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. Author manuscript; available in PMC: 2022 Nov 22.
Published in final edited form as: Dev Cell. 2021 Oct 27;56(22):3052–3065.e5. doi: 10.1016/j.devcel.2021.10.004

A bipartite element with allele-specific functions safeguards DNA methylation imprints at the Dlk1-Dio3 locus

Boaz E Aronson 1,14, Laurianne Scourzic 1,14, Veevek Shah 1, Emily Swanzey 2, Andreas Kloetgen 3,4, Alexander Polyzos 1, Abhishek Sinha 5, Annabel Azziz 6, Inbal Caspi 7, Jiexi Li 1, Bobbie Pelham-Webb 8, Rachel A Glenn 6,7, Thomas Vierbuchen 6,9, Hynek Wichterle 5,10, Aristotelis Tsirigos 3,11, Meelad M Dawlaty 12,13, Matthias Stadtfeld 2,15, Effie Apostolou 1,15,16
PMCID: PMC8628258  NIHMSID: NIHMS1754280  PMID: 34710357

SUMMARY

Loss of imprinting (LOI) results in severe developmental defects, but the mechanisms preventing LOI remain incompletely understood. Here, we dissect the functional components of the imprinting control region of the essential Dlk1-Dio3 locus (called IG-DMR) in pluripotent stem cells. We demonstrate that the IG-DMR consists of two antagonistic elements: a paternally methylated CpG-island that prevents recruitment of TET dioxygenases and a maternally unmethylated non-canonical enhancer that ensures expression of the Gtl2 lncRNA by counteracting de novo DNA methyltransferases. Genetic or epigenetic editing of these elements leads to distinct LOI phenotypes with characteristic alternations of allele-specific gene expression, DNA methylation and 3D chromatin topology. Although repression of the Gtl2 promoter results in dysregulated imprinting, the stability of LOI phenotypes depends on the IG-DMR, suggesting a functional hierarchy. These findings establish the IG-DMR as a bipartite control element that maintains imprinting by allele-specific restriction of the DNA (de)methylation machinery.

Graphical Abstract

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eTOC

Aronson, Scourzic et al. identify gene regulatory elements that ensure epigenetic stability at the major imprinted locus, Dlk1-Dio3, encoding essential developmental regulators. They show that these elements operate by counteracting de novo DNA methylation or demethylation in an allele-specific manner, providing a framework for epigenetic gene expression control.

INTRODUCTION

More than 100 mammalian genes, most of them found within coregulated clusters, are expressed in a monoallelic, parent-of-origin specific manner (Tucci et al., 2019). This phenomenon, referred to as genomic imprinting, is essential for mammalian development. Most imprinted expression is controlled by DNA methylation marks that are established in germ cells in a sex-specific manner at cis-regulatory differentially methylated regions (DMRs) called Imprinting Control Regions (ICR). DMRs within imprinted gene loci are subsequently acted upon by specific transcription factors (TF) and chromatin modifiers to ultimately establish patterns of mono-allelic gene expression (Ferguson-Smith and Bourc'his, 2018). These epigenetic patterns are generally preserved in somatic cells throughout development and in adult tissues and their dysregulation by loss-of-imprinting (LOI) can lead to fetal death or developmental abnormalities as well as other disorders, such as cancer (Kalish et al., 2014). The mechanisms by which ICRs ensure retention of parent-of-origin DNA methylation, known as maintenance of imprinting (MOI), remain incompletely understood.

The Dlk1-Dio3 locus is a paradigmatic imprinted gene cluster that encodes multiple non-coding and coding transcripts within a region that spans almost one megabase of mouse chromosome 12 (da Rocha et al., 2008). LOI at Dlk1-Dio3 is associated with severe developmental defects and aggressive malignancies (da Rocha et al., 2008, Jelinic and Shaw, 2007, Khoury et al., 2010, Manodoro et al., 2014) and can also occur during derivation and culture of induced pluripotent stem cells (iPSCs) (Carey et al., 2011, Liu et al., 2010, Stadtfeld et al., 2012, Mo et al., 2015) and embryonic stem cells (ESCs) (Stelzer et al., 2016, Swanzey et al., 2020) impairing their developmental potential. An intergenic DMR or IG-DMR, which resides between the maternally expressed Gtl2 long non-coding RNA (lncRNA) and the paternally expressed Dlk1 protein coding gene, has been shown to function as the ICR of Dlk1-Dio3 locus (Lin et al., 2003). Similarly to the H19-Igf2 and Rasgrf1 ICRs (Kobayashi et al., 2006, Yoon et al., 2005) the IG-DMR is methylated on the paternal allele and has been suggested to control methylation of the Gtl2 promoter, a secondary DMR (“Gtl2 DMR”) that exhibits paternal-specific methylation in post-implantation embryos and in cultured pluripotent cells (Sato et al., 2011, Stadtfeld et al., 2010) (Figure 1A).

Figure 1: A bipartite Intergenic DMR element regulates genomic imprinting at Dlk1-Dio3.

Figure 1:

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(A) Schematic representation of the murine Dlk1-Dio3 locus highlighting maternally (red) and paternally (blue) expressed genes (not to scale). DMR=Differentially Methylated Region, IG=intergenic, Mat=maternal, Pat=paternal. Lollipops represent DNA methylation state: dark=methylated and white= unmethylated. (B) Distinct chromatin features of IGCGI and IGTRE (see also Figure S1A). IG-DMR coordinates (mm10) are boxed. (C) Schematic representation of distinct phenotypic populations detected by flow cytometry using the Dlk1 reporter system shows an example of normal imprinted (MOI) cells (right). (D) Schematic representation of IGCGI and IGTRE deletions. (E) Relative ratios of specific Dlk1 phenotypes in bulk populations after targeting either IGCGI or IGTRE. N=8 for empty vector control (EV), N=8 for IGCGI and N=6 for IGTRE. Significance relative to EV (dashed lines) was calculated using a two-tailed unpaired Student’s t-test. (F) Frequency of indicated phenotypes in clonal cell lines after targeting either IGCGI or IGTRE. (G) FACS plots of clonal lines with LOI-Dlk1Loss and LOI-BiDlk1 phenotypes and confirmed deletion of IGCGI (ΔCGI) and IGTRE (ΔTRE) respectively. (H) RT-qPCR analysis of Dlk1 and Gtl2 expression relative to Gapdh in Retinoic Acid (RA)-differentiated clones (N=6) with indicated genotypes. Statistical significance relative to wildtype (WT) was calculated using a two-tailed unpaired Student’s t-test. (I) Percent DNA methylation at individual CpG resolution in iPSCs at the IG-DMR and the Gtl2 DMR as assessed by pyrosequencing; ‘Region 2’ (IG-DMRTRE) and ‘Region 3’ (Gtl2 DMR) (see methods for coordinates). Note that Region 2 is ~300bp outside of the region deleted by Cas9. N=2 clones per genotype are represented. Two-tailed Paired Student’s t-test was used to calculate significance relative to MOI levels. For all panels:Asterisks indicate significance: * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001. n.s: not significant. Error bars represent +/− SEM. See statistics summarized in Table S4.

Gtl2 has been shown to repress the maternal Dlk1 gene in cis through recruitment of the Polycomb Repressive Complex II (PRC2) (Zhao et al., 2010, Das et al., 2015, Kaneko et al., 2014, Sanli et al., 2018). This suggests that the control of Gtl2 expression by the IG-DMR (Lin et al., 2003, Kota et al., 2014, Luo et al., 2016, Das et al., 2015) is essential for maintenance of imprinting at Dlk1-Dio3. However, how the IG-DMR achieves this regulation in an allele-specific manner remains elusive. Targeted knockout of the IG-DMR (~4kb region) in mice have shown that transmission of maternal deletion results in LOI, and specifically in paternalization of the maternal allele, including silencing of Gtl2 and bi-allelic expression of Dlk1 (Lin et al., 2003). However, transmission of the paternal deletion results in no phenotype (Lin et al., 2003, Das et al., 2015), which is surprising given that the paternal IG-DMR becomes “imprinted” by DNA methylation in the male germline between embryonic days E15.5 to E16.5 (Sato et al., 2011, Nowak et al., 2011, SanMiguel and Bartolomei, 2018). Different phenotypes were recently reported upon deletion of a 216 bp tandem repeat CpG Island (CGI) within the IG-DMR, where the repressive zinc finger protein Zfp57 binds. Paternally transmitted deletion of this element resulted in maternalization of the paternal allele, whereas maternal transmission of the CGI deletion had no phenotype (Saito et al., 2018, Hara et al., 2019).

These apparently contradictory findings suggested to us that the IG-DMR might be a complex genomic element with multiple cis-regulatory regions that coordinate allele-specific gene expression. We therefore decided to dissect the molecular regulatory logic of imprint maintenance at Dlk1-Dio3 in pluripotent stem cells (PSCs), a cell type that represents a tractable model system to investigate epigenetic mechanisms including imprinting (Swanzey et al., 2020). Our results show that the IG-DMR is a bipartite element that maintains imprinting by stabilizing the germ line-specific DNA methylation state at Dlk1-Dio3. This is achieved by the allele-specific function of two antagonistic cis-regulatory regions within the IG-DMR, which we propose to restrict the activity of DNA methyltransferases and dioxygenases and thereby operate as activator or repressor of the Gtl2 DMR, respectively. Allele-specific modulations of these elements was sufficient to induce specific and opposing expression phenotypes and epigenotypes, both indicative of LOI. Intriguingly, we observed that epigenetic repression of the Gtl2 promoter caused bi-allelic Dlk1 expression without affecting methylation at the IG-DMR. However, this LOI phenotype was unstable and reverted to MOI over time. Therefore, in addition to resolving the complex composition of the IG-DMR, our findings reveal previously unappreciated regulatory principles of the functional hierarchy between genomic elements operational at a gene cluster essential for mammalian development.

RESULTS

The IG-DMR is a bipartite element with two distinct functions

To gain insights into the regulatory mechanisms underlying the maintenance of Dlk1-Dio3 imprinting (Figure 1A), we first assembled in-house (Di Giammartino et al., 2019, Liu et al., 2017, Pelham-Webb et al., 2021) and published datasets (Williams et al., 2011, Shi et al., 2019), including datasets from the CODEX database (Sanchez-Castillo et al., 2015), on DNA methylation, chromatin accessibility, nascent transcription, histone modifications and TF binding profiles at the IG-DMR in mouse ESCs (Takada et al., 2002, Kobayashi et al., 2006, Hiura et al., 2007). This revealed a pronounced dichotomy with respect to the position and nature of epigenomic features within the IG-DMR (chr12: 109,526,778-109,530,083 mm10) (Figures 1A-B and S1A). The 2kb region closer to the Dlk1 gene exhibits high CpG density including a CpG island (CGI) with conserved tandem repeats between human, mouse and sheep (Paulsen et al., 2001), binding of the repressive KRAB domain containing zinc-finger protein Zfp57 (Quenneville et al., 2011, Luo et al., 2016, Riso et al., 2016, Shi et al., 2019) (Figures 1A-B) and components of chromatin-modifying complexes such as PRC2 (Figure S1A). In contrast, the 1kb of the IG-DMR closest to the Glt2 gene is associated with an open chromatin state enriched for the activating H3K27ac mark, nascent bidirectional transcription, the binding of multiple pluripotency-associated TFs and general transcription regulators, consistent with a Transcriptional Regulatory Element (TRE) function (Danko et al., 2015) (Figure S1A). These data suggest that the IG-DMR may consist of two distinct regulatory elements, which we will refer to as IGCGI and IGTRE.

To functionally interrogate potentially distinct roles of the IGCGI and IGTRE in regulating Dlk1-Dio3, we deleted each of these elements in iPSCs harboring a previously described allele-specific Dlk1 reporter system (Swanzey and Stadtfeld, 2016). This transgenic system, in which maternal and paternal Dlk1 alleles are transcriptionally linked to distinct fluorescent reporters (mVenus and tdTomato respectively) (Figure 1C), has been shown to accurately capture the allele-specific expression of Dlk1 and thereby reflecting the imprinting status of the entire Dlk1-Dio3 locus (Swanzey and Stadtfeld, 2016).Since undifferentiated iPSCs do not express appreciable levels of Dlk1, we optimized our flow cytometry analysis approach by combining retinoic acid (RA) induced differentiation with antibody staining against the neuroectodermal marker CD24a and gating on CD24a+ cells (Semrau et al., 2017) (Figure S1B). This strategy allowed us to reliably identify instances of LOI that lead either to bi-allelic expression (LOI-BiDIk1) or complete silencing (LOI-Dlk1Loss) of Dlk1. Reporter iPSCs were transiently transfected with plasmids expressing Cas9 and pairs of guide RNAs (gRNAs) targeting the 5’- and 3’-ends of either IGCGI and IGTRE (Figure 1D, Table S1 and S2). Strikingly, while targeting of the IGCGI resulted in an increased proportion of cells that lost Dlk1 expression (LOI-Dlk1Loss), targeting of the IGTRE led to bi-allelic expression of Dlk1 (LOI-BiDlk1) (Figure 1E). Genotyping of clonal lines obtained by single cell sorting confirmed mono- or bi-allelic deletion of the targeted regions in cells with LOI phenotypes, while clones with MOI had mono-allelic deletions or unedited genomes (Figure 1F, Figure S1C and Table S1). This established LOI-Dlk1Loss as the predominant LOI phenotype in IGCGI targeted clones and LOI-BiDlk1 in IGTRE targeted clones (Figure 1G). These phenotypes were independently validated in the context of directed differentiation towards motor neurons (Wichterle et al., 2002, Novitch et al., 2001) (Figures S1D,E). Quantitative PCR confirmed the Dlk1 expression changes observed with flow cytometry and showed an inverse expression pattern between Dlk1 and the maternally expressed lncRNA Gtl2 (Figure 1H and Table S1). Bisulfite sequencing of ΔIGCGI and ΔIGTRE clones showed that both the IG-DMR itself and the Gtl2 DMR had completely lost methylation upon deletion of IGCGI, while both control elements were fully methylated upon elimination of IGTRE (Figure 1I). We observed the same imprinting phenotypes upon deletion of IGCGI and IGTRE in ESCs carrying the allele-specific reporter systems (Figures S1F-I). Taken together, these data show that the IG-DMR consists of two distinct regulatory elements with essential – but antagonistic – roles in imprinting maintenance in pluripotent cells. Deletion of these elements is sufficient to induce pronounced and specific epigenetic and transcriptional changes throughout the locus and induce either bi-maternal or bi-paternal LOI.

IGCGI and IGTRE are allele-specific regulators of imprinted DNA methylation and gene expression at Dlk1-Dio3

Next, we sought to establish the allelic specificity of IGCGI and IGTRE. To that end, we generated iPSCs carrying the aforementioned allele specific Dlk1 reporter system in a hybrid JF1/B6 F1 background (Figure 2A and Table S2). The JF1 strain carries several SNPs in the IG-DMR compared to the B6 genetic background (Koide et al., 1998) and ESCs from this strain maintain normal imprinting upon culture (Lee et al., 2018). By using gRNAs designed to target sequences with strain-specific SNPs within the first 3 base-pairs downstream of the PAM sequence, we performed allele-specific CRISPR-Cas9 deletion of the IGCGI and IGTRE regions (Figure S2A and Table S1, S2). Derivation and analysis of individual clones by flow cytometry demonstrated that deletion of the paternal IGCGI (ΔPatCGI) was responsible for the Dlk1 loss phenotype, while the BiDIkl phenotype was induced by maternal IGTRE (ΔMatTRE) deletion (Figures 2B, S2B). Genotyping confirmed allele-specific deletions (Figures S2C-F and Table S1). While deletion of MatCGI did not result in a phenotype, some clones (10 out of 56) from the ΔPatTRE targeting did result in the same BiDlk1 phenotype observed upon targeting the maternal allele. However, investigation of these clones revealed indels on the maternal allele (data not shown) indicating allelic promiscuity of Cas9 (Capon et al., 2017), supporting that ΔPatTRE does not result in LOI. Quantitative PCR analysis and subsequent sequencing of clonal amplicons confirmed the expected reciprocal effects of allele-specific PatCGI and MatTRE deletions on Dlk1 and Gtl2 expression while knockout of the allelic counterparts resulted in no such changes (Figures 2C-D, S2G and Table S1). Consistent with these findings and our results obtained via non-allele specific genetic engineering (Figure 1I), CpGs within the IG-DMR and Gtl2 DMR were unmethylated upon paternal IGCGI removal (indicating loss of the paternal imprinting mark) and hypermethylated upon maternal IGTRE removal (indicating aberrant establishment of an imprinting mark on the maternal allele) while control cells exhibited DNA methylation levels typical for epigenetically intact naïve pluripotent cells (Carey et al., 2011, Stadtfeld et al., 2010) (Figure 2E and Table S3).

Figure 2: Dual role of IG-DMR in allele specific regulation of Dlk1-Dio3 locus.

Figure 2:

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(A) Schematic of insertion of an IRES-tdTomato cassette into the maternal Dlk1 allele and validation of MOI in resultant B6xJF1 hybrid reporter iPSCs. OKSM=OCT4, KLF4, SOX2 and MYC; AA= Ascorbic Acid. (B) Representative FACS plots of clones with allele-specific deletions of IGCGI and IGTRE. (C) Allele-specific Gtl2 and Dlk1 expression levels in RA-differentiated clones (N=2 clones). Statistics with two-tailed unpaired Student’s t-test. (D) A heterozygous SNP (arrow) in the Gtl2 RT-qPCR amplicon allows for evaluation of allele-specific expression. (E) Average percentage of DNA methylation in iPSCs at the IG-DMR and the Gtl2 DMR as assessed by pyrosequencing; ‘Region 1’ (IG-DMRCGI), ‘Region 2’ (IG-DMRTRE) and ‘Region 3’ (Gtl2 DMR). N=2 each for clones with maternal IGTRE deletion and paternal IGCGI deletion. Significance with two-tailed Paired Student’s t-test. (F) Schematic of DNA methylation and gene expression changes upon allele-specific deletion of either IGCGI or IGTRE. (G) Allele-specific 4C-seq with IG-DMR as viewpoint. Statistics for IG-DMR interaction (Mat vs Pat) with Gtl2 and Dlk1 are shown at chr12:109540996-109568650 (FDR= 2.3E−64 for MOI and 0.760 for ΔPatCGI) and chr12:109453455-109463336 (FDR= 0.015 for MOI and 0.760 for ΔPatCGI), respectively. For all panels: Asterisks indicate significance: * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001. n.s: not significant. Error bars represent +/− SEM. See statistics summarized in Table S4.

To investigate whether additional chromatin changes occur in the locus, we performed allele-specific chromatin immunoprecipitation (as-ChIP) for the active histone mark H3K27ac followed by Sanger sequencing. While MOI cells strongly enriched for H3K27ac only on the maternal IG-DMR and Gtl2 DMR, ΔPatCGI clones exhibited bi-allelic H3K27ac and ΔMatTRE clones lost H3K27ac almost entirely (Figure S2H and Table S1). Collectively, our results demonstrate that the unmethylated IGTRE is associated with Gtl2 expression and an active chromatin state on the maternal allele, whereas the methylated IGCGI correlates with Gtl2 repression and an inactive chromatin state on the paternal allele (Figure 2F).

We also asked whether the observed epigenetic and transcriptional changes were accompanied by 3D chromatin reorganization around the locus, by using allele-specific 4C-seq. In agreement with recent reports (Lleres et al., 2019), we found that the long-range chromatin contacts around the IG-DMR differ between the maternal and paternal alleles. Specifically, the maternal unmethylated IG-DMR interacts at high frequency with Gtl2, while these interactions are significantly weaker on the paternal allele (Figure 2G). In addition, we found a subtle interaction of the maternal IG-DMR with Dlk1 that, although statistically significant, is marginal and unlikely to be representative of consistent close proximity between these loci (Figure 2G). Deletion of the paternal IGCGI perturbed this allele-specific topology and induced a maternal-like conformation on both alleles (Figures 2G and Table S1). On the other hand, deletion of the maternal IGTRE induced a significant overall decrease of interactivity resembling a bi-paternal topology (Figure S2I). These results suggest that DNA methylation of IGCGI might prevent the physical interaction of the IG-DMR with its target genes and that IGTRE is essential for the maintenance of these long-range chromatin contacts. Taken together, our observations show that the paternal IGCGI and maternal IGTRE have distinct regulatory functions that are required to maintain the respective alleles in epigenetic and conformational states consistent with MOI at Dlk1-Dio3.

The bipartite nature of the IG-DMR results in allele-specific suppression of the DNA methylation machinery

De novo DNA methyltransferases and TET dioxygenases operate at ICRs to establish and erase methylation in germ cells during development (Li and Sasaki, 2011, Bartolomei and Ferguson-Smith, 2011, Plasschaert and Bartolomei, 2014, SanMiguel and Bartolomei, 2018). The allele specific DNA methylation changes that we observed upon IGTRE and IGCGI deletion (Figure 2E) raised the possibility that these enzymes might also be involved in the induction of the distinct LOI phenotypes. In agreement with this, allele-specific chromatin immunoprecipitation (ChIP) experiments documented that DNMT3A, which normally only binds to the paternal allele of the IG-DMR, acquired bi-allelic binding upon deletion of the IGTRE (Figure 3A and S3A). Similarly, we found TET1 to be bound to both alleles of the IG-DMR only upon IGCGI deletion (Figure 3B and S3A). To directly assess the importance of de novo DNA methylation for LOI, we deleted IGTRE in parallel to the gene loci encoding DNMT3A and DNMT3B (Dnmt3a/b) in Dlk1 reporter iPSCs using Cas9 technology (Figure 3C and Table S1). Subsequent differentiation and FACS analysis of bulk populations showed a significant amelioration of the LOI-BiDlk1 phenotype and an increased proportion of cells that retained MOI (Figures 3D-E). Genotyping of targeted regions and surveyor assays in bulk populations confirmed the respective deletions and indels by CRISPR/Cas9 and showed amplicons of equal strength, demonstrating similar degrees of targeting efficiency across experimental conditions (Figure S3B and Table S1). To confirm these observations at the single cell level, we derived and validated clonal ΔIGTRE cell lines from previously characterized DNMT3A/B knockout (DKO) (Lei et al., 1996, Okano et al., 1999) and parental control ESCs. In contrast to WT cells, DKO ESCs lacking the IGTRE did not acquire DNA hypermethylation at either IG-DMR or Gtl2 DMR before (Figure 3F) or after (Figure S3C) after differentiation. This is consistent with the notion that IGTRE antagonizes the activity of canonical de novo methyltransferases on the maternal allele as suggested by ChIP. Nevertheless, Gtl2 expression in ESCs without IGTRE remained low even in absence of DNMT3A/B (Figure S3D). However, upon RA differentiation DNMT3A/B KO ESCs – but not WT ESCs lacking the IGTRE – were able to upregulate Gtl2, which was associated with significantly reduced levels of Dlk1 expression (Figure 3G). Together, these observations suggest that misdirected DNMT3A/B activity contributes to the LOI phenotype observed upon loss of the IGTRE. Of note, ablation of DNMT3A/B in reporter iPSCs without simultaneous removal of IGTRE showed no effects on imprinting stability. This is consistent with prior reports indicating that these two enzymes are not required for maintenance of imprinting at Dlk1-Dio3 in mouse embryos with an intact IG-DMR (Hirasawa et al., 2008).

Figure 3: Allele-specific prevention of DNA methyltransferase and TET protein function by the IG-DMR.

Figure 3:

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(A) Increased DNMT3A occupancy of the IGCGI upon maternal IGTRE deletion (left) due to bi-allelic binding (right) as measured by ChIP-qPCR and sequencing of PCR products. (B) Increased TET1 occupancy of the IGTRE upon paternal IGCGI deletion (left) due to bi-allelic binding (right) as measured by ChIP-qPCR and sequencing of PCR products. N=2 clones for (A,B ). (C) Schematic of combined IG-DMR editing and Dnmt3a/b deletion. (D) Ratios of phenotypic populations in pooled populations upon targeting Dnmt3a/b and IGTRE alone or in combination N=6 experiments. Statistical significance compared to EV (dashed line) with two-tailed unpaired Student’s t-test. (E) Representative bulk populations FACS plots upon indicated deletions. (F) Average percentage of DNA methylation in RA-differentiated clones at IG-DMR and Gtl2 DMR as assessed by pyrosequencing; ‘Region 1’ (IG-DMRCGI), ‘Region 2’ (IG-DMRTRE) and ‘Region 3’ (Gtl2 DMR). (G) Dlk1 and Gtl2 expression relative to Gapdh in six WT and three DNMT3A/B Knock out (DKO) RA-differentiated IGTRE clones. (H) Strategy of targeting dCas9-TET1 to the IGCGI or dCas9-DNMT3A to the IGTRE and (I) representative bulk population FACS plots after implementation. (J) DNA methylation in iPSCs after indicated dCas9 targeting; ‘Region 1’ (IG-DMRCGI), ‘Region 2’ (IG-DMRTRE) and ‘Region 3’ (Gtl2 DMR). Statistical significance with two-tailed paired Student’s t-test relative to the respective EV clone (see also Table S2-4). (K) Gtl2 RNA expression levels in dCas9-TET1 and dCas9-DNMT3A iPSCs. Statistical significance with two-tailed unpaired Student’s t-test. For all panels: Asterisks indicate significance: * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001. n.s: not significant. Error bars represent +/− SEM. See statistics summarized in Table S4.

Similar to our observations with DNMT3A/B and the IGTRE, flow cytometric analysis of iPSCs after simultaneous deletion of the IGCGI and the genes encoding all TET proteins (Tet1, Tet2 and Tet3) (Dawlaty et al., 2014) suggested amelioration of the LOI-Dlk1 phenotype (Figures S3E-H). However, analysis of clones derived from either TET1/2/3-deficient or WT ESCs several weeks after IGCGI deletion showed loss of DNA methylation at the IG-DMR in almost all cell lines (Figure S3I) and comparable Dlk1/Gtl2 expression levels (Figure S3J), suggesting that LOI upon IGCGI deletion is inevitable even in absence of TET enzymes, possibly by a passive DNA demethylation.

Together the experiments further highlight the essential roles of IGTRE and IGCGI in preventing de novo DNA methylation or demethylation, respectively, and thus maintaining Dlk1-Dio3 imprinting.

Forced gain or loss of DNA methylation at the IG-DMR is sufficient to induce LOI

The results described above demonstrate that the protection of allele-specific DNA methylation states by the IGCGI and IGTRE are important to maintain imprinting at Dlk1-Dio3. We therefore asked whether local DNA methylation changes at either of these respective elements could phenocopy the effect of the respective genetic deletions on imprint stability. We generated Dlk1 reporter iPSCs that express a catalytically deactivated Cas9 (dCas9) fused to the catalytic domain of either TET1 or DNMT3A (Tables S1 and S2) (Xu et al., 2016, Choudhury et al., 2016, Morita et al., 2016, Verma et al., 2018). We then stably expressed multiplexed gRNAs targeting either three ZFP57 binding sites within the IGCGI or the sites of nascent transcription of the IGTRE (Figure 3H and Table S1). We observed that targeting of dCas9-TET1 to the IGCGI resulted in conversion of almost all cells to the LOI-Dlk1Loss phenotype (Figure 3I), recapitulating our observations upon deletion of the PatCGI (Figure 2B). This phenotype was accompanied by loss of DNA methylation on both IG-DMR and Gtl2 DMR along with Gtl2 upregulation (Figures 3J, K and Table S1). On the other hand, targeting of dCas9-DNMT3A to the IGTRE induced a LOI-BiDlk1 population with respective hypermethylation and loss of Gtl2 expression (Figures 3H-K). Taken together, these experiments demonstrate that local changes in DNA methylation at the IGCGI and IGTRE are sufficient to affect the methylation status at the distal Gtl2 DMR and perturb imprinted gene expression.

DMR-specific targeting of CRISPRi results in either transient or irreversible LOI

So far, our results have shown that allele-specific genetic or epigenetic manipulation of the specific elements within the IG-DMR induces LOI phenotypes that correlate with methylation and transcriptional changes at the Gtl2. To test whether the regulatory function of the IG-DMR can be overridden by direct silencing of Gtl2, we generated stable reporter iPSCs expressing the transcriptional repressor dCas9-BFP-KRAB (CRISPRi) and gRNAs that target either the IGcGI, the IGTRE or the Gtl2 DMR (Figure 4A and Table S1). Of note, the KRAB domain used in our experiments was shown to mediate stable de novo DNA methylation in early embryonic cells (Quenneville et al., 2012, Ying et al., 2015). Consistent with dCas9-mediated epigenetic editing of the IG-DMR (Figures 3H-K), targeting of CRISPRi to either the IGCGI or IGTRE resulted in hypermethylation of the IG-DMR and Gtl2 DMR as well as Gtl2 repression (Figures 4B-D and S4A, B). Targeting of the Gtl2 DMR similarly triggered a high degree of LOI-BiDlk1, but in contrast to IG-DMR targeting, only induced local Gtl2 DMR hypermethylation without affecting the methylation status of the IG-DMR (Figure 4D). This demonstrates that targeted repression of Gtl2 is sufficient to activate maternal Dlk1 without acquisition of DNA methylation at the IG-DMR. To test whether the absence of IG-DMR hypermethylation might affect the stability of this LOI phenotype, we made use of the observation that our lentiviral CRISPRi, without continuous resorting or reselection, is silenced over time in PSCs. We established BFP+ and BFP− subclones from originally BFP+ (i.e. dCas9-BFP-KRAB expressing) cells, followed by extensive passaging allowing for potential reversal of epigenetic effects after dCas9-KRAB (Mandegar et al., 2016). This was done in cells where CRISPRi was targeted either to the IGCGI or to the Gtl2 DMR. FACS analysis showed that all CGI-targeted clones retained their LOI-BiDlk1 phenotype and hypermethylation of both IG-DMR and Gtl2 DMR, independently of their BFP expression status (Figures 4E-F). This demonstrates that the change in imprinting status was irreversible. In contrast, three out of six Gtl2-targeted BFP− clones partially reverted to MOI (Figures 4E-F and S4C). This partial rescue was not seen in clones that retained CRISPRi expression (BFP+). Bisulfite sequencing of the MOI population of partially rescued clones showed reestablishment of normal methylation levels (~50%) at the Gtl2 promoter. Interestingly, the remaining BiDlk1 population of rescued clones showed intermediate levels of methylation that were ~10% reduced compared to clones still expressing CRISPRi, suggesting an ongoing DNA demethylation that could reach MOI levels upon further passaging (Figure 4G). Furthermore, qPCR analysis confirmed that Gtl2 expression was significantly increased in rescued Gtl2-targeted clones compared to CGI-targeted clones that had lost CRISPRi expression (Figure 4H and Table S1). These results suggest that although modulating the activity or methylation of the Gtl2 promoter can transiently alter gene expression at Dlk1-Dio3, the DNA methylation status of the IG-DMR is the determining factor for long-term imprinting stability of this cluster.

Figure 4: IG-DMR methylation status dictates Dlk1-Dio3 imprinting stability.

Figure 4:

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(A) Strategy to element-specific targeting of dCas9-BFP-KRAB. (B) Representative FACS plots showing dominant LOI-BiDlk1 populations after bi-allelic KRAB targeting to either IGCGI or Gtl2 promoter (see also Figure S4A). (C) RT-qPCR analysis of Gtl2 expression in indicated cells. Statistical significance was calculated by two-tailed unpaired Student’s t-test. (D) Average percentage of methylated CpGs at IG-DMR and Gtl2 DMR as determined by bisulfite sequencing analysis. Note that targeting dCas9-BFP-KRAB to the Gtl2 DMR does not result in hypermethylation at the IG-DMR. Statistical analysis by comparison to the EV controls using two-tailed paired Student’s t-test. (E) FACS plots of dCas9-BFP-KRAB CGI-targeted clone and a rescued Gtl2-targeted clone at passage 15 (p15). (F) Percentage of the LOI-BiDlk1 population in N=4 dCas9-BFP-KRAB CGI-targeted clones and N=6 Gtl2-targeted clones at p15. (G) Bisulfite sequencing analysis in iPSCs of MOI and BiDIk1 populations of two rescued dCas9-BFP-KRAB targeted clones; ‘Region 1’ (IG-DMRCGI) and ‘Region 3’ (Gtl2 DMR). Statistical significance with two-tailed paired Student’s t-test using CGI clones #27 and #28 as controls (see Table S2). (H) RT-qPCR analysis of Gtl2 expression in the BFP− populations of Gtl2-targeted and IGCGI-targeted clones. Statistical analysis by comparison within each BFP− or BFP+ population and normalized to EV using two-tailed paired Student’s t-test. For all panels: Asterisks indicate significance: * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001. n.s: not significant. Error bars represent +/− SEM. See statistics summarized in Table S4.

DISCUSSION

In this study, we combine genetic engineering with targeted epigenetic editing in mouse pluripotent stem cells to dissect the regulatory mechanisms that protect imprint stability at Dlk1-Dio3, a gene cluster essential for mammalian development. Our findings extend prior studies that have identified the IG-DMR as the critical control element of Dlk1-Dio3 (Lin et al., 2003, Nowak et al., 2011, Kota et al., 2014, Luo et al., 2016, Wang et al., 2017, Saito et al., 2018) and reconcile previously divergent in vivo phenotypes upon knockout of different parts of the IG-DMR (Hara et al., 2019, Lin et al., 2003, Saito et al., 2018) . More importantly, our study provides insights into the IG-DMR structure and mechanisms of action that can be exploited to modulate Dlk1-Dio3 imprinting status in a predictable fashion.

By allele-specific CRISPR knockout experiments, we revealed that the IG-DMR is a bipartite element, composed of a repressive element (IGCGI) that functions on the paternal allele and an activating element (IGTRE) on the maternal allele, both of which are necessary to maintain allele-specific gene expression over distance (Figure S4D). Deletion of each element from the respective allele results in either bi-paternal or bi-maternal LOI, while reciprocal deletions show normal imprinting. Furthermore, we provide evidence that the two elements of IG-DMR independently safeguard the imprinted DNA methylation state of Dlk1-Dio3 by restricting the competing de novo DNA methylation and demethylation activities on the locus in an allele-specific manner. All these features document that the IG-DMR is distinct from other previously described composite ICRs, which serve as methylation-sensitive insulator boundaries, as in the case of Rasgrf1 and Snrpn (Yoon et al., 2005, Bartolomei, 2009, Rabinovitz et al., 2012, Hsiao et al., 2019). Intriguingly, some of the molecular characteristics of the IG-DMR resemble the features of so-called orphan CGI, which are distal to promoters and are characterized by active enhancer marks, such as H3K27ac, topological interactions with nearby target genes and overlapping ZFP57 and MLL1 consensus motifs (Quenneville et al., 2011, Anvar et al., 2016, Bae et al., 2016, Bina, 2017, Bell and Vertino, 2017, Mendizabal and Yi, 2016). Importantly, similarly to the IG-DMR, the activity of orphan CGIs is controlled by DNA methylation (Illingworth et al., 2010). Orphan CGIs were also recently shown to mediate physical and functional communication between poised enhancers and target developmental genes (Pachano et al., 2021). Future experiments will be required to identify functional commonalities between IG-DMR and orphan CGI enhancers.

It has previously been suggested that the IG-DMR functions as an enhancer for Gtl2 expression (Lin et al., 2003, Kota et al., 2014, Das et al., 2015, Luo et al., 2016, Wang et al., 2017). Consistent with these studies, we found that deletion of the IG-DMR, and specifically the maternal unmethylated IGTRE, resulted in transcriptional silencing of Gtl2 in PSCs, but also in DNA hypermethylation of both IG-DMR and Gtl2 DMR. Deletion of IGTRE in the absence of DNA methyltransferase activity allowed us to functionally uncouple these molecular effects. This supported a dual function of the IGTRE both as transcriptional enhancer of Gtl2 in PSCs, and – more importantly – as a guardian of the unmethylated status of the maternal Gtl2 DMR, thereby maintaining proper imprinting of the locus. The activation of Gtl2 expression upon differentiation of DNMT3A/B-deficient ESCs even in absence of the IGTRE suggests the presence of additional cell-type specific enhancers and/or of trans-acting factors that can act directly upon the Gtl2 promoter in differentiated cells when the latter is unmethylated. Whether this non-canonical enhancer function is present in other ICRs or developmental loci, remains to be shown. The factors and mechanisms that counteract DNA methyltransferase activity on IGTRE and the distal Gtl2 DMR remain elusive. Our CRISPRi experiments support that the active chromatin and transcriptional status of IGTRE is necessary for protecting the maternal allele from de novo DNA methylation both locally and on the Gtl2 DMR – potentially through looping (Figure S4D). Indeed, active histone marks (Rose and Klose, 2014) as well as binding of transcription factors or the activating complex CBP/p300 have been shown to protect CpG sites from DNMT3A/B activity (Gebhard et al., 2010, Lienert et al., 2011, Straussman et al., 2009, Zhang et al., 2017). Similarly, the nascent non-coding transcripts of the locus (enhancer RNA and/or Gtl2 non-coding RNA) may also play protective roles, as has been previously suggested (Kota et al., 2014), by either interacting with PRC2 (Das et al., 2015) or DNMT3A/B and DNMT1 (Zhao et al., 2016, Morlando and Fatica, 2018). Finally, recent studies demonstrated that a class of transcription factors, including key pluripotency regulators such as SOX2 and KLF4 which bind on IGTRE, can directly prevent DNA methylation, or even induce demethylation of their target sites (Vanzan et al., 2021). Further investigation will be required to dissect the relative contribution of each of these factors in maintenance of imprinting at Dlk1-Dio3.

In sharp contrast to IGTRE which blocks de novo DNA methylation on the maternal allele, our experiments showed that IGCGI is essential for preventing both TET-dependent and independent DNA demethylation of the paternal DMRs. Several trans-acting factors could mediate this function (Figure S4D). A candidate is the KRAB domain containing zinc finger ZFP57, which has been shown to bind to the methylated IG-DMR in the region of the IGCGI (Riso et al., 2016, Luo et al., 2016, Strogantsev et al., 2015, Shi et al., 2019) and maintains closed chromatin by recruiting repressive complexes, such as DNA and H3K9 methyltransferases (Quenneville et al., 2011, Riso et al., 2016). Moreover, knockdown of Zfp57 has been shown to cause accumulation of 5-hydroxymethylcytosine (5hmC) at the IG-DMR (Coluccio et al., 2018), which suggests that this protein is involved in preventing TET binding and activity, as well as passive DNA demethylation on the paternal IG-DMR.

To further dissect the regulatory logic of the Dlk1-Dio3 locus, we harnessed the power of dCas9 epigenetic editing (Hsu et al., 2014). These experiments revealed a hierarchical and unidirectional regulation between IG-DMR and Gtl2 DMR that is substantiated by the following key observations. First, although any targeted modulation of the IG-DMR (either by dCas9-TET1, dCas9-DNMT3A or CRISPRi) resulted in methylation changes on both DMRs, targeting of the Gtl2 DMR affected only local DNA methylation without any effects on IG-DMR, suggesting a “one-way” communication. In addition, although targeting of the Gtl2 DMR can cause LOI, normal imprinting can be restored by the IG-DMR, suggesting that this element controls long-term imprinting stability. These observations are consistent with the observation that the Gtl2 promoter acquires allele-specific DNA methylation during embryonic development to match the status of the IG-DMR (Sato et al., 2011). Dissecting the molecular mechanisms (e.g. physical proximity or spreading) underlying this regulatory hierarchy and their degree of conservation among cell types (Alexander et al., 2019) and species would be interesting areas for future investigation, in particular in the context of imprinting disorders.

In conclusion, our study refined the mechanistic understanding of the regulatory logic that safeguards imprinting stability at Dlk1-Dio3 and revealed ways to perturb this logic in a rationale and predictable manner. Although previously reported in vivo phenotypes are consistent with a bi-partite nature of the IG-DMR, additional work will be required to dissect the precise function of IGCGI and IGTRE during establishment and maintenance of Dlk1-Dio3 imprinting in the organism. We envision that the molecular principles operational at the IG-DMR may serve as paradigms to better understand the epigenetic regulation of other developmental gene loci.

Limitations of the Study

While this work provides a detailed investigation of the molecular function of the IG-DMR in maintaining genomic imprinting in mouse pluripotent cells, additional work will be required to confirm the bipartite nature of this control element in other cell types and during the initial establishment of Dlk1-Dio3 imprinting in male germ cells in vivo. In addition, the precise molecular mechanisms of LOI downstream of inactivation of IGCGI and IGTRE – such as the involvement of additional protein and non-protein factors and their relationship to DNMT3A/B and TET proteins – remain to be elucidated.

STAR METHODS

RESOURCE AVAILABILITY

Lead Contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Dr. Effie Apostolou (efa2001@med.cornell.edu).

Materials Availability

Plasmids and cell lines uniquely generated in this study (see Key Resource Table) are available from the lead contact upon request.

KEY RESOURCES TABLE

REAGENT or RESOURCE SOURCE IDENTIFIER
Antibodies
Rabbit monoclonal anti-H3K27ac Abcam Cat# ab4729, RRID: AB_2118291
Mouse monoclonal anti-CD24 APC-eFluor 780 Thermo Fisher Scientific Cat# 47-0242-82, RRID:AB_10853172
Chicken polyclonal anti-GFP Thermo Fisher Scientific Cat# A10262, RRID:AB_2534023
Rabbit polyclonal anti-RFP Abcam Cat# ab62341, RRID:AB_945213
Rabbit polyclonal anti-Tet1 GeneTex Cat# GTX125888, RRID:AB_11164485
Mouse polyclonal anti-Dnmt3a Novus Biological Cat# NB120-13888, RRID:AB_789607
Mouse anti-Isl1/2 Jessell Lab/HHMI CU RRID: AB_2314683
Chemicals, peptides, and recombinant proteins
Lipofectamine 2000 Invitrogen Cat# 11668019
T7 endonuclease NEB Cat# M0302L
Protein G Dynabeads Invitrogen Cat# 10004D
Ampure XP beads Beckman Coulter Cat# A63880
Di(N-succinimidyl) glutarate (DSG) Sigma Cat# 80424
Retinoic acid Sigma Cat#R2625
Deposited Data
Raw and analyzed allele-specific 4C-seq data This paper GSE148315
Mouse reference genome GRCm38 (mm10) Genome Reference Consortium https://www.ncbi.nlm.nih.gov/assembly/GCF_000001635.20/
Experimental models: Cell lines
Mouse B6J: iPSC Dlk1matVenus/patTomato, clone #6aa Swanzey et al., 2016 N/A
Mouse matJF1/patB6N/J: iPSC Dlk1matTomato/patVenus, clone #104 This paper N/A
Dnmt3ab KO ESCs, clone DKO#9 Lei et al.,1996 N/A
Tet1/2/3 KO ESCs, clone TKO#9 Dawlaty et al., 2014 N/A
Tet1/2/3 WT ESCs, clone WT#8 Dawlaty et al., 2014 N/A
Oligonucleotides
See Table S1 gRNAs and primer sequences
Recombinant DNA
pdCas9-Dnmt3a-puro Vojta et al., 2016 Addgene Plasmid #71667
pPGKENTRY-dCas9-Tet1CD Verma et al., 2018. N/A
pMIGR1-IRES-BSD This paper N/A
pBS-Dlk1-tomato Swanzey et al., 2016 N/A
pHR-SFFV-dCAS9-BFP-KRAB Gilbert et al., 2013 Addgene Plasmid #46911
pLKO5.sgRNA.EFS.PAC Heckl et al., 2014 Addgene Plasmid #57825
pSpCas9(BB)-2A-GFP (pX458) Ran et al., 2013 Addgene Plasmid #48138
pHR-EF1a-dCAS9-Dnmt3a-BSD This paper N/A
pHR-EF1a-dCAS9-Tet1-BSD This paper N/A
pLKO5.sgRNA.EFS.PAC - F+E - Neo This paper N/A
pHR-EF1a-dCAS9-BFP-KRAB This paper N/A
pX330-PGK-NeoR Le Cong et al, 2013 Addgene Plasmid #42230
Software and algorithms
FlowJo (v10.7.0) BD Biosciences http://v9docs.flowjo.com/html/index.html
RRID:SCR_008520
IGV browser Robinson et al., 2011 https://software.broadinstitute.org/software/igv/
RRID:SCR_011793
Samtools (v1.8) Li et al., 2009 http://samtools.sourceforge.net/
RRID:SCR_002105
ImageJ Schindelin et al., 2015 https://imagej.nih.gov/ij/
RRID:SCR_003070
GraphPad Prism GraphPad http://www.graphpad.com/
RRID:SCR_002798
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Data and code availability

  • 4C-seq data generated in this study have been deposited at GEO under the accession number GSE148315 and are available as of the date of publication.

  • This study did not generate a new code, but the scripts used in the study are available from the Lead Contact upon request.

  • Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.

EXPERIMENTAL MODELS AND SUBJECT DETAILS

Cell lines

Primary irradiated mouse embryonic fibroblasts (MEF’s) were derived from either male or female embryos between ED13.5-14.5. Dnmt3ab KO (Lei et al., 1996, Okano et al., 1999) as well as Tet1/2/3 WT and KO ESCs (Dawlaty et al., 2014) were obtained as described previously. Allele-specific iPSC reporter (Swanzey and Stadtfeld, 2016) were derived from male Tail Tip Fibroblasts 1 to 2 days post-natal and all experiments were conducted at low passage unless otherwise noted.

Animals

Mouse strain colony maintenance and crosses were performed according to protocols approved by the Institutional Animal Care and Use Committee of Weill Cornell Medical College, in compliance with the ARRIVE guidelines. Mice were housed in a specific pathogen-free, temperature-controlled facility with a 12-h light/dark cycle in individually ventilated cages. Animals were provided food (standard rodent chow diet) and water ad libitum. B6 mice used to generate JF1/B6 iPSCs were on a mixed background between C57BL/6NJ (Jax 005304) and C57BL/6J (Jax 000664).

METHOD DETAILS

ESC and iPSC cell culture

All cells were cultured at 37°C with 5% CO2. C57BL/6J and C57BI/6J-JF1 iPSC’s or V6.5 ESC’s were cultured on plates coated with 0.2% gelatin on irradiated mouse embryonic fibroblasts (MEF’s) with ES KO medium supplemented with 10% FBS, 10mg recombinant leukemia inhibitory factor (LIF), 0.1 mM beta-mercaptoethanol (Sigma-Aldrich), penicillin/streptomycin, 1mM L-glutamine and 1% nonessential amino acids (all from Invitrogen).

Generating allele-specific iPSC Dlk1-reporter in hybrid maternal JF1/paternal B6 background

4μg total of IRES-tdTomato-Neomycin donor (Swanzey and Stadtfeld, 2016) and CRISPR/Cas9 vectors were transfected into Mat-JF1 X Pat-B6-Venus Tail Tip Fibroblasts-derived IPSCs using Lipofectamine 2000 (Invitrogen #11668019). Transfected cells were selected for 4 days with 1mg/ml Geniticin (Invitrogen #10131-035) and plated on DR4 MEFs. Individual clones were picked, expanded and screened for the proper integration of tdTomato and persistence of GFP sequence by PCR. Sole expression of Pat-Venus was confirmed by flow cytometry and proper monoallelic Gtl2 expression was confirmed by RT-qPCR and Sanger sequencing (see complete list of expression primers in Table S1).

PSC with CRISPR/Cas9 mediated deletions

sgRNA sequences were cloned either into pX330-U6-Chimeric_BB-CBh-hSpCas9 (pX330) or into the pSpCas9(BB)-2A-GFP (pX458) vector under the U6 promoter by digesting the vector with Bbsl (NEB #0539) and ligating annealed sgRNA oligonucleotides into the vector with T4 ligase (NEB, M0202L) under standard conditions. pX458-sgRNA’s were transfected using Lipofectamine 2000 (Invitrogen #11668019) into 300k iPSC’s on plates coated with 0.2% gelatin. 800 ng DNA was transfected per gRNA supplemented to 4μg total DNA with scrambled pX458 vector. iPSC’s were sorted for GFP 48-72 hours post-transfection and plated on MEF feeder plates. Subsequent clonal populations were obtained by single-cell sorting the bulk on 96-well plates on the FACS Aria II (BD biosciences).

Confirmation of deletions was performed by genotyping using a 3-primer strategy with 2 primers flanking and 1 primer inside the region of interest. As all primers have a different distance to the ‘breakpoint’, deletions and inversions could be detected by gel electrophoresis after PCR. For allele-specific genotyping, the primers were positioned on a SNP on the 3’ end and contained 1 mismatch in the first 5 nucleotides upstream of the SNP. Indels, generated by transfecting Cas9 with a single gRNA, were confirmed with T7 surveyor assays as described previously (Guschin et al., 2010). All sgRNA oligonucleotides, genotyping and Surveyor primers are listed in supplemental Table S1.

Cloning of multiple gRNA expression vectors for dCas9 expressing iPSC’s

sgRNA’s were cloned into the pLKO5.sgRNA.FCS.PAC-NEO under the U6 promoter as described above. Next, the U6 promoter, sgRNA and scaffold combinations were PCR amplified with primers containing overhangs with recognition sites of various restriction enzymes (see below). These enzymes were chosen in such a manner that a digested amplicon would have a complementary overhang with the next amplicon. Digestion and subsequent ligation of all guides into the vector thus resulted in simultaneous insertion of multiple gRNA’s that are each under the control of a single U6 promoter.

To insert three guides, the vector was digested with XbaI (NEB, R0145), amplicon-1 was digested with XbaI and MfeI (NEB, R0589), amplicon −2 was digested with EcoRI (NEB, R0101) and Sall (NEB, R0138) and amplicon −3 was digested with XhoI (NEB, R0146) and AvrII (NEB, R0174). To insert four guides, the vector was digested with XbaI, amplicon −1 was digested with XbaI and MfeI, amplicon −2 was digested with EcoRI and BgIII (NEB, R0144), amplicon 3 with BamH1 (NEB, R3136) and Sail and amplicon −4 was digested with XhoI and AvrII (Dow et al., 2015). Ligation was performed in T4 ligation buffer (NEB B0202) and incubated with T4 Ligase (5 μl, 400U/μl; NEB M0202L).

Cloning of dCas9 plasmids

To create a vector for stable dCas9 expression in iPSC’s, we chose the lentiviral pHR-SFFV-dCAS9-BFP-KRAB vector (Gilbert et al., 2013) and performed an EcoRI digestion to remove the SFFV promoter. An Ef1α promoter sequence was PCR amplified from pdCas9-VP64 (Addgene #61425) using primers with EcoRI restriction site overhangs. The amplicon was EcoRI digested and ligated to replace the SFFV promoter.

To create pHR-dCas9-TET1-BSD, we digested the pHR-Ef1α-dCAS9-BFP-KRAB with Sbfl-HF (NEB, R3642) and Mlul-HF (NEB, R0198) to remove dCas9-BFP-KRAB. We then PCR-amplified a dCas9-TET1 sequence from pENTRY-dCas9-TET1CD (Verma et al., 2018) and an IRES-BSD sequence from pMIGR-IRES-BSD. The purified IRES-BSD product was digested with EcoRI. The fragments were ligated using Gibson assembly (NEB E2611L).

To create pHR-dCas9-DNMT3A-BSD, we PCR amplified the dCas9-DNMT3A sequence from pdCas9-DNMT3A-puro (Vojta et al., 2016) and digested pMIGR-IRES-BSD with EcoRI to produce an IRES-BSD fragment. The fragments were ligated using Gibson assembly.

To create gRNA delivery vectors, sgRNAs were cloned into the pLKO5.sgRNA.FCS.PAC (Heckl et al., 2014) with adjustments made to the scaffold according to (Chen et al., 2013). The original puromycin cassette was removed by BamHI-MluI digestion and a neomycin sequence was PCR amplified from a pHR-Dlk1-IRES-tomato vector and inserted with Gibson assembly. Primers used for cloning have been listed in Table S1. All used and constructed plasmids have been assembled in Table S2.

Lentiviral production and infection.

293T cells were transfected with overexpression constructs along with the packaging vectors VSV-G and Delta8.9 using PEI reagent (PEI MAX, Polyscience, 24765-2). The supernatant was collected after 48 and 72 h, and the virus was concentrated using polyethylglycol (Sigma, P4338). V6.5 cells or iPSC’s were infected in medium containing 5 μg/ml polybrene (Millipore, TR-1003-G), followed by centrifugation at 2,500 r.p.m. for 90 min at 32 °C. Media was changed after 8-12h following spinfection.

Generation of stable dCas9 expressing cell lines

To generate stable dCas9 and gRNA expressing cell lines, IPSCs were transduced with concentrated lentivirus (as described above). 5x 3000 cells were pre-plated on gelatin 0.2% on 5 wells of 96-well plates and transduced each with 10ul concentrated virus in 100ul KO-DMEM with polybrene 1mg/ml. The cells were passaged and selected with 10 μg/ml Blasticidin (Life Tech, A11139-03) (dCas9/DNMT3A and dCas9-TET1) for 4 days or sorted on the FACS Aria II (dCas9-KRAB-BFP). The selected populations were then transduced again with the pLKO5 lentivirus, containing sgRNA’s against the target of interest, and selected with 1 mg/ml geneticin (Invitrogen 10131-035) for 5 days.

Retinoic acid differentiation assays

For differentiation assays, 10K cells/cm2 were plated in standard DMEM supplemented with 10% FBS, 0.1 mM beta-mercaptoethanol (Sigma-Aldrich), penicillin/streptomycin, 1mM L-glutamine and 1% nonessential amino acids (all from Invitrogen). 0.4 μg/ml retinoic acid (Sigma-Aldrich, R2625) was added followed by daily media changes for six days.

Flow cytometry

To assess the proportion of mVenus and tdTomato in the established reporter cell lines, a single-cell suspension was filtered, stained with anti-mouse CD24a/APC-eFluo780 antibody (Affymetrix 47-0242-82) and assessed on the BD Aria or FACS Canto II. Analysis was done in FlowJo (BD Biosciences).

qPCR

Total mRNA was extracted from pre-plated iPSC’s and RA differentiated cells using Qiagen RNeasy kit (Qiagen 74106). On-column DNA digestion was performed on all samples with RNAse free DNAse set (Qiagen 79254). RNA was reverse transcribed with random hexamers using iScript™ Reverse Transcription Supermix for RT-qPCR (Biorad, 1708841). Total expression of transcripts was quantified by qRT-PCR using Powerup SYBR Green Master Mix (Life Technologies, A25778) and amplification performed on a QuantStudio3 (Applied Biosystems). Expression primers can be found in Table S1.

Bisulfite conversion and pyrosequencing

To assess methylation, genomic DNA was extracted from pre-plated iPSC’s or sorted RA differentiated populations. The cells were lysed overnight at 50 °C in Lysis buffer (10mM Tris pH 7.5, 150mM NaCI, 0.2% SDS) followed by isopropanol precipitation. Genomic DNA was sent to EpigenDx Inc. for bisulfite conversion and pyrosequencing. The genomic location (mm10) of the three assessed regions were:Region 1:chr12:109528391-109528471, Region 2: chr12:109530388-109530457 and Region 3: chr12:109541718-109541777. See full list of DNA methylation quantifications summarized in Table S3.

ChIP-qPCR

ChIP was performed as previously described (Liu et al., 2017). Specifically, cells were crosslinked in 1% formaldehyde at room temperature for 10 min and quenched with 125 mM glycine for 5 min at room temperature. A 50 min incubation with 2mM Disuccinimidyl glutarate (DSG) was used in addition to formaldehyde for TET1 ChIP two-step crosslinking. The cells were used for DNMT3A and TET1 (30 × 106 cells) and H3K27ac ChIP (10 × 106 cells). The cell pellets were washed twice in PBS and resuspended in 400 μl lysis buffer (10 mM Tris pH 8, 1 mM EDTA and 0.5% SDS) per 20 × 106 cells. The cells were sonicated using the Bioruptor (Diagenode) (30 cycles of 30 s on/off; high setting) and spun down at the maximum speed for 10 min at 4°C. The supernatants were diluted five times with dilution buffer (0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris pH 8 and 167 mM NaCI) and incubated overnight with antibodies against histone H3K27ac (Abcam, ab4729), TET1 (GeneTex 125888) and DNMT3A (Novus Biological 120-13888) with rotation at 4°C. Protein G Dynabeads (Invitrogen, 10004D) pre-blocked with BSA protein (100 ng per 10 μl Dynabeads) were added (10 μl blocked Dynabeads per 10 × 106 cells) the following day and incubated for 2–3h at 4°C. An additional pre-clearing step was performed whereby the lysed cells were mixed with beads alone for 3h and removed prior to adding the DNMT3A antibody. The beads were immobilized on a magnet and washed twice in low-salt buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 150 mM NaCI and 20 mM Tris pH 8), twice in high-salt buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 500 mM NaCI and 20 mM Tris pH 8), twice in LiCI buffer (0.25 M LiCI, 1% NP-40, 1% deoxycholic acid (sodium salt), 1 mM EDTA and 10 mM Tris pH 8) and once in TE buffer. The DNA was then eluted from the beads by incubating with 150 μl elution buffer (1% SDS and 100 mM NaHCO3) for 20 min at 65°C (vortexing every 10 min). The supernatants were collected and reverse crosslinked by incubation overnight at 65°C in the presence of proteinase K. After RNase A treatment for 1 h at 37°C, the DNA was purified using a minElute kit (Qiagen, 28004). Enrichment of protein binding to defined DNA sequences was assessed by qPCR. Primer sequences can be found in supplemental Table S1.

4C-seq

Experiments were performed in duplicate to generate two technical replicates per sample. JF1/B6 iPSC’s (2 × 106) were crosslinked in 1% formaldehyde at room temperature for 10 min and quenched with 125 mM glycine for 5 min at room temperature. The cell pellets were washed twice in PBS and resuspended in 300 μl lysis buffer (10mM Tris-HCI (pH 8.0), 10mM NaCI, 0.2% Igepal CA630 (Sigma I8896), on ice for 20 min. Following centrifugation at 2,500g for 5 min at 4°C, the pellet was resuspended in in 50uL of 0.5% SDS and incubated for 10 min at 65°C. SDS was quenched with 145uL water and 25uL of 10% TritonX-100. 25ul of Cutsmart buffer was added with Dpnll enzyme (10 μl; NEB, R0543M) and the samples were incubated overnight at 37 °C with 700rpm rotation. The samples were then diluted with 663μl Milli-Q water, 120 μl T4 ligation buffer (NEB B0202), 60ul ATP 10mM, 120 μl Triton X-100, 12ul BSA 10mg/ml and incubated with Ligase (5 μl, 400U/μl; NEB M0202L) for 3h on a rotor at room temperature. The samples were then treated with proteinase K and reverse crosslinked overnight. Following RNAse treatment, phenol/chloroform extraction and DNA precipitation, the pellets were dissolved in 100 μl of 10 mM Tris pH 7.5 and digested overnight at 37°C by adding 20 μl Cutsmart buffer (NEB), 10ul μl NlaIII (NEB, R0125) and 70 μl Milli-Q water. Following enzyme inactivation, the samples were diluted in 2345 ul Milli-Q water, 300 ul 10×ligation buffer (NEB), 150ul ATP 10mM and incubated with, 5 μl T4 DNA ligase 2M U/ul (NEB, M0202M) overnight at 16°C. The DNA was purified by phenol/chloroform extraction, ethanol precipitation and Zymo columns (D4014).

For allele-specific 4C-seq library preparation, primers were designed upstream of a SNP within IG-DMR. Due to lack of suitable restriction fragments on ΔMatTRE samples, non-allele specific libraries were prepared with the Gtl2 DMR as viewpoint. Library preparation was further performed by using a PCR strategy as previously described (Krijger et al., 2020). Briefly, 4x200 ng of 4C-template DNA was used to PCR amplify the libraries using the Roche Expand long template PCR system (Roche, 11681842001). Primers were removed using Ampure XP beads (Beckman Coulter, A63880). A second round of PCR was performed using the initial PCR library as a template, with overlapping primers to add the full adapters. The libraries were sequenced on a miSeq platform in SE150 mode. All primer sequences can be found in Table S1.

Motor neuron differentiation assays

Motor neuron differentiations were performed as described (Wichterle et al., 2002). Briefly, iPSC colonies were dissociated and plated at a 23K cells/cm2 in ADFNK medium (Neurobasal medium (Thermofisher 21103049), 10% KnockOut Serum Replacement (Thermofisher 10828028), 0.1 mM beta-mercaptoethanol, 2mM L-glutamine, and penicillin/streptomycin). Two days post-dissociation, embryoid bodies were treated with 1μM retinoic acid and 0.5μM SAG for four days. Embryoid bodies were collected for further analysis at day 6 post dissociation.

Immunocytochemistry

Embryoid bodies were fixed, sectioned, and processed for immunocytochemistry with antibodies as previously described (Novitch et al., 2001). Briefly, embryoid bodies were fixed for 30 minutes at 4°C with 4% Paraformaldehyde, 0.1% Triton-X-100, and 10% Horse Serum in PBS. Then, following 3 washes in 1x PBS, embryoid bodies were equilibrated in 30% sucrose/PBS for 30 minutes, embedded in OCT, and sectioned on a crystostat (15 μM). Primary antibody incubation of sections was performed in PBS with 10% Horse Serum overnight at 4°C. Following 3 washes in 1X PBS, secondary antibody incubation of sections was performed in PBS with 10% Horse Serum for 1 hour at room temperature. Sections were then imaged on an inverted microscope (Zeiss Axio Observer Z1 Inverted Microscope).

QUANTIFICATION AND STATISTICAL ANALYSIS

Analysis of 4C-seq data

The 4C-seq data was analyzed in a similar fashion as recently described (Raviram et al., 2016, Di Giammartino et al., 2019). Viewpoint primers were trimmed off from all sequencing reads using seqtk (version 1.3.0). Then, allele-specific reads were identified by perfectly matching the respective allele-specific primer sequence against each read, and raw fastq files were split to represent maternal and paternal alleles as individual samples separately. The read-sequence was aligned using bowtie2 v2.3.4.1 (Langmead and Salzberg, 2012) against a reduced genome that consists only of reference genome sequences adjacent to DpnII/NlaIII cut-sites (following the 4C-ker pipeline) (Raviram et al., 2016). Next, the genome was binned into 5kb bins shifted by 500bp (overlapping by 90% with adjacent bins). Reads were counted by unique alignment position in all overlapping bins. Read counts per bin were normalized by sequencing depth per replicate using edgeR (version 3.14.0) (Nikolayeva and Robinson, 2014), resulting in counts per million (CPM). Significance of differential interaction was determined by glmQLFit and glmQLFTest functions from edgeR followed by mutliple testing correction and displaying false-discovery rate (FDR). Visualization was done using average CPM signals per condition.

Statistical analysis

Results were displayed as mean ± SEM and statistically analyzed using the GraphPad Prism software (version 8.4.1). Statistical significance of differences between the results was assessed mostly using a two-tailed unpaired Student’s t-test. Welch’s correction was applied when comparing samples with significantly different variances. Two-tailed Paired Student’s t-test was used specifically for CpG methylation analysis. Statistically significant p-values were indicated as follows: * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001. No statistical method was used to predetermine sample size, nor blinding or randomization of samples were applied.

Supplementary Material

Supplemental Information
Table S3

Table S3: Percentage of DNA methylation for clones analyzed in this study (excel) (related to STAR methods)

Table S4

Table S4. Raw values and statistics for all graphs included in this study (excel) (related to STAR methods)

HIGHLIGHTS.

  • The IG-DMR is a bipartite element with distinct allele-specific functions

  • A non-canonical enhancer within the IG-DMR prevents DNA methyltransferase activity

  • Targeted epigenome editing allows induction of specific imprinting phenotypes

  • CRISPRi reveals a functional hierarchy between DMRs that dictates imprint stability

ACKNOWLEDGEMENTS

We are grateful to A. Melnick, R. Chaligne and the members of the Apostolou, Tsirigos and Stadtfeld laboratories for critical reading of the manuscript, to N. Ben Chetrit for advice on microscopy experiments and to D. Huangfu and L. Dow for sharing CRISPR–Cas9 vectors. B.E.A. is supported by the National Health Institutes, National Cancer Institute (NCI) Grant T32 CA203702, L.S. is supported by the Lady TATA Memorial Fund and the Lymphoma Research Foundation, A.T. is supported by the NCI (P01CA229086 and R01CA252239), B.P.W. is supported by the NICHD with a T32 (T32HD060600) and an F30 (F30HD097926), as well as by a Medical Scientist Training Program grant from the NIGMS under award number T32GM007739 to the Weill Cornell/Rockefeller/Sloan Kettering Tri-lnstitutional MD-PhD Program, M.S. is supported by grants from the NIH (R01GM121994) and the Tri-Institutional Stem Cell Initiative by the Starr Foundation (Tri-SCI). E.A. is supported by the Edward Mallinckrodt Jr. Foundation, the NIH (DP2DA043813 and R01GM138635) and Tri-SCI.

Footnotes

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DECLARATION OF INTERESTS

The authors declare no competing interests.

REFERENCES

  1. ALEXANDER KA & GARCIA-GARCIA MJ Imprinted gene expression at the Dlk1-Dio3 cluster is controlled by both maternal and paternal IG-DMRs in a tissue-specific fashion. 10.1101/536102 [DOI] [Google Scholar]
  2. ANVAR Z, CAMMISA M, RISO V, BAGLIVO I, KUKREJA H, SPARAGO A, GIRARDOT M, LAD S, DE FEIS I, CERRATO F, ANGELINI C, FEIL R, PEDONE PV, GRIMALDI G & RICCIO A 2016. ZFP57 recognizes multiple and closely spaced sequence motif variants to maintain repressive epigenetic marks in mouse embryonic stem cells. Nucleic Acids Res, 44, 1118–32. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. BAE MG, KIM JY & CHOI JK 2016. Frequent hypermethylation of orphan CpG islands with enhancer activity in cancer. BMC Med Genomics, 9 Suppl 1, 38. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. BARTOLOMEI MS 2009. Genomic imprinting: employing and avoiding epigenetic processes. Genes Dev, 23, 2124–33. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. BARTOLOMEI MS & FERGUSON-SMITH AC 2011. Mammalian genomic imprinting. Cold Spring Harb Perspect Biol, 3. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. BELL JSK & VERTINO PM 2017. Orphan CpG islands define a novel class of highly active enhancers. Epigenetics, 12, 449–464. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. BINA M 2017. Imprinted control regions include composite DNA elements consisting of the ZFP57 binding site overlapping MLL1 morphemes. Genomics, 109, 265–273. [DOI] [PubMed] [Google Scholar]
  8. CAPON SJ, BAILLIE GJ, BOWER NI, DA SILVA JA, PATERSON S, HOGAN BM, SlMONS C & SMITH KA 2017. Utilising polymorphisms to achieve allele-specific genome editing in zebrafish. Biol Open, 6, 125–131. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. CAREY BW, MARKOULAKI S, HANNA JH, FADDAH DA, BUGANIM Y, KIM J, GANZ K, STEINE EJ, CASSADY JP, CREYGHTON MP, WELSTEAD GG, GAO Q & JAENISCH R 2011. Reprogramming factor stoichiometry influences the epigenetic state and biological properties of induced pluripotent stem cells. Cell Stem Cell, 9, 588–98. [DOI] [PubMed] [Google Scholar]
  10. CHEN B, GILBERT LA, CIMINI BA, SCHNITZBAUER J, ZHANG W, LI GW, PARK J, BLACKBURN EH, WEISSMAN JS, QI LS & HUANG B 2013. Dynamic imaging of genomic loci in living human cells by an optimized CRISPR/Cas system. Cell, 155, 1479–91. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. CHOUDHURY SR, CUI Y, LUBECKA K, STEFANSKA B & IRUDAYARAJ J 2016. CRISPR-dCas9 mediated TET1 targeting for selective DNA demethylation at BRCA1 promoter. Oncotarget, 7, 46545–46556. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. COLUCCIO A, ECCO G, DUC J, OFFNER S, TURELLI P & TRONO D 2018. Individual retrotransposon integrants are differentially controlled by KZFP/KAP1-dependent histone methylation, DNA methylation and TET-mediated hydroxymethylation in naive embryonic stem cells. Epigenetics Chromatin, 11, 7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. DA ROCHA ST, EDWARDS CA, ITO M, OGATA T & FERGUSON-SMITH AC 2008. Genomic imprinting at the mammalian Dlk1-Dio3 domain. Trends Genet, 24, 306–16. [DOI] [PubMed] [Google Scholar]
  14. DANKO CG, HYLAND SL, CORE LJ, MARTINS AL, WATERS CT, LEE HW, CHEUNG VG, KRAUS WL, LIS JT & SIEPEL A 2015. Identification of active transcriptional regulatory elements from GRO-seq data. Nat Methods, 12, 433–8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. DAS PP, HENDRIX DA, APOSTOLOU E, BUCHNER AH, CANVER MC, BEYAZ S, LJUBOJA D, KUINTZLE R, KIM W, KARNIK R, SHAO Z, XIE H, XU J, DE LOS ANGELES A, ZHANG Y, CHOE J, JUN DL, SHEN X, GREGORY RI, DALEY GQ, MEISSNER A, KELLIS M, HOCHEDLINGER K, KIM J & ORKIN SH 2015. PRC2 Is Required to Maintain Expression of the Maternal Gtl2-Rian-Mirg Locus by Preventing De Novo DNA Methylation in Mouse Embryonic Stem Cells. Cell Rep, 12, 1456–70. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. DAWLATY MM, BREILING A, LE T, BARRASA MI, RADDATZ G, GAO Q, POWELL BE, CHENG AW, FAULL KF, LYKO F & JAENISCH R 2014. Loss of Tet enzymes compromises proper differentiation of embryonic stem cells. Dev Cell, 29, 102–11. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. DI GIAMMARTINO DC, KLOETGEN A, POLYZOS A, LIU Y, KIM D, MURPHY D, ABUHASHEM A, CAVALIERE P, ARONSON B, SHAH V, DEPHOURE N, STADTFELD M, TSIRIGOS A & APOSTOLOU E 2019. KLF4 is involved in the organization and regulation of pluripotency-associated three-dimensional enhancer networks. Nat Cell Biol, 21, 1179–1190. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. DOW LE, FISHER J, O'ROURKE KP, MULEY A, KASTENHUBER ER, LIVSHITS G, TSCHAHARGANEH DF, SOCCI ND & LOWE SW 2015. Inducible in vivo genome editing with CRISPR-Cas9. Nat Biotechnol, 33, 390–394. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. FERGUSON-SMITH AC & BOURC'HIS D 2018. The discovery and importance of genomic imprinting. Elite, 7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. GEBHARD C, BENNER C, EHRICH M, SCHWARZFISCHER L, SCHILLING E, KLUG M, DIETMAIER W, THIEDE C, HOLLER E, ANDREESEN R & REHLI M 2010. General transcription factor binding at CpG islands in normal cells correlates with resistance to de novo DNA methylation in cancer cells. Cancer Res, 70, 1398–407. [DOI] [PubMed] [Google Scholar]
  21. GILBERT LA, LARSON MH, MORSUT L, LIU Z, BRAR GA, TORRES SE, STERN-GINOSSAR N, BRANDMAN O, WHITEHEAD EH, DOUDNA JA, LIM WA, WEISSMAN JS & QI LS 2013. CRISPR-mediated modular RNA-guided regulation of transcription in eukaryotes. Cell, 154, 442–51. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. GUSCHIN DY, WAITE AJ, KATIBAH GE, MILLER JC, HOLMES MC & REBAR EJ 2010. A rapid and general assay for monitoring endogenous gene modification. Methods Mol Biol, 649, 247–56. [DOI] [PubMed] [Google Scholar]
  23. HARA S, TERAO M, MURAMATSU A & TAKADA S 2019. Efficient production and transmission of CRISPR/Cas9-mediated mutant alleles at the IG-DMR via generation of mosaic mice using a modified 2CC method. Sci Rep, 9, 20202. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. HECKL D, KOWALCZYK MS, YUDOVICH D, BELIZAIRE R, PURAM RV, MCCONKEY ME, THIELKE A, ASTER JC, REGEV A & EBERT BL 2014. Generation of mouse models of myeloid malignancy with combinatorial genetic lesions using CRISPR-Cas9 genome editing. Nat Biotechnol, 32, 941–6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. HIRASAWA R, CHIBA H, KANEDA M, TAJIMA S, LI E, JAENISCH R & SASAKI H 2008. Maternal and zygotic Dnmt1 are necessary and sufficient for the maintenance of DNA methylation imprints during preimplantation development. Genes Dev, 22, 1607–16. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. HIURA H, KOMIYAMA J, SHIRAI M, OBATA Y, OGAWA H & KONO T 2007. DNA methylation imprints on the IG-DMR of the Dlk1-Gtl2 domain in mouse male germline. FEBS Lett, 581, 1255–60. [DOI] [PubMed] [Google Scholar]
  27. HSIAO JS, GERMAIN ND, WILDERMAN A, STODDARD C, WOJENSKI LA, VILLAFANO GJ, CORE L, COTNEY J & CHAMBERLAIN SJ 2019. A bipartite boundary element restricts UBE3A imprinting to mature neurons. Proc Natl Acad Sci U S A, 116, 2181–2186. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. HSU PD, LANDER ES & ZHANG F 2014. Development and applications of CRISPR-Cas9 for genome engineering. Cell, 157, 1262–78. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. ILLINGWORTH RS, GRUENEWALD-SCHNEIDER U, WEBB S, KERR AR, JAMES KD, TURNER DJ, SMITH C, HARRISON DJ, ANDREWS R & BIRD AP 2010. Orphan CpG islands identify numerous conserved promoters in the mammalian genome. PLoS Genet, 6, e1001134. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. JELINIC P & SHAW P 2007. Loss of imprinting and cancer. J Pathol, 211, 261–8. [DOI] [PubMed] [Google Scholar]
  31. KALISH JM, JIANG C & BARTOLOMEI MS 2014. Epigenetics and imprinting in human disease. Int J Dev Biol, 58, 291–8. [DOI] [PubMed] [Google Scholar]
  32. KANEKO S, SON J, BONASIO R, SHEN SS & REINBERG D 2014. Nascent RNA interaction keeps PRC2 activity poised and in check. Genes Dev, 28, 1983–8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. KHOURY H, SUAREZ-SAIZ F, WU S & MINDEN MD 2010. An upstream insulator regulates DLK1 imprinting in AML. Blood, 115, 2260–3. [DOI] [PubMed] [Google Scholar]
  34. KOBAYASHI H, SUDA C, ABE T, KOHARA Y, IKEMURA T & SASAKI H 2006. Bisulfite sequencing and dinucleotide content analysis of 15 imprinted mouse differentially methylated regions (DMRs): paternally methylated DMRs contain less CpGs than maternally methylated DMRs. Cytogenet Genome Res, 113, 130–7. [DOI] [PubMed] [Google Scholar]
  35. KOIDE T, MORIWAKI K, UCHIDA K, MITA A, SAGAI T, YONEKAWA H, KATOH H, MIYASHITA N, TSUCHIYA K, NIELSEN TJ & SHIROISHI T 1998. A new inbred strain JF1 established from Japanese fancy mouse carrying the classic piebald allele. Mamm Genome, 9, 15–9. [DOI] [PubMed] [Google Scholar]
  36. KOTA SK, LLERES D, BOUSCHET T, HIRASAWA R, MARCHAND A, BEGON-PESCIA C, SANLI I, ARNAUD P, JOURNOT L, GIRARDOT M & FEIL R 2014. ICR noncoding RNA expression controls imprinting and DNA replication at the Dlk1-Dio3 domain. Dev Cell, 31, 19–33. [DOI] [PubMed] [Google Scholar]
  37. KRIJGER PHL, GEEVEN G, BIANCHI V, HILVERING CRE & DE LAAT W 2020. 4C-seq from beginning to end: A detailed protocol for sample preparation and data analysis. Methods, 170, 17–32. [DOI] [PubMed] [Google Scholar]
  38. LANGMEAD B & SALZBERG SL 2012. Fast gapped-read alignment with Bowtie 2. Nat Methods, 9, 357–9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. LEE J, MATSUZAWA A, SHIURA H, SUTANI A & ISHINO F 2018. Preferable in vitro condition for maintaining faithful DNA methylation imprinting in mouse embryonic stem cells. Genes Cells, 23, 146–160. [DOI] [PubMed] [Google Scholar]
  40. LEI H, OH SP, OKANO M, JUTTERMANN R, GOSS KA, JAENISCH R & LI E 1996. De novo DNA cytosine methyltransferase activities in mouse embryonic stem cells. Development, 122, 3195–205. [DOI] [PubMed] [Google Scholar]
  41. LI Y & SASAKI H 2011. Genomic imprinting in mammals: its life cycle, molecular mechanisms and reprogramming. Cell Res, 21, 466–73. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. LIENERT F, WIRBELAUER C, SOM I, DEAN A, MOHN F & SCHUBELER D 2011. Identification of genetic elements that autonomously determine DNA methylation states. Nat Genet, 43, 1091–7. [DOI] [PubMed] [Google Scholar]
  43. LIN SP, YOUNGSON N, TAKADA S, SEITZ H, REIK W, PAULSEN M, CAVAILLE J & FERGUSON-SMITH AC 2003. Asymmetric regulation of imprinting on the maternal and paternal chromosomes at the Dlk1-Gtl2 imprinted cluster on mouse chromosome 12. Nat Genet, 35, 97–102. [DOI] [PubMed] [Google Scholar]
  44. LIU L, LUO GZ, YANG W, ZHAO X, ZHENG Q, LV Z, LI W, WU HJ, WANG L, WANG XJ & ZhOu Q 2010. Activation of the imprinted Dlk1-Dio3 region correlates with pluripotency levels of mouse stem cells. J Biol Chem, 285, 19483–90. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. LIU Y, PELHAM-WEBB B, DI GIAMMARTINO DC, LI J, KIM D, KITA K, SAIZ N, GARG V, DOANE A, GIANNAKAKOU P, HADJANTONAKIS AK, ELEMENTO O & APOStOlOU E 2017. Widespread Mitotic Bookmarking by Histone Marks and Transcription Factors in Pluripotent Stem Cells. Cell Rep, 19, 1283–1293. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. LLERES D, MOINDROT B, PATHAK R, PIRAS V, MATELOT M, PIGNARD B, MARCHAND A, PONCELET M, PERRIN A, TELLIER V, FEIL R & NOORDERMEER D 2019. CTCF modulates allele-specific sub-TAD organization and imprinted gene activity at the mouse Dlk1-Dio3 and Igf2-H 19 domains. Genome Biol, 20, 272. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. LUO Z, LIN C, WOODFIN AR, BARTOM ET, GAO X, SMITH ER & SHILATIFARD A 2016. Regulation of the imprinted Dlk1-Dio3 locus by allele-specific enhancer activity. Genes Dev, 30, 92–101. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. MANDEGAR MA, HUEBSCH N, FROLOV EB, SHIN E, TRUONG A, OLVERA MP, CHAN AH, MIYAOKA Y, HOLMES K, SPENCER CI, JUDGE LM, GORDON DE, ESKILDSEN TV, VILLALTA JE, HORLBECK MA, GILBERT LA, KROGAN NJ, SHEIKH SP, WEISSMAN JS, QI LS, SO PL & CONKLIN BR 2016. CRISPR Interference Efficiently Induces Specific and Reversible Gene Silencing in Human iPSCs. Cell Stem Cell, 18, 541–53. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. MANODORO F, MARZEC J, CHAPLIN T, MIRAKI-MOUD F, MORAVCSIK E, JOVANOVIC JV, WANG J, IQBAL S, TAUSSIG D, GRIMWADE D, GRIBBEN JG, YOUNG BD & DEBERNARDI S 2014. Loss of imprinting at the 14q32 domain is associated with microRNA overexpression in acute promyelocytic leukemia. Blood, 123, 2066–74. [DOI] [PubMed] [Google Scholar]
  50. MENDIZABAL I & YI SV 2016. Whole-genome bisulfite sequencing maps from multiple human tissues reveal novel CpG islands associated with tissue-specific regulation. Hum Mol Genet, 25, 69–82. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. MO CF, WU FC, TAI KY, CHANG WC, CHANG KW, KUO HC, HO HN, CHEN HF & LIN SP 2015. Loss of non-coding RNA expression from the DLK1-DIO3 imprinted locus correlates with reduced neural differentiation potential in human embryonic stem cell lines. Stem Cell Res Ther, 6, 1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. MORITA S, NOGUCHI H, HORII T, NAKABAYASHI K, KIMURA M, OKAMURA K, SAKAI A, NAKASHIMA H, HATA K, NAKASHIMA K & HATADA I 2016. Targeted DNA demethylation in vivo using dCas9-peptide repeat and scFv-TET1 catalytic domain fusions. Nat Biotechnol, 34, 1060–1065. [DOI] [PubMed] [Google Scholar]
  53. MORLANDO M & FATICA A 2018. Alteration of Epigenetic Regulation by Long Noncoding RNAs in Cancer. Int J Mol Sci, 19. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. NIKOLAYEVA O & ROBINSON MD 2014. edgeR for differential RNA-seq and ChIP-seq analysis: an application to stem cell biology. Methods Mol Biol, 1150, 45–79. [DOI] [PubMed] [Google Scholar]
  55. NOVITCH BG, CHEN AI & JESSELL TM 2001. Coordinate regulation of motor neuron subtype identity and pan-neuronal properties by the bHLH repressor Olig2. Neuron, 31, 773–89. [DOI] [PubMed] [Google Scholar]
  56. NOWAK K, STEIN G, POWELL E, HE LM, NAIK S, MORRIS J, MARLOW S & DAVIS TL 2011. Establishment of paternal allele-specific DNA methylation at the imprinted mouse Gtl2 locus. Epigenetics, 6, 1012–20. [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. OKANO M, BELL DW, HABER DA & LI E 1999. DNA methyltransferases Dnmt3a and Dnmt3b are essential for de novo methylation and mammalian development. Cell, 99, 247–57. [DOI] [PubMed] [Google Scholar]
  58. PACHANO T, SANCHEZ-GAYA V, EALO T, MARINER-FAULI M, BLECKWEHL T, ASENJO HG, RESPUELA P, CRUZ-MOLINA S, MUNOZ-SAN MARTIN M, HARO E, VAN IWFJ, LANDEIRA D & RADA-IGLESIAS A 2021. Orphan CpG islands amplify poised enhancer regulatory activity and determine target gene responsiveness. Nat Genet, 53, 1036–1049. [DOI] [PMC free article] [PubMed] [Google Scholar]
  59. PAULSEN M, TAKADA S, YOUNGSON NA, BENCHAIB M, CHARLIER C, SEGERS K, GEORGES M & FERGUSON-SMITH AC 2001. Comparative sequence analysis of the imprinted Dlk1-Gtl2 locus in three mammalian species reveals highly conserved genomic elements and refines comparison with the Igf2-H19 region. Genome Res, 11, 2085–94. [DOI] [PMC free article] [PubMed] [Google Scholar]
  60. PELHAM-WEBB B, POLYZOS A, WOJENSKI L, KLOETGEN A, LI J, DI GIAMMARTINO DC, SAKELLAROPOULOS T, TSIRIGOS A, CORE L & APOSTOLOU E 2021. H3K27ac bookmarking promotes rapid post-mitotic activation of the pluripotent stem cell program without impacting 3D chromatin reorganization. Mol Cell, 81, 1732–1748 e8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  61. PLASSCHAERT RN & BARTOLOMEI MS 2014. Genomic imprinting in development, growth, behavior and stem cells. Development, 141, 1805–13. [DOI] [PMC free article] [PubMed] [Google Scholar]
  62. QUENNEVILLE S, TURELLI P, BOJKOWSKA K, RACLOT C, OFFNER S, KAPOPOULOU A & TRONO D 2012. The KRAB-ZFP/KAP1 system contributes to the early embryonic establishment of site-specific DNA methylation patterns maintained during development. Cell Rep, 2, 766–73. [DOI] [PMC free article] [PubMed] [Google Scholar]
  63. QUENNEVILLE S, VERDE G, CORSINOTTI A, KAPOPOULOU A, JAKOBSSON J, OFFNER S, BAGLIVO I, PEDONE PV, GRIMALDI G, RICCIO A & TRONO D 2011. In embryonic stem cells, ZFP57/KAP1 recognize a methylated hexanucleotide to affect chromatin and DNA methylation of imprinting control regions. Mol Cell, 44, 361–72. [DOI] [PMC free article] [PubMed] [Google Scholar]
  64. RABINOVITZ S, KAUFMAN Y, LUDWIG G, RAZIN A & SHEMER R 2012. Mechanisms of activation of the paternally expressed genes by the Prader-Willi imprinting center in the Prader-Willi/Angelman syndromes domains. Proc Natl Acad Sci U S A, 109, 7403–8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  65. RAVIRAM R, ROCHA PP, MULLER CL, MIRALDI ER, BADRI S, FU Y, SWANZEY D , PROUDHON C, SNETKOVA V, BONNEAU R & SKOK JA 2016. 4C-ker: A Method to Reproducibly Identify Genome-Wide Interactions Captured by 4C-Seq Experiments. PLoS Comput Biol, 12, e1004780. [DOI] [PMC free article] [PubMed] [Google Scholar]
  66. RISO V, CAMMISA M, KUKREJA H, ANVAR Z, VERDE G, SPARAGO A, ACURZIO B, LAD S, LONARDO E, SANKAR A, HELIN K, FEIL R, FICO A, ANGELINI C, GRIMALDI G & RICCIO A 2016. ZFP57 maintains the parent-of-origin-specific expression of the imprinted genes and differentially affects non-imprinted targets in mouse embryonic stem cells. Nucleic Acids Res, 44, 8165–78. [DOI] [PMC free article] [PubMed] [Google Scholar]
  67. ROSE NR & KLOSE RJ 2014. Understanding the relationship between DNA methylation and histone lysine methylation. Biochim Biophys Acta, 1839, 1362–72. [DOI] [PMC free article] [PubMed] [Google Scholar]
  68. SAITO T, HARA S, KATO T, TAMANO M, MURAMATSU A, ASAHARA H & TAKADA S 2018. A tandem repeat array in IG-DMR is essential for imprinting of paternal allele at the Dlk1-Dio3 domain during embryonic development. Hum Mol Genet, 27, 3283–3292. [DOI] [PubMed] [Google Scholar]
  69. SANCHEZ-CASTILLO M, RUAU D, WILKINSON AC, NG FS, HANNAH R, DIAMANTI E, LOMBARD P, WILSON NK & GOTTGENS B 2015. CODEX: a next-generation sequencing experiment database for the haematopoietic and embryonic stem cell communities. Nucleic Acids Res, 43, D1117–23. [DOI] [PMC free article] [PubMed] [Google Scholar]
  70. SANLI I, LALEVEE S, CAMMISA M, PERRIN A, RAGE F, LLERES D, RICCIO A, BERTRAND E & FEIL R 2018. Meg3 Non-coding RNA Expression Controls Imprinting by Preventing Transcriptional Upregulation in cis. Cell Rep, 23, 337–348. [DOI] [PubMed] [Google Scholar]
  71. SANMIGUEL JM & BARTOLOMEI MS 2018. DNA methylation dynamics of genomic imprinting in mouse development. Biol Reprod, 99, 252–262. [DOI] [PMC free article] [PubMed] [Google Scholar]
  72. SATO S, YOSHIDA W, SOEJIMA H, NAKABAYASHI K & HATA K 2011. Methylation dynamics of IG-DMR and Gtl2-DMR during murine embryonic and placental development. Genomics, 98, 120–7. [DOI] [PubMed] [Google Scholar]
  73. SEMRAU S, GOLDMANN JE, SOUMILLON M, MIKKELSEN TS, JAENISCH R & VAN OUDENAARDEN A 2017. Dynamics of lineage commitment revealed by single-cell transcriptomics of differentiating embryonic stem cells. Nat Commun, 8, 1096. [DOI] [PMC free article] [PubMed] [Google Scholar]
  74. SHI H, STROGANTSEV R, TAKAHASHI N, KAZACHENKA A, LORINCZ MC, HEMBERGER M & FERGUSON-SMITH AC 2019. ZFP57 regulation of transposable elements and gene expression within and beyond imprinted domains. Epigenetics Chromatin, 12, 49. [DOI] [PMC free article] [PubMed] [Google Scholar]
  75. STADTFELD M, APOSTOLOU E, AKUTSU H, FUKUDA A, FOLLETT P, NATESAN S, KONO T, SHIODA T & HOCHEDLINGER K 2010. Aberrant silencing of imprinted genes on chromosome 12qF1 in mouse induced pluripotent stem cells. Nature, 465, 175–81. [DOI] [PMC free article] [PubMed] [Google Scholar]
  76. STADTFELD M, APOSTOLOU E, FERRARI F, CHOI J, WALSH RM, CHEN T, OOI SS, KIM SY, BESTOR TH, SHIODA T, PARK PJ & HOCHEDLINGER K 2012. Ascorbic acid prevents loss of Dlk1-Dio3 imprinting and facilitates generation of all-iPS cell mice from terminally differentiated B cells. Nat Genet, 44, 398–405, S1-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
  77. STELZER Y, WU H, SONG Y, SHIVALILA CS, MARKOULAKI S & JAENISCH R 2016. Parent-of-Origin DNA Methylation Dynamics during Mouse Development. Cell Rep, 16, 3167–3180. [DOI] [PMC free article] [PubMed] [Google Scholar]
  78. STRAUSSMAN R, NEJMAN D, ROBERTS D, STEINFELD I, BLUM B, BENVENISTY N, SIMON I, YAKHINI Z & CEDAR H 2009. Developmental programming of CpG island methylation profiles in the human genome. Nat Struct Mol Biol, 16, 564–71. [DOI] [PubMed] [Google Scholar]
  79. STROGANTSEV R, KRUEGER F, YAMAZAWA K, SHI H, GOULD P, GOLDMAN-ROBERTS M, MCEWEN K, SUN B, PEDERSEN R & FERGUSON-SMITH AC 2015. Allele-specific binding of ZFP57 in the epigenetic regulation of imprinted and non-imprinted monoallelic expression. Genome Biol, 16, 112. [DOI] [PMC free article] [PubMed] [Google Scholar]
  80. SWANZEY E, MCNAMARA TF, APOSTOLOU E, TAHILIANI M & STADTFELD M 2020. A Susceptibility Locus on Chromosome 13 Profoundly Impacts the Stability of Genomic Imprinting in Mouse Pluripotent Stem Cells. Cell Rep, 30, 3597–3604 e3. [DOI] [PubMed] [Google Scholar]
  81. SWANZEY E & STADTFELD M 2016. A reporter model to visualize imprinting stability at the Dlk1 locus during mouse development and in pluripotent cells. Development, 143, 4161–4166. [DOI] [PMC free article] [PubMed] [Google Scholar]
  82. TAKADA S, PAULSEN M, TEVENDALE M, TSAI CE, KELSEY G, CATTANACH BM & FERGUSON-SMITH AC 2002. Epigenetic analysis of the Dlk1-Gtl2 imprinted domain on mouse chromosome 12: implications for imprinting control from comparison with Igf2-H19. Hum Mol Genet, 11, 77–86. [DOI] [PubMed] [Google Scholar]
  83. TUCCI V, ISLES AR, KELSEY G, FERGUSON-SMITH AC & ERICE IMPRINTING G 2019. Genomic Imprinting and Physiological Processes in Mammals. Cell, 176, 952–965. [DOI] [PubMed] [Google Scholar]
  84. VANZAN L, SOLDATI H, YTHIER V, ANAND S, BRAUN SMG, FRANCIS N & MURR R 2021. High throughput screening identifies SOX2 as a super pioneer factor that inhibits DNA methylation maintenance at its binding sites. Nat Commun, 12, 3337. [DOI] [PMC free article] [PubMed] [Google Scholar]
  85. VERMA N, PAN H, DORE LC, SHUKLA A, LI QV, PELHAM-WEBB B, TEIJEIRO V, GONZALEZ F, KRIVTSOV A, CHANG CJ, PAPAPETROU EP, HE C, ELEMENTO O & HUANGFU D 2018. TET proteins safeguard bivalent promoters from de novo methylation in human embryonic stem cells. Nat Genet, 50, 83–95. [DOI] [PMC free article] [PubMed] [Google Scholar]
  86. VOJTA A, DOBRINIC P, TADIC V, BOCKOR L, KORAC P, JULG B, KLASIC M & ZOLDOS V 2016. Repurposing the CRISPR-Cas9 system for targeted DNA methylation. Nucleic Acids Res, 44, 5615–28. [DOI] [PMC free article] [PubMed] [Google Scholar]
  87. WANG Y, SHEN Y, DAI Q, YANG Q, ZHANG Y, WANG X, XIE W, LUO Z & LIN C 2017. A permissive chromatin state regulated by ZFP281-AFF3 in controlling the imprinted Meg3 polycistron. Nucleic Acids Res, 45, 1177–1185. [DOI] [PMC free article] [PubMed] [Google Scholar]
  88. WICHTERLE H, LIEBERAM I, PORTER JA & JESSELL TM 2002. Directed differentiation of embryonic stem cells into motor neurons. Cell, 110, 385–97. [DOI] [PubMed] [Google Scholar]
  89. WILLIAMS K, CHRISTENSEN J, PEDERSEN MT, JOHANSEN JV, CLOOS PA, RAPPSILBER J & HELIN K 2011. TET1 and hydroxymethylcytosine in transcription and DNA methylation fidelity. Nature, 473, 343–8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  90. XU X, TAO Y, GAO X, ZHANG L, LI X, ZOU W, RUAN K, WANG F, XU GL & HU R 2016. A CRISPR-based approach for targeted DNA demethylation. Cell Discov, 2, 16009. [DOI] [PMC free article] [PubMed] [Google Scholar]
  91. YING Y, YANG X, ZHAO K, MAO J, KUANG Y, WANG Z, SUN R & FEI J 2015. The Kruppel-associated box repressor domain induces reversible and irreversible regulation of endogenous mouse genes by mediating different chromatin states. Nucleic Acids Res, 43, 1549–61. [DOI] [PMC free article] [PubMed] [Google Scholar]
  92. YOON B, HERMAN H, HU B, PARK YJ, LINDROTH A, BELL A, WEST AG, CHANG Y, STABLEWSKI A, PIEL JC, LOUKINOV DI, LOBANENKOV VV & SOLOWAY PD 2005. Rasgrf1 imprinting is regulated by a CTCF-dependent methylation-sensitive enhancer blocker. Mol Cell Biol, 25, 11184–90. [DOI] [PMC free article] [PubMed] [Google Scholar]
  93. ZHANG YW, WANG Z, XIE W, CAI Y, XIA L, EASWARAN H, LUO J, YEN RC, LI Y & BAYLIN SB 2017. Acetylation Enhances TET2 Function in Protecting against Abnormal DNA Methylation during Oxidative Stress. Mol Cell, 65, 323–335. [DOI] [PMC free article] [PubMed] [Google Scholar]
  94. ZHAO J, OHSUMI TK, KUNG JT, OGAWA Y, GRAU DJ, SARMA K, SONG JJ, KINGSTON RE, BOROWSKY M & LEE JT 2010. Genome-wide identification of polycomb-associated RNAs by RIP-seq. Mol Cell, 40, 939–53. [DOI] [PMC free article] [PubMed] [Google Scholar]
  95. ZHAO Y, SUN H & WANG H 2016. Long noncoding RNAs in DNA methylation: new players stepping into the old game. Cell Biosci, 6, 45. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplemental Information
NIHMS1754280-supplement-Supplemental_Information.pdf (1.4MB, pdf)
Table S3

Table S3: Percentage of DNA methylation for clones analyzed in this study (excel) (related to STAR methods)

NIHMS1754280-supplement-Table_S3.xlsx (20.7KB, xlsx)
Table S4

Table S4. Raw values and statistics for all graphs included in this study (excel) (related to STAR methods)

NIHMS1754280-supplement-Table_S4.xlsx (60.3KB, xlsx)

Data Availability Statement

  • 4C-seq data generated in this study have been deposited at GEO under the accession number GSE148315 and are available as of the date of publication.

  • This study did not generate a new code, but the scripts used in the study are available from the Lead Contact upon request.

  • Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.

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