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Cited by 302Genomics and Chromatin DynamicsChromatin Remodeling and CancerPluripotent Stem Cells Research

YY1 and CTCF orchestrate a 3D chromatin looping switch during early neural lineage commitment

Open access
Beagan, Jonathan A., Duong, Michael T., Titus, Katelyn R., Zhou, Linda, Cao, Zhendong, Ma, Jingjing, Lachanski, Caroline V., Gillis, Daniel R. +1 more·2017·Genome Research
DOI 10.1101/gr.215160.116PMC PMC5495066
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Abstract

CTCF is an architectural protein with a critical role in connecting higher-order chromatin folding in pluripotent stem cells. Recent reports have suggested that CTCF binding is more dynamic during development than previously appreciated. Here, we set out to understand the extent to which shifts in genome-wide CTCF occupancy contribute to the 3D reconfiguration of fine-scale chromatin folding during early neural lineage commitment. Unexpectedly, we observe a sharp decrease in CTCF occupancy during the transition from naïve/primed pluripotency to multipotent primary neural progenitor cells (NPCs). Many pluripotency gene-enhancer interactions are anchored by CTCF, and its occupancy is lost in parallel with loop decommissioning during differentiation. Conversely, CTCF binding sites in NPCs are largely preexisting in pluripotent stem cells. Only a small number of CTCF sites arise de novo in NPCs. We identify another zinc finger protein, Yin Yang 1 (YY1), at the base of looping interactions between NPC-specific genes and enhancers. Putative NPC-specific enhancers exhibit strong YY1 signal when engaged in 3D contacts and negligible YY1 signal when not in loops. Moreover, siRNA knockdown of Yy1 specifically disrupts interactions between key NPC enhancers and their target genes. YY1-mediated interactions between NPC regulatory elements are often nested within constitutive loops anchored by CTCF. Together, our results support a model in which YY1 acts as an architectural protein to connect developmentally regulated looping interactions; the location of YY1-mediated interactions may be demarcated in development by a preexisting topological framework created by constitutive CTCF-mediated interactions.

AI snapshot

Written from the paper’s full text.

TL;DR

During early neural lineage commitment, CTCF occupancy is globally pruned from pluripotent stem cells to multipotent neural progenitor cells (NPCs), while NPC-specific enhancer–gene loops are instead anchored by YY1. The paper proposes that YY1-mediated NPC looping interactions arise within a preexisting constitutive CTCF-anchored topological framework.

Key findings

  • CTCF binding decreases stepwise across ES 2i, ES serum, and NPCs: 56,138, 50,185, and 28,860 sites, respectively, with 60,688 unique sites total. Of these, 26,435 were constitutive, 8,832 were “2i only,” 20,068 were “2i+Serum,” and only 1,119 were “NPC-only”; CTCF gene expression and protein levels also decreased in NPCs.
  • 5C analysis identified 141 constitutive loops, 46 “2i+Serum” loops lost in NPCs, and 75 NPC-only loops. “2i+Serum” loops were enriched for “2i+Serum” CTCF sites, and the pluripotency-specific Sox2–ES enhancer loop broke concurrently with CTCF loss.
  • NPC-only loops were not enriched for NPC-only CTCF binding: almost 40% of NPC-only interactions lacked CTCF anchoring, versus <5% of constitutive and <10% of “2i+serum” interactions. Instead, YY1 was strongly enriched at NPC-only loops and at NPC-specific enhancers engaged in 3D contacts, with negligible YY1 signal at NPC-specific enhancers not in loops.
  • YY1 sites were less constitutive than CTCF sites (36%, 39%, and 25% constitutive across ES serum, NPCs, and pro-B cells; n = 3,474). NPC-specific enhancers were enriched in NPC-only loops when bound by YY1 without CTCF, but not when bound by CTCF without YY1. Yy1 siRNA knockdown produced >50% reduction in Yy1 expression/protein, disrupted the Sox2–enhancer loop, decreased Sox2 expression, and also reduced Klf4 and Zfp462 looping and Nes/Olig1/Olig2 expression.

Methods

  • ChIP-seq was performed for CTCF in mouse ES 2i, ES serum, and NPCs, with ENCODE mouse brain CTCF data reanalyzed; RNA-seq and Western blot assessed CTCF expression/protein. YY1 and H3K27ac ChIP-seq datasets were also analyzed.
  • 5C with high-throughput sequencing generated fine-scale chromatin architecture maps at ~4- to 12-kb matrix resolution across >7 Mb of the mouse genome, using n = 2 biological replicates and reanalyzed published 5C libraries. A new computational pipeline used donut and lower-left background filters, logistic-distribution P-values, interaction scores, and seven looping classes; top five dynamic classes had empirical FDR <15%.
  • Yy1 knockdown in NPCs used Dharmacon siRNA pools at 20 nM for 78 h, followed by Western blot, qPCR, and 3C/5C analysis.

Limitations

  • Stated: the authors cannot rule out that heterogeneity in ENCODE brain tissues contributed to the aggregate CTCF decrease, though their NPC cultures were >90% SOX2 positive. The role of YY1 in ES cells remains unclear because of possible greater ES reliance on CTCF, different ChIP methods between ES and NPC YY1 datasets, or a different regulatory role for YY1 in the two cell types.
  • Stated: technical issues with poor library complexity prevented high-complexity 5C maps at Olig1, Olig2, and Nes after Yy1 knockdown; for those loci, only expression reductions were observed.
  • Evident: 5C covered selected regions (>7 Mb) rather than the whole genome, and the Yy1 perturbation was an siRNA knockdown with >50% reduction rather than a complete knockout, so some conclusions about YY1’s architectural role remain correlative or partial.

Model-written summary. Check it against the paper before relying on it.

Integrity signals

Registry

Crossref · 10.1101/gr.215160.116

Cites

66 of 66 references verified0 citation errors · 0 not checkable

Statistics

Nothing to recompute.

Claims the paper makes

0 of 59 cited sources checked · 0 supported · 0 flagged

  • Findingown data

    YY1 is present at the base of looping interactions between NPC-specific genes and enhancers.

    “We identify another zinc finger protein, Yin Yang 1 (YY1), at the base of looping interactions between NPC-specific genes and enhancers.”
    Quote in the paperSubstantial claim
  • Findingown data

    CTCF binding sites in NPCs are largely preexisting in pluripotent stem cells.

    “Conversely, CTCF binding sites in NPCs are largely preexisting in pluripotent stem cells.”
    Quote in the paperSubstantial claim
  • Findingown data

    The number of CTCF binding sites decreased in a stepwise manner during the transition from naïve pluripotency to multipotency, with the sharpest drop between ES serum and NPC conditions.

    “We first noticed that the number of CTCF binding sites decreased in a stepwise manner during the transition from naïve pluripotency to multipotency, with the sharpest drop in binding sites between ES serum and NPC conditions (Fig. 1A).”
    Quote in the paperSubstantial claim
  • Findingown data

    CTCF occupancy decreases sharply during the transition from naïve/primed pluripotency to multipotent primary neural progenitor cells (NPCs).

    “Unexpectedly, we observe a sharp decrease in CTCF occupancy during the transition from naïve/primed pluripotency to multipotent primary neural progenitor cells (NPCs).”
    Quote in the paperSubstantial claim
  • Findingown data

    YY1-mediated interactions between NPC regulatory elements are often nested within constitutive loops anchored by CTCF.

    “YY1-mediated interactions between NPC regulatory elements are often nested within constitutive loops anchored by CTCF.”
    Quote in the paperSubstantial claim
  • May combine two claims. 32
  • Classified as probably not a substantial claim. 35
  • The quote only partly states this claim. 1

  • May combine two claims.

    Findingown data

    Many pluripotency gene-enhancer interactions are anchored by CTCF, and CTCF occupancy is lost in parallel with loop decommissioning during differentiation.

    “Many pluripotency gene-enhancer interactions are anchored by CTCF, and its occupancy is lost in parallel with loop decommissioning during differentiation.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Putative NPC-specific enhancers exhibit strong YY1 signal when engaged in 3D contacts and negligible YY1 signal when not in loops.

    “Putative NPC-specific enhancers exhibit strong YY1 signal when engaged in 3D contacts and negligible YY1 signal when not in loops.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingown data

    CTCF binding can display the opposite trend in other developmental lineages, in some cases increasing between embryonic and adult stages.

    “Notably, when we investigated other developmental lineages, we found that CTCF binding can display the opposite trend, in some cases increasing between the embryonic and adult stages (Supplemental Fig. 1A,B).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    CTCF gene expression decreased between pluripotent stem cell states and multipotent NPCs and between embryonic mouse brain and mature adult brain regions.

    “We observed a general accordance between the ChIP-seq and RNA-seq results in our cellular models: CTCF gene expression decreased between the pluripotent stem cell states and multipotent NPCs (Fig. 1C) and also between the embryonic mouse brain and mature adult brain regions (Fig. 1D).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Almost 40% of "NPC-only" interactions were not anchored by CTCF binding, whereas only <5% and <10% of "constitutive" and "2i+serum" interactions lacked CTCF binding, respectively.

    “Notably, when we explored the percentage of key looping classes anchored by CTCF, we found that almost 40% of “NPC-only” interactions were not anchored by CTCF binding, whereas only <5% and <10% of “constitutive” and “2i+serum” interactions lacked CTCF binding, respectively (Fig. 4E, black bars).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    During the departure from pluripotency, CTCF binding is lost, and the looping interaction connecting the Sox2 gene to the putative ES-specific enhancer concurrently breaks apart.

    “During the departure from pluripotency, CTCF binding is lost, and the looping interaction connecting the Sox2 gene to the putative ES-specific enhancer concurrently breaks apart (Fig. 4B,C; Supplemental Fig. 10A,B, red arrow).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    CTCF signal was negligible at all classes of YY1 binding, suggesting YY1 and CTCF do not, for the most part, directly colocalize.

    “CTCF signal was negligible at all classes of YY1 binding, suggesting that YY1 and CTCF do not, for the most part, directly colocalize (Supplemental Fig. 12F,G).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Strong YY1 signal at NPC-specific enhancers engaged in "NPC-only" looping interactions and negligible YY1 binding at NPC-specific enhancers that do not loop.

    “We found strong YY1 signal at NPC-specific enhancers engaged in “NPC-only” looping interactions and negligible YY1 binding at NPC-specific enhancers that do not loop.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    We identified 141 loops present in all three cell types ("Constitutive"), 46 loops present in both ES 2i and ES serum but lost in NPCs ("2i+Serum"), and 75 loops specific to the NPC state ("NPC-only").

    “Altogether, we identified several classes of 3D interactions (Fig. 3I) and elected to focus our analysis on three main groups: (1) 141 loops present in all three cell types (“Constitutive,” gray class); (2) 46 loops present in both ES 2i and ES serum but lost in NPCs (“2i+Serum,” purple class); and (3) 75 loops specific to the NPC state (“NPC-only,” green class).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    A markedly lower proportion of classified YY1 sites were constitutive among ES serum, NPCs, and primary pro-B cells (36%, 39%, and 25%, respectively; n = 3474), indicating YY1 might exhibit more cell-type-specific binding than CTCF.

    “In contrast, a markedly lower proportion of classified YY1 sites were constitutive among ES serum, NPCs, and primary pro-B cells (36%, 39%, and 25%, respectively; n = 3474), indicating that YY1 might exhibit more cell-type–specific binding than CTCF (Fig. 5H).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    CTCF occupancy is predominantly lost in the transition from ES cells to multipotent NPCs, and CTCF occupancy is highest in the naïve pluripotent stem cell state and globally decreases during commitment to multipotent NPCs.

    “First, we find that CTCF occupancy is predominantly lost in the transition from ES cells to multipotent NPCs, suggesting that the CTCF occupancy landscape might be saturated in naïve pluripotency and regulated primarily through selective pruning of CTCF binding sites.”
    • Also stated in: § Discussion · plain
      “Here, we find that CTCF occupancy is highest in the naïve pluripotent stem cell state and globally decreases in parallel with its expression during the commitment to multipotent NPCs.”
    Quote in the paperSubstantial claim

  • May combine two claims. The quote only partly states this claim.

    Findingown data

    Upon knockdown of Yy1 in NPCs, many 3D interactions break apart, including several key NPC-specific enhancer-gene looping interactions.

    “Several key NPC-specific enhancer-gene looping interactions were ablated upon Yy1 knockdown.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    A large cohort of roughly 8000 and roughly 20,000 CTCF sites are lost during the transition from ES 2i to ES serum and ES serum to NPCs, respectively.

    “A large cohort of roughly 8000 and roughly 20,000 CTCF sites are lost during the transition from ES 2i to ES serum and ES serum to NPCs, respectively.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    High levels of YY1 occupancy were detected at NPC-specific genes and enhancers when engaged in NPC-specific 3D interactions, and negligible YY1 levels when these regulatory elements did not interact.

    “Rather, we detected high levels of occupancy of the zinc finger protein YY1 at NPC-specific genes and enhancers when engaged in NPC-specific 3D interactions and negligible YY1 levels when these regulatory elements did not interact.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    "2i+serum" looping interactions were enriched for ES-specific enhancers regardless of CTCF and YY1 occupancy, whereas ES-specific genes were only enriched in "2i+serum" looping interactions when bound by CTCF without YY1.

    “Additionally, “2i+serum” looping interactions were enriched for ES-specific enhancers regardless of CTCF and YY1 occupancy, whereas ES-specific genes were only enriched in “2i+serum” looping interactions when bound by CTCF without YY1 (Supplemental Fig. 12J,L).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    YY1 occupancy signal was low and diffuse across putative ES-specific regulatory elements and did not show a clear preference between those engaged in loops versus nonloops.

    “YY1 occupancy signal was low and diffuse across putative ES-specific regulatory elements and did not show a clear preference between those engaged in loops versus nonloops (Supplemental Fig. 12D,E).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The majority of architectural protein-bound NPC-specific enhancers were bound by YY1 without CTCF, whereas the majority of ES-specific enhancers were bound by CTCF without YY1.

    “By focusing on distal cell-type–specific regulatory elements that overlap binding sites of CTCF, YY1, or both (and not considering those bound by neither), we observed that the majority of architectural protein-bound NPC-specific enhancers were bound by YY1 without CTCF, whereas the majority of ES-specific enhancers were bound by CTCF without YY1 (Fig. 5J).”
    Quote in the paperSubstantial claim

  • Classified as probably not a substantial claim. May combine two claims.

    Premisecitation

    Looping interactions and sub-TADs often link genes to developmentally regulated enhancers and are markedly reorganized between cellular states.

    “Looping interactions and sub-TADs often link genes to developmentally regulated enhancers and are markedly reorganized between cellular states (Tolhuis et al. 2002; Sanyal et al. 2012; Phillips-Cremins et al. 2013; Dowen et al. 2014; Smith et al. 2016).”
    Quote in the paperSubstantial claim0 of 3 sources checked
    • cites Tolhuis et al. 2002

      Not checked in the free run.

    • cites Sanyal et al. 2012

      Not checked in the free run.

    • cites Phillips-Cremins et al. 2013

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  • May combine two claims.

    Interpretationreasoning

    Although CTCF occupancy appears to decrease during the transition from pluripotency to early neuronal lineage commitment, it is not a pervasive trend across all developmental lineages.

    “Thus, although CTCF occupancy appears to decrease during the transition from pluripotency to early neuronal lineage commitment, it is not a pervasive trend across all developmental lineages.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Premisecitation

    CTCF is found at the base of looping interactions, and knockdown of CTCF abrogates chromatin connections.

    “CTCF is found at the base of looping interactions, and knockdown of the protein abrogates chromatin connections (Kurukuti et al. 2006; Hadjur et al. 2009; Handoko et al. 2011; Splinter et al. 2012).”
    Quote in the paperSubstantial claim0 of 3 sources checked
    • cites Kurukuti et al. 2006

      Not checked in the free run.

    • cites Hadjur et al. 2009

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    • cites Handoko et al. 2011

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  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    An independent study reported a decrease in CTCF protein levels in whole mouse brains during the transition from E15 to post-natal week 1.

    “Corroborating our results, while this manuscript was under review, an independent study also reported a decrease in CTCF protein levels in whole mouse brains during the transition from E15 to post-natal week 1 (Sams et al. 2016).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Sams et al. 2016

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    CTCF-mediated interactions can be disrupted by mutation, inversion, and/or deletion of the CTCF motif, indicating that consensus orientation is a critical contributing factor in loop establishment and/or maintenance.

    “CTCF-mediated interactions can be disrupted by mutation, inversion, and/or deletion of the CTCF motif, indicating that consensus orientation is a critical contributing factor in loop establishment and/or maintenance (de Wit et al. 2015; Guo et al. 2015; Sanborn et al. 2015).”
    Quote in the paperNot a substantial claim0 of 3 sources checked
    • cites de Wit et al. 2015

      Not checked in the free run.

    • cites Guo et al. 2015

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    • cites Sanborn et al. 2015

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  • May combine two claims.

    Interpretationown data

    YY1 acts as an architectural protein to connect developmentally regulated looping interactions, and the location of YY1-mediated interactions may be demarcated in development by a preexisting topological framework created by constitutive CTCF-mediated interactions.

    “Together, our results support a model in which YY1 acts as an architectural protein to connect developmentally regulated looping interactions; the location of YY1-mediated interactions may be demarcated in development by a preexisting topological framework created by constitutive CTCF-mediated interactions.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Of the subset of loops bound by CTCF with clear consensus sequences, 92% were anchored by consensus sequences pointed toward each other in a convergent orientation.

    “Importantly, of the subset of loops bound by CTCF with clear consensus sequences, 92% were anchored by consensus sequences pointed toward each other in a convergent orientation (Rao et al. 2014; Vietri Rudan et al. 2015).”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites Rao et al. 2014

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    • cites Vietri Rudan et al. 2015

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  • Classified as probably not a substantial claim.

    Premisecitation

    CTCF functions as an architectural protein to connect higher-order chromatin configurations.

    “A leading hypothesis is that CTCF's diverse regulatory roles can be explained by a unifying mechanism in which it functions as an architectural protein to connect higher-order chromatin configurations (Phillips and Corces 2009).”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites Phillips and Corces 2009

      Not checked in the free run.

  • Classified as probably not a substantial claim.

    Study design and conductown data

    Thresholds were chosen so that the top five largest dynamic looping classes achieved an empirical false discovery rate less than 15%.

    “Thresholds were chosen so that our top five largest dynamic looping classes achieved an empirical false discovery rate less than 15% (Supplemental Methods; Supplemental Fig. S8B–D).”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    CTCF has a well-established role in connecting long-range looping interactions.

    “CTCF has a well-established role in connecting long-range looping interactions (de Wit et al. 2015; Guo et al. 2015).”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites de Wit et al. 2015

      Not checked in the free run.

    • cites Guo et al. 2015

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  • May combine two claims.

    Premisecitation

    Sox2 forms a pluripotency-specific loop with a putative ES-specific enhancer ∼120 kb downstream that is essential for proper expression of the gene in ES cells.

    “Sox2 forms a pluripotency-specific loop with a putative ES-specific enhancer ∼120 kb downstream that is essential for proper expression of the gene in ES cells (Fig. 4A,B, magenta arrowhead; Li et al. 2014).”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites Li et al. 2014

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Interpretationcitation

    Heterogeneity in cells derived from ENCODE tissues may contribute to the aggregate decrease in CTCF levels.

    “Sams et al. also identified differential CTCF levels across neurons, astrocytes, and oligodendrocytes from the hippocampus, highlighting that we cannot rule out the possibility that heterogeneity in cells derived from ENCODE tissues may contribute to the aggregate decrease in CTCF levels.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Sams et al. 2016

      Not checked in the free run.

  • Classified as probably not a substantial claim. May combine two claims.

    Study design and conductown data

    ChIP-seq experiments were conducted in the same batch, sequenced on the same flow cell, and down-sampled to the same read depth to attenuate technical artifacts.

    “ChIP-seq experiments were conducted in the same batch, sequenced on the same flow cell, and down-sampled to the same read depth to attenuate technical artifacts that might influence our observed results (Fig. 2E).”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Interpretationcitation

    There is a larger class of dynamically occupied CTCF sites than previously appreciated.

    “These results confirm and extend recent reports suggesting that there is a larger class of dynamically occupied CTCF sites than previously appreciated (Shen et al. 2012; Maurano et al. 2015).”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites Shen et al. 2012

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    • cites Maurano et al. 2015

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  • May combine two claims.

    Study design and conductcitation

    NPC cultures exhibited a highly consistent morphology throughout the population, and >90% were SOX2 positive.

    “Our NPC cultures exhibited a highly consistent morphology throughout the population, and >90% were SOX2 positive (Beagan et al. 2016), suggesting that our NPC preparations were substantially less heterogeneous than brain tissue lysates.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites Beagan et al. 2016

      Not checked in the free run.

  • May combine two claims.

    Interpretationown data

    CTCF gene and protein expression levels decrease in the transition from pluripotency to multipotent neural progenitor cells in parallel with a global decrease in the number of genome-wide CTCF binding sites.

    “Our data indicate that CTCF gene and protein expression levels decrease in the transition from pluripotency to multipotent neural progenitor cells in parallel with a global decrease in the number of genome-wide CTCF binding sites.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    A large proportion of CTCF sites are preestablished at least as early in development as naïve pluripotency and selectively lost in early neural lineage commitment.

    “Rather, a large proportion of CTCF sites are preestablished at least as early in development as naïve pluripotency and selectively lost in early neural lineage commitment (Fig. 2D, orange and purple classes).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    YY1 is present at NPC regulatory elements engaged in 3D interactions and might serve as an architectural protein to connect NPC-specific genes and enhancers.

    “These data suggest that YY1 is present at NPC regulatory elements engaged in 3D interactions and support our working hypothesis that YY1 might serve as an architectural protein to connect NPC-specific genes and enhancers.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S1 (asserted), S3 (load-bearing). May combine two claims.

    Interpretationreasoning

    A notable proportion of the large class (n = ∼20,000) of '2i+serum' CTCF sites might be methylated during the initial establishment of the neural lineage and subsequently remain methylated and unbound through terminal differentiation in the developing/maturing brain.

    “Because CTCF binding is anti-correlated with DNA methylation, we posit that a notable proportion of the large class (n = ∼20,000) of “2i+serum” CTCF sites might be methylated during the initial establishment of the neural lineage and subsequently remain methylated and unbound through terminal differentiation in the developing/maturing brain.”
    Quote in the paperNot a substantial claim
    • Rests on an assumption: CTCF binding is anti-correlated with DNA methylation.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductown data

    Due to technical issues related to poor library complexity across all conditions in this batch of experiments, high complexity 5C maps could not be obtained at Olig1, Olig2, and Nes regions.

    “Due to technical issues related to poor library complexity across all conditions in this batch of experiments, we were unable to obtain high complexity 5C maps at Olig1, Olig2, and Nes regions.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationown data

    Developmentally regulated genes such as Nes, Olig1, Olig2, and Sox2 form de novo connections to their target enhancers via YY1 within a larger topological framework preexisting from naïve pluripotency and connected by constitutive CTCF.

    “Together, these results support a working model of 3D genome folding in which developmentally regulated genes such as Nes, Olig1, Olig2, and Sox2 form de novo connections to their target enhancers via YY1 within a larger topological framework preexisting from naïve pluripotency and connected by constitutive CTCF.”
    • Also stated in: § Discussion · plain
      “Our data support a model in which YY1-anchored looping interactions arise de novo in NPCs within a larger topological framework established prior to or during naïve pluripotency and connected by constitutively bound CTCF (Fig. 7E).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    The CTCF occupancy landscape might be saturated in naïve pluripotency and regulated primarily through selective pruning of CTCF binding sites.

    “suggesting that the CTCF occupancy landscape might be saturated in naïve pluripotency and regulated primarily through selective pruning of CTCF binding sites.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    The Olig1 and Olig2 genes encode bHLH transcription factors involved in differentiation along the oligodendrocyte lineage.

    “The Olig1 and Olig2 genes encode bHLH transcription factors involved in differentiation along the oligodendrocyte lineage (Zhou et al. 2000).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Zhou et al. 2000

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    YY1 is necessary for the formation of specific 3D interactions in B cells.

    “(2) it is necessary for the formation of specific 3D interactions in B cells (Medvedovic et al. 2013);”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Medvedovic et al. 2013

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  • Classified as probably not a substantial claim.

    Premisecitation

    Cells in the naïve pluripotency state (ES 2i) have consistently been found to exhibit an extreme state of hypomethylation across the genome.

    “in which cells have consistently been found to exhibit an extreme state of hypomethylation across the genome (Ficz et al. 2013; Habibi et al. 2013; Leitch et al. 2013).”
    Quote in the paperSubstantial claim0 of 3 sources checked
    • cites Ficz et al. 2013

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    • cites Habibi et al. 2013

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    • cites Leitch et al. 2013

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  • Probably not a substantial claim: may not pass S4 (based).

    Interpretationnone given

    Architectural proteins such as YY1 might cooperatively build upon a constitutive CTCF architectural 'seed' scaffold to connect nearby developmentally regulated genes and enhancers.

    “Thus we posit that architectural proteins such as YY1 might cooperatively build upon a constitutive CTCF architectural “seed” scaffold to connect nearby developmentally regulated genes and enhancers.”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim.

    Interpretationown data

    It is not clear to what extent observed differences between ES and NPC YY1 are due to increased reliance by ES cells on CTCF, different ChIP methods, or a different regulatory role for YY1.

    “It is not clear to what extent our observed differences between ES and NPC YY1 are due to (1) an increased reliance by ES cells on CTCF as the primary architectural protein; (2) different ChIP methods between the ES and NPC YY1 data sets (Supplemental Methods); or (3) a different regulatory role for YY1 in the two cell types.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    Although data indicate YY1 might be important for developmentally regulated looping in somatic cells, we cannot conclusively define or rule out any role for YY1 in mediating loops in ES cells.

    “Thus, although our data indicate that YY1 might be important for developmentally regulated looping in somatic cells, we cannot conclusively define or rule out any role for YY1 in mediating loops in ES cells.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    The REPO domain of YY1 physically interacts with cohesin.

    “Previous studies have shown that the REPO domain of YY1 physically interacts with cohesin (Pan et al. 2013).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Pan et al. 2013

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    Up to 30% of YY1 sites bind at locations directly adjacent to CTCF and might work together to cooperatively influence occupancy.

    “Furthermore, a recent genomics analysis showed that up to 30% of YY1 sites bind at locations directly adjacent to CTCF and might work together to cooperatively influence occupancy (Schwalie et al. 2013).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Schwalie et al. 2013

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductcitation

    5C primer biases were corrected by adapting and modifying the Express matrix balancing algorithm.

    “5C primer biases were corrected by adapting and modifying the Express matrix balancing algorithm (Sauria et al. 2015).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Sauria et al. 2015

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    CTCF-mediated looping interactions might function to coordinate nearby interactions involving RNA Pol II.

    “In agreement with this idea, Ruan and colleagues reported evidence that CTCF-mediated looping interactions might function to coordinate nearby interactions involving RNA Pol II (Tang et al. 2015).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Tang et al. 2015

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    A chromatin molecular motor, such as cohesin/condensin, might extrude chromatin until blocked by architectural proteins, resulting in looped out chromatin.

    “Marko, Mirny, Lieberman-Aiden and colleagues have posited that a chromatin molecular motor, such as cohesin/condensin, might extrude chromatin until blocked by architectural proteins, resulting in looped out chromatin (Alipour and Marko 2012; Sanborn et al. 2015; Fudenberg et al. 2016).”
    Quote in the paperNot a substantial claim0 of 3 sources checked
    • cites Alipour and Marko 2012

      Not checked in the free run.

    • cites Sanborn et al. 2015

      Not checked in the free run.

    • cites Fudenberg et al. 2016

      Not checked in the free run.

  • Classified as probably not a substantial claim.

    Premisecitation

    The largest rearrangement of DNA methylation during neural development occurs upon the departure from pluripotency.

    “Recent reports suggest that the largest rearrangement of DNA methylation during neural development occurs upon the departure from pluripotency (Ziller et al. 2015; Sharma et al. 2016).”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites Ziller et al. 2015

      Not checked in the free run.

    • cites Sharma et al. 2016

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Seminal genome-wide CTCF occupancy studies based on two to three cell types initially suggested that the CTCF binding landscape remains largely unchanged across mammalian lineages.

    “Seminal genome-wide CTCF occupancy studies based on two to three cell types initially suggested that the CTCF binding landscape remains largely unchanged across mammalian lineages (Kim et al. 2007; Cuddapah et al. 2009).”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites Kim et al. 2007

      Not checked in the free run.

    • cites Cuddapah et al. 2009

      Not checked in the free run.

  • Classified as probably not a substantial claim.

    Premisecitation

    DNA methylation is a critical regulator of neural lineage commitment.

    “DNA methylation is a critical regulator of neural lineage commitment (Meissner et al. 2008) and CTCF binding (Renda et al. 2007).”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites Meissner et al. 2008

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    YY1 can connect a long-range interaction in B cells in the absence of its transcriptional activation domain.

    “(3) it can connect a long-range interaction in B cells in the absence of its transcriptional activation domain (Mehra et al. 2016);”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Mehra et al. 2016

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    The zinc fingers of YY1 may interact with the N terminus of CTCF.

    “Biochemical studies have indicated that the zinc fingers of YY1 may interact with the N terminus of CTCF (Donohoe et al. 2007), suggesting that YY1 could anchor loops via homodimerization (Supplemental Fig. 16A) or heterodimerization (Supplemental Fig. 16B) mechanisms.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Donohoe et al. 2007

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S1 (asserted).

    Premisecitation

    Sub-TAD/TAD boundaries, anchored by constitutive CTCF, might constrain developmentally regulated enhancers from aberrantly looping to off-target genes.

    “A leading hypothesis is that sub-TAD/TAD boundaries, anchored by constitutive CTCF, might constrain developmentally regulated enhancers from aberrantly looping to off-target genes (Dowen et al. 2014; Symmons et al. 2014; Lupiáñez et al. 2015; Flavahan et al. 2016; Hnisz et al. 2016).”
    Quote in the paperNot a substantial claim0 of 3 sources checked
    • cites Dowen et al. 2014

      Not checked in the free run.

    • cites Symmons et al. 2014

      Not checked in the free run.

    • cites Lupiáñez et al. 2015

      Not checked in the free run.

  • Classified as probably not a substantial claim.

    Premisecitation

    Pluripotency genes connect to enhancers in smaller looping interactions nested within larger constitutive structures.

    “We and others have previously reported that pluripotency genes connect to enhancers in smaller looping interactions nested within larger constitutive structures (Phillips-Cremins et al. 2013; Dowen et al. 2014).”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites Phillips-Cremins et al. 2013

      Not checked in the free run.

    • cites Dowen et al. 2014

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    A more recent comparison of CTCF occupancy across 40 cell types revealed that at least 80% of CTCF sites are dynamic across cellular states.

    “A more recent comparison of CTCF occupancy across 40 cell types revealed that at least 80% of CTCF sites are dynamic across cellular states (Maurano et al. 2015).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Maurano et al. 2015

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    YY1 is required for proper neural development.

    “(4) it is required for proper neural development (He et al. 2007).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites He et al. 2007

      Not checked in the free run.

  • Classified as probably not a substantial claim.

    Study design and conductreasoning

    To facilitate comparison of ChIP-seq libraries across cell types, mapped reads were filtered to remove optical and PCR duplicates and then down-sampled to equivalent read numbers across cellular states.

    “To facilitate the comparison of ChIP-seq libraries across cell types, the mapped reads were filtered to remove optical and PCR duplicates and then down-sampled to equivalent read numbers across cellular states.”
    Quote in the paperSubstantial claim

  • Classified as probably not a substantial claim.

    Study design and conductcitation

    Binned 5C counts were normalized for the expected background interaction frequency with local 'donut' and 'lower left' filters.

    “Binned 5C counts were normalized for the expected background interaction frequency with local “donut” and “lower left” filters (Rao et al. 2014) as detailed in the Supplemental Methods.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites Rao et al. 2014

      Not checked in the free run.

  • Classified as probably not a substantial claim. May combine two claims.

    Study design and conductnone given

    Background-corrected interaction frequencies for each independent replicate and region were fitted with a logistic distribution, and P-values were assigned to each bin-bin pair.

    “Background-corrected interaction frequencies for each independent replicate and region were fitted with a logistic distribution, and P-values were assigned to each bin–bin pair.”
    Quote in the paperSubstantial claim
62 of 62

  • The paper relies on this without support.

    The 5C interrogated regions are representative of genome-wide 3D chromatin architecture.

    “We generated fine-scale chromatin architecture maps (∼4- to12-kb matrix resolution) across >7 Mb of the mouse genome surrounding key developmentally regulated genes with Chromosome-Conformation-Capture-Carbon-Copy (5C) and high-throughput sequencing.”

  • The paper relies on this without support.

    Putative NPC-specific enhancers are bona fide regulatory enhancers.

    “we parsed our putative genome-wide NPC-specific enhancers (Supplemental Methods) into those that engage in “NPC-only” loops (Fig. 5F) and those that do not participate in long-range interactions (Fig. 5G).”

  • The paper relies on this without support.

    CTCF binding is anti-correlated with DNA methylation.

    “Because CTCF binding is anti-correlated with DNA methylation, we posit that a notable proportion of the large class (n = ∼20,000) of “2i+serum” CTCF sites might be methylated during the initial establishment of the neural lineage and subsequently remain methylated and unbound through terminal differentiation in the developing/maturing brain.”

  • The paper relies on this without support.

    The putative NPC-specific enhancers marked by H3K27ac are functional enhancers that regulate the associated genes.

    “In NPCs, putative enhancer(s) marked by NPC-specific H3K27ac connect to both Olig1 and Olig2 in a rosette-like structure, and these genes show markedly increased expression in NPCs compared to ES 2i/ES serum (Fig. 6B–D, magenta arrows).”

  • The paper relies on this without support.

    The Yy1-targeting siRNA specifically reduces YY1 without off-target effects.

    “Transfection of the Yy1-targeting siRNA pool produced a >50% decrease in Yy1 expression and protein levels compared to a control nontargeting pool condition (Fig. 7A,B).”
5 of 5

Claims other papers make about this one

Each is checked against this paper’s own text: does it say what the citing paper says it does?

  • YY1 has a global role in connecting cell type-specific active enhancers and promoters in a range of mammalian cell types including mESCs and neural progenitor cells.

    “Recent studies have confirmed its global role in connecting cell type-specific active enhancers and promoters in a range of mammalian cell types including mESCs [295] and neural progenitor cells [365].”

    From Transcriptional enhancers and their communication with gene promoters (2021) · cites this paper as [365]

    The Results section reports YY1 enrichment at looping NPC-specific enhancers and genes, and loss of several such loops after Yy1 knockdown. But the paper describes YY1 as a candidate architectural protein and does not establish the claimed global role; it also says it cannot conclusively define YY1’s looping role in ES cells. In the citing sentence, the ES-cell example is attached to a different citation. (LLM, conf=0.86)

    Experimental — accuracy not yet measured

  • YY1 and ZNF143 are zinc finger TFs with DNA binding domains that have been implicated as looping factors mediating interactions between active regulatory elements.

    “YY1 and ZNF143 are both zinc finger TFs with DNA binding domains that have recently been implicated as looping factors mediating interactions between active regulatory elements [295, 362–365].”

    From Transcriptional enhancers and their communication with gene promoters (2021) · cites this paper as [365]

    The Results section identifies YY1 as a zinc-finger protein enriched at enhancer–gene looping interactions, and the Discussion describes it as a candidate architectural protein. Our copy does not address ZNF143, so it supports the YY1 part of the claim but not the assertion about both factors. (LLM, conf=0.88)

    Experimental — accuracy not yet measured

  • No statistical methods were used to pre-determine sample sizes, but the sample sizes are similar to those reported in previous publications.

    “No statistical methods were used to pre-determine sample sizes but our sample sizes are similar to those reported in previous publications17,27,28.”

    From Three-dimensional genome restructuring across timescales of activity-induced neuronal gene expression (2020) · cites this paper as [27]

Self-check

Percentages, sums and reported differences re-computed from the paper’s own numbers.

Each contradiction between two passages listed here was confirmed by an independent check.

  • An siRNA knockdown is called a knockout

    Minor

    The described intervention uses siRNA to reduce Yy1 levels, but the results later call it a Yy1 knockout.

1 of 1

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Authors
Beagan, J. A., Duong, M. T., Titus, K. R., Zhou, L., Cao, Z., Ma, J., Lachanski, C. V., Gillis, D. R., & Phillips-Cremins, J. E.
Affiliations
University of Pennsylvania (US)
ORCID
Venue
Genome Research
Publisher
Cold Spring Harbor Laboratory
Published
2017-05-23
Volume
27
Issue
7
Pages
1139-1152
ISSN
  • 1088-9051 (ISSN-L)
  • 1549-5469
DOI
10.1101/gr.215160.116
PMC
PMC5495066
OpenAlex
W2616964127
Semantic Scholar
ef47c2402c2ca8c046dfd6f1c16e7268ea95d80a
Cited by
299
References
66
Citations by year
2026: 19 · 2025: 32 · 2024: 24 · 2023: 33 · 2022: 36 · 2021: 46 · 2020: 38 · 2019: 30 · 2018: 36 · 2017: 5
Type
posted content
Language
en
Open access
Licence
cc-by-nc
Locations
  • Genome Research (publishedVersion)
  • PubMed Central (publishedVersion, cc-by-nc)
  • PubMed (publishedVersion)
  • HighWire Press Open Archive (submittedVersion)
Funders
  • The New York Stem Cell Foundation
  • Alfred P. Sloan Foundation
  • NIH Director's New Innovator Award from the National Institute of Mental Health (1DP2MH11024701)
  • 4D Nucleome (NIH) Common (1U01HL12999801)
  • NSF-NIGMS (1562665)
  • National Science Foundation (DGE-1321851)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Genomics and Chromatin Dynamics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Chromatin Remodeling and Cancer
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Pluripotent Stem Cells Research
Keywords
CTCF, YY1, neural progenitor cells, enhancer-promoter interactions, chromatin structure, pluripotency, zinc finger proteins, chromatin looping
MeSH terms
Human Embryonic Stem Cells, CCCTC-Binding Factor, Cell Differentiation, Cell Line, Chromatin, Enhancer Elements, Genetic, Humans, Chromatin Assembly and Disassembly, YY1 Transcription Factor, Genome-Wide Association Study, Neural Stem Cells

About these checks

Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.

Claims read by deepseek-v4.1-flash under definition version 3 (extract-v3) on 2026-10-10.

Classified by jev-1.13-20260917 (gold-guideline-v0.2) on 2026-10-10.

Statistics and arithmetic are recomputed, not judged.

Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.

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