNAs and is capable of acting in trans, did not phenocopy gene expression changes following enhancer deletion. Rather, our work supports a model whereby eRNAs have critical roles in organizing three-dimensional nuclear interactions, thereby activating a select subset of interacting genes regulated by a super-enhancer (Li et al., 2013; Lai et al., 2013; Hsieh et al., 2014). Under commonly used in vitro serum and leukemia inhibitory factor (LIF) culture conditions, Nanog and Dppa3 are two of the most heterogeneously expressed genes on a cell-to-cell basis (Galonska et al., 2015). ESCs maintained in serum-free defined conditions containing two kinase inhibitors to suppress differentiation cues (2i; Mek and Gsk3 inhibitors) have higher and more homogenous levels of Nanog as well as Dppa3 (Silva et al., 2009; Galonska et al., 2015). These ESCs are in an epigenetically and transcriptionally distinct ‘‘naive’’ pluripotent state, likely as a result of dynamic NOS binding at enhancer elements (Galonska et al., 2015). Interestingly, gene expression differences in Nanog and Dppa3 in these two in vitro conditions parallel expression changes observed in WT and 45-enhancer-deleted ESCs. This suggests that dynamic epigenetic changes in the 45 enhancer landscape in the two ESC states result in differential regulation of Nanog and Dppa3. In addition to driving expression of lineage-critical factors during development, super-enhancers play a critical role in pathologic processes, including malignancies (Hnisz et al., 2013). Inhibition of transcriptional co-activators and chromatin regulators that densely occupy super-enhancers has been shown to decrease aberrant expression of oncogenes in tumor cells, including Myc (Loveń et al., 2013). However, current approaches using small molecules to target co-activators such as Brd4 lack specificity and disrupt most genes regulated by super-enhancers. Our work points to a method to selectively target a subset of genes regulated by super-enhancers in pathologic states. ASOs, which are used as RNA-targeting therapies for a variety of diseases (Arun et al., 2016), offer an intriguing alternative to small molecules, as they allow direct pharmacological targeting of activating eRNAs at oncogenic loci. EXPERIMENTAL PROCEDURES Cell Culture, RNA Isolation, and Real-Time qRT-PCR ESCs were cultured under previously described conditions (Rao et al., 2010). Total RNA isolation, cDNA conversion, and qPCR was performed as previously described (Pulakanti et al., 2013; Stelloh et al., 2016). Primers used for qRT-PCR, ChIP-qPCR, and 3C are listed in Table S1 (all primers in this study were designed using mm9).

Chromosome Conformation Capture 3C libraries for ESC and NIH/3T3 crosslinked chromatin were generated as described in Stelloh et al., (2016) using a modified protocol from Hage`ge et al., (2007). Interaction between the anchoring point and distal fragments was determined by SYBR Green qPCR and normalized to bacterial artificial chromosome/clone (BAC) templates. Fold changes were divided by relative interaction at the Ercc3 locus (a housekeeping gene known to loop in all cell types) to account for differences in library generation (Table S1). At least two independent libraries were used for each cell line, and statistical analysis was calculated relative to WT ESCs. For each genomic region tested, the peak normalized interaction for each ESC library was set to one. Genome Editing To generate 45 Nanog super-enhancer-deleted ESC clones, a single gRNA designed using the CRISPR design tool (http://crispr.mit.edu/) was cloned into the Cas9-expressing vector px459 (Cong et al., 2013). An HDR vector (pL452) containing a floxed Neo cassette flanked by homology regions was co-transfected along with the gRNA in WT ESCs. Individual clones that were resistant to both puromycin and G418 were isolated and expanded for genotyping (Table S1). To generate 5 and +60 Nanog super-enhancer-deleted ESC clones, we used two gRNAs (designed same as above) as described in Zhou et al., (2014) and Huang et al., (2016) (Table S1). Antisense Oligonucleotides ASOs targeting eRNAs produced at the 45 Nanog super-enhancer were designed by Integrated DNA Technologies (Table S1). ASOs were designed with phosphorothioate bonds and a 10-bp gapmer flanked by 5-bp blocks of 20 -Omethyl modified ribonucleotides that protect the ASO from nuclease degradation. ASOs were transiently transfected at 100 nM with Lipofectamine 2000. SUPPLEMENTAL INFORMATION Supplemental Information includes Supplemental Experimental Procedures, five figures, and one table and can be found with this article online at http:// dx.doi.org/10.1016/j.celrep.2016.09.002. AUTHOR CONTRIBUTIONS S.B. performed all experiments, while M.H.R. provided assistance with specific experiments. K.P. assisted with computational analysis. S.B. and S.R. conceived the study and designed experiments. ACKNOWLEDGMENTS We thank Christopher Glass and Minna Kaikkonen for the suggestion of using ASOs for eRNA depletion, Kim Lennox (Integrated DNA Technologies) for designing ASOs, and Sergey Tarima for assistance with statistical analysis. This work was supported in part by funding from the NIDDK (grant DK103502) to S.B. and the MCW MSTP T32 (NIGMS, grant GM080202). The majority of support came from Midwest Athletes against Childhood Cancer, the American Society of Hematology, and Hyundai Hope on Wheels (all to S.R.). Received: February 5, 2016 Revised: June 13, 2016 Accepted: August 30, 2016 Published: September 27, 2016 REFERENCES

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