Insulator dysfunction and oncogene activation in IDH mutant gliomas

Mutant IDH protein generates the oncometabolite 2-hydroxyglutarate.

“Mutant IDH protein produces a novel onco-metabolite, 2-hydroxyglutarate (2-HG), that interferes with iron-dependent hydroxylases, including the TET family of 5′-methylcytosine hydroxylases3–7.”

• cites [3] Dang, L. et al. Cancer-associated IDH1 mutations produce 2-hydroxyglutarate. Nature 462, 739–744 (2009) sourceSupported
• cites [4] Figueroa, M. E. et al. Leukemic IDH1 and IDH2 mutations result in a hypermethylation phenotype, disrupt TET2 function, and impair hematopoietic differentiation. Cancer Cell 18, 553–567 (2010) sourceSupported
• cites [5] Xu, W. et al. Oncometabolite 2-hydroxyglutarate is a competitive inhibitor of α-ketoglutarate-dependent dioxygenases. Cancer Cell 19, 17–30 (2011) sourceSupported
• cites [6] Lu, C. et al. IDH mutation impairs histone demethylation and results in a block to cell differentiation. Nature 483, 474–478 (2012) sourceSupported
• cites [7] Cairns, R. A. & Mak, T. W. Oncogenic isocitrate dehydrogenase mutations: mechanisms, models, and clinical opportunities. Cancer Dis. 3, 730–741 (2013) sourceNot checked — no full text of the source

IDH mutant gliomas show a CpG island methylator phenotype.

“IDH mutant gliomas thus manifest a CpG island methylator phenotype (G-CIMP)10,11, though the functional significance of this altered epigenetic state remains unclear.”

• cites [10] Noushmehr, H. et al. Identification of a CpG island methylator phenotype that defines a distinct subgroup of glioma. Cancer Cell 17, 510–522 (2010) sourcePartly supported

  The Results sections report a strong association between IDH1 mutation and G-CIMP in gliomas. But in an independent set, only 35 of 48 IDH1-mutant tumors (72.9%) were G-CIMP-positive, and the paper observed no IDH2 mutations in its TCGA set. The claim is therefore broader than the paper’s finding: not all IDH1-mutant gliomas showed the phenotype, and it does not establish the association for IDH mutations generally. (LLM, conf=0.84)

  Experimental — accuracy not yet measured
• cites [11] Turcan, S. et al. IDH1 mutation is sufficient to establish the glioma hypermethylator phenotype. Nature 483, 479–483 (2012) sourceSupported

Genes in the same topological domain correlate across normal brain samples.

“Consistent with prior studies16, we found that genes in the same domain correlate across samples, but that genes separated by a boundary show lower correlation (Fig. 1e).”

• cites [16] Nora, E. P. et al. Spatial partitioning of the regulatory landscape of the X-inactivation centre. Nature 485, 381–385 (2012) sourcePartly supported

  The transcriptome-analysis passage (labeled Abstract, Fig. 4b) reports that genes within the same TAD had more correlated expression profiles than genes in different domains. But it measured 17 time points during female mouse embryonic stem-cell differentiation, not correlations across normal brain samples. (LLM, conf=0.88)

  Experimental — accuracy not yet measured

2-hydroxyglutarate interferes with iron-dependent hydroxylases, including the TET family of 5-methylcytosine hydroxylases.

“Mutant IDH protein produces a novel onco-metabolite, 2-hydroxyglutarate (2-HG), that interferes with iron-dependent hydroxylases, including the TET family of 5′-methylcytosine hydroxylases3–7.”

• cites [3] Dang, L. et al. Cancer-associated IDH1 mutations produce 2-hydroxyglutarate. Nature 462, 739–744 (2009) sourcePartly supported

  The section “Mutant IDH1 directly converts αKG to 2HG” confirms that mutant IDH1 produces 2HG. The Discussion suggests that 2HG may inhibit αKG-dependent prolyl hydroxylases, but presents this as a possibility, not an established effect. Our copy does not identify TET 5-methylcytosine hydroxylases as targets. (LLM, conf=0.88)

  Experimental — accuracy not yet measured
• cites [4] Figueroa, M. E. et al. Leukemic IDH1 and IDH2 mutations result in a hypermethylation phenotype, disrupt TET2 function, and impair hematopoietic differentiation. Cancer Cell 18, 553–567 (2010) sourcePartly supported

  The Results section (page unavailable) identifies TET enzymes as iron-dependent 5-methylcytosine hydroxylases and shows that mutant IDH inhibits TET2-mediated hydroxylation. The paper proposes 2-HG as the mechanism, but does not establish interference with the TET family as a whole or with other iron-dependent hydroxylases; the Discussion treats effects on other enzyme families as a possibility for future study. (LLM, conf=0.83)

  Experimental — accuracy not yet measured
• cites [5] Xu, W. et al. Oncometabolite 2-hydroxyglutarate is a competitive inhibitor of α-ketoglutarate-dependent dioxygenases. Cancer Cell 19, 17–30 (2011) sourceSupported
• cites [6] Lu, C. et al. IDH mutation impairs histone demethylation and results in a block to cell differentiation. Nature 483, 474–478 (2012) sourcePartly supported

  The paper’s Abstract (page unavailable) states that mutant IDH produces 2HG, reports mutant IDH-induced inhibition of the α-ketoglutarate-dependent enzyme TET2, and shows that 2HG inhibits a histone demethylase. It does not explicitly establish inhibition of the TET family as a whole or describe these enzymes as iron-dependent hydroxylases. (LLM, conf=0.82)

  Experimental — accuracy not yet measured
• cites [7] Cairns, R. A. & Mak, T. W. Oncogenic isocitrate dehydrogenase mutations: mechanisms, models, and clinical opportunities. Cancer Dis. 3, 730–741 (2013) sourceNot checked — no full text of the source

This boundary contains a strong CTCF binding site over a canonical CTCF motif with a CpG dinucleotide in a position previously linked to methylation sensitivity.

“Our ChIP-seq data confirm that this boundary contains a strong CTCF binding site over a canonical CTCF motif with a CpG dinucleotide in a position previously linked to methylation-sensitivity25 (Fig. 3b).”

• cites [25] Wang, H. et al. Widespread plasticity in CTCF occupancy linked to DNA methylation. Genome Res. 22, 1680–1688 (2012) sourceSupported

IDH mutant gliomas share the proneural transcriptional program characteristic of PDGFRA-amplified tumors.

“Nonetheless, IDH mutant gliomas strongly express PDGFRA (Fig. 2b), and share the proneural transcriptional program characteristic of PDGFRA-amplified tumors23,24.”

• cites [23] Brennan, C. W. et al. The somatic genomic landscape of glioblastoma. Cell 155, 462–477 (2013) sourcePartly supported

  In Results, under “Molecular subclasses defined by global mRNA expression and DNA Methylation” (page unavailable), the paper describes G-CIMP and non-G-CIMP proneural glioblastomas as transcriptomically similar and associates PDGFRA amplification with the non-G-CIMP proneural subgroup. It reports that all IDH1-mutant cases had the G-CIMP pattern. These findings support the shared proneural program in the glioblastomas studied, but do not establish it for IDH-mutant gliomas more broadly. (LLM, conf=0.82)

  Experimental — accuracy not yet measured
• cites [24] Verhaak, R. G. et al. Integrated genomic analysis identifies clinically relevant subtypes of glioblastoma characterized by abnormalities in PDGFRA, IDH1, EGFR, and NF1 . Cancer Cell 17, 98–110 (2010) sourcePartly supported

  In Results > Functional Annotation of Subtypes (page unavailable), the paper reports that 11 of 12 IDH1 mutations occurred in the Proneural class, which was also enriched for PDGFRA amplification and had a proneural expression signature. This supports the shared-program claim for the glioblastomas studied, but not its extension to IDH-mutant gliomas generally. (LLM, conf=0.85)

  Experimental — accuracy not yet measured

Genes separated by a domain boundary show lower correlation across normal brain samples.

“Consistent with prior studies16, we found that genes in the same domain correlate across samples, but that genes separated by a boundary show lower correlation (Fig. 1e).”

• cites [16] Nora, E. P. et al. Spatial partitioning of the regulatory landscape of the X-inactivation centre. Nature 485, 381–385 (2012) sourcePartly supported

  The passage on gene expression dynamics during early differentiation reports that genes within the same TAD had more correlated expression profiles than genes in different domains. It measured female mouse embryonic stem cells over a differentiation time course, not normal brain samples, so it does not support that setting. (LLM, conf=0.88)

  Experimental — accuracy not yet measured

IDH mutant gliomas have increased methylation at CTCF binding sites.

“Here we show that IDH mutant gliomas exhibit hyper-methylation at CTCF binding sites, compromising binding of this methylation-sensitive insulator protein.”

Treating IDH mutant gliomaspheres with a demethylating agent partially restores insulator function.

“Treatment of IDH mutant gliomaspheres with demethylating agent partially restores insulator function and down-regulates PDGFRA.”

The 625 loci with reduced CTCF binding show higher DNA methylation in IDH mutant gliomas than in IDH wildtype gliomas.

“We found that these loci have higher GC content, and exhibit significantly higher levels of DNA methylation in IDH mutant gliomas, relative to IDH wildtype (Fig. 1c–d).”

CRISPR disruption of the CTCF motif in IDH wildtype gliomaspheres increases PDGFRA expression.

“Conversely, CRISPR-mediated disruption of the CTCF motif in IDH wildtype gliomaspheres up-regulates PDGFRA and increases proliferation.”

CRISPR disruption of the CTCF motif in IDH wildtype gliomaspheres increases proliferation.

“Conversely, CRISPR-mediated disruption of the CTCF motif in IDH wildtype gliomaspheres up-regulates PDGFRA and increases proliferation.”

Loss of CTCF at a domain boundary allows a constitutive enhancer to interact abnormally with PDGFRA.

“We specifically demonstrate that loss of CTCF at a domain boundary permits a constitutive enhancer to aberrantly interact with the receptor tyrosine kinase gene PDGFRA, a prominent glioma oncogene.”

Treating IDH mutant gliomaspheres with a demethylating agent lowers PDGFRA expression.

“Treatment of IDH mutant gliomaspheres with demethylating agent partially restores insulator function and down-regulates PDGFRA.”

In IDH mutant gliomas, more CTCF binding sites are commonly lost than gained, with 625 lost versus 300 gained and p<10^-12.

“Significantly more sites were commonly lost than gained (625 vs 300, p<10−12).”

CTCF binding is reduced at CTCF binding sites in IDH mutant gliomas.

“Here we show that IDH mutant gliomas exhibit hyper-methylation at CTCF binding sites, compromising binding of this methylation-sensitive insulator protein.”

The 625 loci with reduced CTCF binding in mutant tumors have higher GC content.

“We found that these loci have higher GC content, and exhibit significantly higher levels of DNA methylation in IDH mutant gliomas, relative to IDH wildtype (Fig. 1c–d).”

PDGFRA expression in insulator CRISPR gliomaspheres increased further after extended culture, to 2-fold over control.

“Notably, PDGFRA expression in insulator CRISPR gliomaspheres increased further after extended culture (to 2-fold over control), potentially due to selection of effectively edited clones.”

In TCGA lower-grade gliomas, an intervening boundary reduces correlation between neighboring genes.

“Here again we found that the presence of an intervening boundary reduces correlation between neighboring genes.”

IDH mutant gliomas strongly express PDGFRA.

“Nonetheless, IDH mutant gliomas strongly express PDGFRA (Fig. 2b), and share the proneural transcriptional program characteristic of PDGFRA-amplified tumors23,24.”

The putatively disrupted boundaries have higher DNA methylation in IDH mutant tumors than in wildtype.

“The putatively disrupted boundaries exhibit higher DNA methylation and lower CTCF binding in IDH mutant tumors, relative to wildtype (Extended Data Fig. 1).”

In that same repeat analysis, 54% of gene pairs that lost correlation were within the same domain, versus 29% expected (p<3×10−8).

“This yielded similar results: 84% pairs gaining correlation cross boundary vs. 71% expected (p<8*10−3), 54% pairs losing correlation are within the same domain vs. 29% expected (p<3*10−8).”

In wildtype gliomas, 3C showed a strong interaction between the PDGFRA promoter and its intragenic enhancer, which are about 50 kb apart.

“In wildtype gliomas, 3C revealed a strong interaction between the PDGFRA promoter and its intragenic enhancer, which are ~50 kb apart (Fig. 3j,k).”

In the BT142 mutant line, the CpG dinucleotide in the CTCF motif has higher methylation than wildtype models, about 13% versus 2%.

“In this mutant line, the CpG dinucleotide in the CTCF motif exhibits higher methylation than wildtype models (~13% vs ~2% per bisulfite sequencing), and CTCF binding is ~3-fold lower.”

5-aza treatment reduced methylation of the CTCF motif by about 2.5-fold.

“5-aza treatment reduced methylation of the CTCF motif by ~2.5-fold, increased CTCF occupancy by ~1.7-fold and down-regulated PDGFRA expression by ~5-fold (Fig. 4b–d).”

Gene pairs that correlate less strongly in IDH mutants are more likely to be in the same domain, at 52% versus 31% expected and p<10^-5.

“Conversely, gene pairs that correlate less strongly in IDH mutants are more likely to reside in the same domain (52% vs 31% expected at random; p<10−5).”

Combined expression of PDGFRA and FIP1L1 is associated with poorer outcome in IDH mutant lower-grade gliomas.

“Moreover, combined expression of PDGFRA and FIP1L1 is associated with poorer outcome in IDH mutant lower-grade gliomas (Extended Data Fig. 2b).”

In that restricted analysis, 73% of pairs losing correlation were within the same domain, versus 31% expected (p<8×10−4).

“Repeating the analysis with only the 14,055 genes that have expressed over 1 TPM in at least half the samples also yielded similar results (Extended Data Fig. 7): 92% pairs gaining correlation cross boundary vs. 69% expected (p<2*10−3), 73% pairs losing correlation are within the same domain vs. 31% expected (p<8*10−4).”

CTCF occupancy at this site is reduced by 30% to 50% in IDH mutant tumors and gliomasphere models compared with wildtype.

“Quantitative ChIP-PCR reveals that CTCF occupancy at this site is reduced between 30% and 50% in IDH mutant tumors and gliomasphere models, relative to wildtype (Fig. 3c,d).”

Repeating the analysis with boundaries defined from GM12878, K562 and NHEK again identified PDGFRA as an over-expressed gene next to a disrupted boundary.

“This yielded very similar results, and again highlighted PDGFRA as an over-expressed gene adjacent to a disrupted boundary.”

A large enhancer about 50 kb upstream of FIP1L1 has strong acetylation in wildtype and mutant tumors.

“We identified a large enhancer ~50 kb upstream of FIP1L1 with strong acetylation in wildtype and mutant tumors (Fig. 3a; Extended Data Fig. 4).”

In the repeat analysis using K562 and NHEK boundaries, 84% of gene pairs that gained correlation crossed a boundary, versus 71% expected (p<8×10−3).

“This yielded similar results: 84% pairs gaining correlation cross boundary vs. 71% expected (p<8*10−3), 54% pairs losing correlation are within the same domain vs. 29% expected (p<3*10−8).”

Fisher exact testing showed that the shared set of lost peaks in IDH-mutant tumors was highly significant (p<10−100).

“Fisher exact test confirmed that the overlap between peaks lost in the IDH mutant tumors is highly significant (p<10−100).”

In IDH mutant tumors, the FIP1L1 enhancer-PDGFRA promoter interaction is stronger than the FIP1L1 enhancer-FIP1L1 promoter interaction.

“Remarkably, we found that the FIP1L1 enhancer-PDGFRA promoter interaction is stronger than the FIP1L1 enhancer-FIP1L1 promoter interaction in IDH mutant tumors.”

Insulator CRISPR gliomaspheres have about a 2-fold growth advantage over control GFP CRISPR gliomaspheres.

“In support, the insulator CRISPR gliomaspheres have a ~2-fold growth advantage over the control GFP CRISPR gliomaspheres (Fig. 4k).”

In the BT142 mutant line, CTCF binding is about 3-fold lower than in wildtype models.

“In this mutant line, the CpG dinucleotide in the CTCF motif exhibits higher methylation than wildtype models (~13% vs ~2% per bisulfite sequencing), and CTCF binding is ~3-fold lower.”

CHD4 was among the top-ranking genes in the analysis, with p<10−32.

“In addition to PDGFRA, top ranking genes include CHD4 (p<10−32), a driver of glioblastoma tumor initiation38, L1CAM (p<10−8), a regulator of the glioma stem cells and tumor growth39, and other candidate regulators (Supplemental Table 1).”

IDH mutations cause hyper-methylation of CTCF binding sites genome-wide.

“We specifically find that, in addition to familiar effects on CpG islands, IDH mutations cause hyper-methylation of CTCF binding sites genome-wide.”

This growth advantage depends on PDGFRa signaling, as it is abrogated by dasatinib or crenolanib.

“This growth advantage is dependent on PDGFRa signaling, as it is abrogated by treatment with PDGFR inhibitors, dasatinib or crenolanib (Fig. 4k, Extended Data Fig. 6).”

This interaction is about 5-fold stronger than the interaction between the PDGFRA promoter and its intragenic enhancer.

“For comparison, this interaction is ~5-fold stronger than that between PDGFRA promoter and its intragenic enhancer.”

Genes in top scoring domains include PDGFRA, with p<10^-21.

“Genes in top scoring domains include PDGFRA (p<10−21), an established glioma oncogene22, and other candidate regulators of gliomagenesis (Table S1).”

Figure 2a shows that genes in disrupted domains are upregulated in IDH mutant tumors compared with the median expression in wild-type.

“Figure 2a depicts significance of disrupted domains and the fold-change of genes in them that are upregulated in IDH mutant tumors (compared to median expression in wild-type).”

The putatively disrupted boundaries have lower CTCF binding in IDH mutant tumors than in wildtype.

“The putatively disrupted boundaries exhibit higher DNA methylation and lower CTCF binding in IDH mutant tumors, relative to wildtype (Extended Data Fig. 1).”

The PDGFRA promoter does not interact with the FIP1L1 enhancer in wildtype tumors.

“In contrast, the PDGFRA promoter does not interact with the FIP1L1 enhancer in wildtype tumors, consistent with retention of the intervening boundary (Fig 3h,i).”

Disruption of a specific boundary bordering PDGFRA allows a potent enhancer to contact and activate this canonical glioma oncogene abnormally.

“Disruption of a specific boundary bordering PDGFRA allows a potent enhancer to aberrantly contact and activate this canonical glioma oncogene.”

In IDH mutant gliomas, PDGFRA and FIP1L1 expression are strongly correlated despite an intervening boundary.

“Closer examination of expression patterns in IDH mutant gliomas reveals a striking correlation between PDGFRA and FIP1L1, despite an intervening boundary (Fig. 2c).”

The CpG in this motif becomes highly methylated in IDH mutants.

“Moreover, the CpG in this motif becomes highly methylated in IDH mutants (Fig. 3e,f).”

In all six cell types examined, PDGFRA and FIP1L1 are in distinct domains separated by one CTCF-anchored constitutive boundary.

“In all six cell types examined, PDGFRA and FIP1L1 reside in distinct domains, separated by one CTCF-anchored constitutive boundary (Fig. 3a, Extended Data Fig. 3).”

L1CAM was among the top-ranking genes, with p<10−8.

“In addition to PDGFRA, top ranking genes include CHD4 (p<10−32), a driver of glioblastoma tumor initiation38, L1CAM (p<10−8), a regulator of the glioma stem cells and tumor growth39, and other candidate regulators (Supplemental Table 1).”

5-aza treatment increased CTCF occupancy by about 1.7-fold.

“5-aza treatment reduced methylation of the CTCF motif by ~2.5-fold, increased CTCF occupancy by ~1.7-fold and down-regulated PDGFRA expression by ~5-fold (Fig. 4b–d).”

When the analysis was restricted to 14,055 genes expressed above 1 TPM in at least half the samples, 92% of pairs gaining correlation crossed a boundary, versus 69% expected (p<2×10−3).

“Repeating the analysis with only the 14,055 genes that have expressed over 1 TPM in at least half the samples also yielded similar results (Extended Data Fig. 7): 92% pairs gaining correlation cross boundary vs. 69% expected (p<2*10−3), 73% pairs losing correlation are within the same domain vs. 31% expected (p<8*10−4).”

Flow cytometry showed about a 1.8-fold increase in the fraction of cells with PDGFRa surface expression.

“Similarly, flow cytometry revealed a ~1.8-fold increase in the fraction of cells with PDGFRa surface expression (Fig. 4j).”

Gene pairs separated by less than 180 kb that correlate more strongly in IDH mutants than wildtype gliomas are strongly enriched for crossing domain boundaries, at 90% versus 69% expected and p<10^-4.

“Remarkably, the resulting set is strongly enriched for gene pairs that cross domain boundaries (90% vs 69% expected at random; p<10−4).”

In IDH mutant tumors, 3C showed a strong interaction between the PDGFRA promoter and the FIP1L1 enhancer despite a separation of about 900 kb.

“Here, 3C revealed a strong interaction between the PDGFRA promoter and the FIP1L1 enhancer, despite a separation of ~900 kb (Fig. 3i).”

5-aza treatment down-regulated PDGFRA expression by about 5-fold.

“5-aza treatment reduced methylation of the CTCF motif by ~2.5-fold, increased CTCF occupancy by ~1.7-fold and down-regulated PDGFRA expression by ~5-fold (Fig. 4b–d).”

RT-PCR showed about a 1.6-fold increase in PDGFRA mRNA in insulator CRISPR cells relative to control.

“RT-PCR revealed a ~1.6-fold increase in PDGFRA mRNA in the insulator CRISPR cells, relative to control (Fig. 4i).”

CTCF binding is sensitive to DNA methylation.

“Since CTCF binding is methylation-sensitive18,19, its localization might be altered by DNA hyper-methylation in IDH mutant gliomas.”

• cites [18] Bell, A. C. & Felsenfeld, G. Methylation of a CTCF-dependent boundary controls imprinted expression of the Igf2 gene. Nature 405, 482–485 (2000) sourceNot checked — no full text of the source
• cites [19] Hark, A. T. et al. CTCF mediates methylation-sensitive enhancer-blocking activity at the H19/Igf2 locus. Nature 405, 486–489 (2000) sourceNot checked — no full text of the source

IDH mutations promote glioma formation by disrupting chromosomal topology and permitting abnormal regulatory interactions that induce oncogene expression.

“Our study suggests that IDH mutations promote gliomagenesis by disrupting chromosomal topology and allowing aberrant regulatory interactions that induce oncogene expression.”

Widespread disruption of CTCF binding and boundary element function could provide many opportunities for oncogene deregulation and subsequent selection of proliferative progeny that inherit the altered epigenetic state.

“The widespread disruption of CTCF binding and boundary element function could provide many opportunities for oncogene deregulation, and subsequent selection of proliferative progeny that inherit the altered epigenetic state.”

Altered expression patterns in IDH mutant gliomas may reflect reduced CTCF binding and consequent disruption of domain boundaries and topologies.

“Thus, altered expression patterns in IDH mutant gliomas may reflect reduced CTCF binding and consequent disruption of domain boundaries and topologies.”

CRISPR-mediated disruption of the amplified DNA was confirmed by Surveyor Assay.

“CRISPR-mediated disruption of this amplified DNA was confirmed via Surveyor Assay (Transgenomic), with amplified uninfected GSC6 genomic DNA being added to each annealing reaction as the unmodified control.”

The methylator phenotype disrupts CTCF binding and domain boundaries, thereby affecting gene expression in IDH mutant gliomas.

“These data suggest that the methylator phenotype disrupts CTCF binding and domain boundaries, thereby affecting gene expression in IDH mutant gliomas.”

Disruption of a boundary element by IDH mutation and hyper-methylation allows a potent constitutive enhancer to interact abnormally with and up-regulate PDGFRA.

“This suggests that disruption of a boundary element by IDH mutation and hyper-methylation allows a potent constitutive enhancer to aberrantly interact with, and up-regulate PDGFRA.”

Reduced CTCF binding may compromise the boundary flanking PDGFRA in IDH mutant, hyper-methylated tumors.

“This suggests that reduced CTCF binding may compromise the boundary flanking PDGFRA in IDH mutant, hyper-methylated tumors.”

An abnormal interaction with this constitutive locus may drive PDGFRA expression in IDH mutant tumors.

“This suggests that an aberrant interaction with this constitutive locus may drive PDGFRA expression in IDH mutant tumors.”

In insulator CRISPR gliomaspheres, about 25% of alleles contain a deletion within the CTCF motif expected to disrupt binding, compared with less than 0.1% in the GFP control.

“Direct sequencing of the target locus revealed that ~25% of alleles in the insulator CRISPR gliomaspheres contain a deletion within the CTCF motif expected to disrupt binding, compared to <0.1% in the GFP control (Fig. 4g,h).”

• AssumptionThe authors state this as an assumption: The CRISPR-induced deletion within the CTCF motif disrupts CTCF binding.