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Cited by 213Acute Myeloid Leukemia ResearchGenomics and Chromatin DynamicsChromatin Remodeling and Cancer

Subtype-specific 3D genome alteration in acute myeloid leukaemia

Open access
Xu, Jie, Song, Fan, Lyu, Huijue, Kobayashi, Mikoto, Zhang, Baozhen, Zhao, Ziyu, Hou, Ye, Wang, Xiaotao +21 more·2022·Nature
DOI 10.1038/s41586-022-05365-xPMC PMC10060167
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Abstract

Acute myeloid leukaemia (AML) represents a set of heterogeneous myeloid malignancies, and hallmarks include mutations in epigenetic modifiers, transcription factors and kinases 1 – 5 . The extent to which mutations in AML drive alterations in chromatin 3D structure and contribute to myeloid transformation is unclear. Here we use Hi-C and whole-genome sequencing to analyse 25 samples from patients with AML and 7 samples from healthy donors. Recurrent and subtype-specific alterations in A/B compartments, topologically associating domains and chromatin loops were identified. RNA sequencing, ATAC with sequencing and CUT&Tag for CTCF, H3K27ac and H3K27me3 in the same AML samples also revealed extensive and recurrent AML-specific promoter–enhancer and promoter–silencer loops. We validated the role of repressive loops on their target genes by CRISPR deletion and interference. Structural variation-induced enhancer-hijacking and silencer-hijacking events were further identified in AML samples. Hijacked enhancers play a part in AML cell growth, as demonstrated by CRISPR screening, whereas hijacked silencers have a downregulating role, as evidenced by CRISPR-interference-mediated de-repression. Finally, whole-genome bisulfite sequencing of 20 AML and normal samples revealed the delicate relationship between DNA methylation, CTCF binding and 3D genome structure. Treatment of AML cells with a DNA hypomethylating agent and triple knockdown of DNMT1 , DNMT3A and DNMT3B enabled the manipulation of DNA methylation to revert 3D genome organization and gene expression. Overall, this study provides a resource for leukaemia studies and highlights the role of repressive loops and hijacked cis elements in human diseases. Extensive genomic analyses of the chromatin architecture in acute myeloid leukaemia reveals several characteristics, including subtype-specific distal enhancers and silencers, that may represent new anticancer therapeutic targets.

AI snapshot

Written from the paper’s full text.

TL;DR

This study maps 3D genome organization in primary acute myeloid leukemia (AML) and identifies subtype-specific A/B compartment switches, TAD alterations, chromatin loops, and architectural stripes. It further shows that promoter-silencer loops and structural-variant-induced enhancer/silencer hijacking contribute to gene dysregulation, and that DNA hypomethylating treatment or DNMT triple knockdown can partially reverse AML-associated chromatin topology.

Key findings

  • In 25 AML and 7 healthy donor samples, Hi-C identified recurrent and subtype-specific 3D alterations: on average 3.2% A-to-B and 2.9% B-to-A compartment switches versus HSPC; 391 of 622 curated COSMIC cancer/AML-related genes fell in altered TADs in at least one sample, with >100 recurrent.
  • AML-specific loops averaged ~200 per AML sample; 42.4% were promoter-enhancer (P-E) and 11.2% promoter-silencer (P-S), with P-S loops accounting for 9.2% of all loops and averaging 169 kb. CRISPR deletion of an RTTN silencer increased RTTN expression 3-fold (p=5E-5), and CRISPRi de-repression of IKZF2-linked silencers increased IKZF2 expression >35-fold (P=0.00017).
  • SV-induced neo-loops included enhancer hijacking (17.2% of neo-loops) and silencer hijacking (5.7%); CRISPRi screening of 74 neo P-E loops in Kasumi-1 cells identified 14 sgRNAs targeting 13 enhancers as significantly depleted, and hijacked silencer de-repression increased EXD1 and ALG10 expression by 5.97-fold and 2.70-fold, respectively.
  • 5-AZA treatment reduced global methylation from 74% to 39%; 31.6% of AML-specific loops dissociated after treatment versus 2.5% of shared loops, and 55% of A-to-B altered compartments in an AML cell line were reversed to the A compartment.

Methods

  • Primary samples: in situ Hi-C and RNA-seq in 32 primary samples (25 AML, 4 normal CD34+ HSPC, 3 PBMC), with ~600 million paired-end reads per Hi-C library; WGS at ~40x in 25 AML; ATAC-seq and CUT&Tag for H3K27ac, H3K27me3, and CTCF; WGBS in 18 AML and 2 PBMC at ~30X; 199 total genomic datasets.
  • Functional validation: 4C-seq, 3D DNA FISH, HiChIP for H3K27me3, CRISPR deletion, CRISPRi/CRISPR activation, luciferase reporter assays, and a CRISPRi screen in Kasumi-1 cells; cell-line experiments in Kasumi-1, THP-1, HL60, U937, and K562.
  • Perturbation: U937 cells treated with 0.5 μM 5-AZA for 12 days or subjected to inducible DNMT3A/3B/1 triple knockdown, followed by WGBS and Hi-C; computational analyses included Hi-C compartment/TAD/loop calling, NeoLoopFinder, stripe detection, and differential loop/compartment analyses.

Limitations

  • The paper states that future experiments should aim to dissolve chromatin interactions without changing local chromatin status at cis-regulatory elements, and that phenotypic effects need deeper study, with animal models desirable.
  • Evident from the text: the primary AML cohort is moderate in size (25 samples), and subtype-specific conclusions rest on small subgroups; functional validation of loops and hijacking is largely performed in AML cell lines rather than in vivo.
  • Evident from the text: although the authors report that <4.8% of AML-specific loops were in CNV regions, structural variation and copy-number effects remain potential confounders in 3D genome analyses.

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

Integrity signals

Registry

Crossref · 10.1038/s41586-022-05365-x

Cites

82 of 82 references verified0 citation errors · 0 not checkable

Statistics

Nothing to recompute.

Claims the paper makes

0 of 15 cited sources checked · 0 supported · 0 flagged

  • Findingown data

    A cluster of AML samples in the Hi-C clustering all contained mutations in the KMT2B gene.

    “We noticed that a cluster of samples all contained mutations in the KMT2B gene, which has not been used as a dominant AML subtyping classifier before.”
    Quote in the paperNot a substantial claim
  • Findingown data

    The majority of TAD alterations did not affect gene expression.

    “However, as shown in Extended Data Fig. 3c, the majority of TAD alterations did not affect the expression of genes, consistent with a recent study in multiple cancer types26.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [26] Akdemir, K. C. et al. Disruption of chromatin folding domains by somatic genomic rearrangements in human cancer. Nat. Genet. 52, 294–305 (2020). source

      Not checked in the free run.

  • Findingown data

    Unsupervised hierarchical clustering of Hi-C PC1 separated AML samples into groups that accurately reflected AML genetic subtypes, including RUNX1 and CEBPA mutations.

    “The samples were clustered into different groups, which accurately reflected the AML genetic subtypes (Fig. 1c), such as RUNX1 and CEBPA mutations.”
    Quote in the paperSubstantial claim
  • Findingown data

    Other compartment switch regions unique to AML or AML subgroups contain known AML genes such as POUA2F1, FGF13, and BCL11.

    “Similarly, we observed other compartment switch regions unique to AML or AML subgroups, containing known AML gens such as POUA2F1, FGF13, and BCL11 (Extended Data Fig. 2c).”
    Quote in the paperNot a substantial claim
  • Findingown data

    A/B compartment switching regions grouped by genetic mutation profile.

    “Moreover, the A/B compartment switching regions also grouped by their genetic mutation profile (Fig. 1f).”
    Quote in the paperSubstantial claim
  • May combine two claims. 34
  • Classified as probably not a substantial claim. 29
  • The quote only partly states this claim. 1

  • May combine two claims.

    Findingown data

    391 out of 622 curated COSMIC cancer-related and AML-related genes were located inside altered TADs in at least one sample, and more than 100 were recurrent across multiple samples.

    “We identified 391 out of the 622 curated COSMIC cancer-related and AML-related genes located inside the altered TADs in at least one sample, and more than 100 are recurrent across multiple samples (Extended Data Fig. 3b)4,24,25.”
    Quote in the paperSubstantial claim0 of 3 sources checked
    • cites [4] Papaemmanuil, E. et al. Genomic classification and prognosis in acute myeloid leukemia. N. Engl. J. Med. 374, 2209–2221 (2016). source

      Not checked in the free run.

    • cites [24] Sondka, Z. et al. The COSMIC Cancer Gene Census: describing genetic dysfunction across all human cancers. Nat. Rev. Cancer 18, 696–705 (2018). source

      Not checked in the free run.

    • cites [25] Metzeler, K. H. et al. Spectrum and prognostic relevance of driver gene mutations in acute myeloid leukemia. Blood 128, 686–698 (2016). source

      Not checked in the free run.

  • May combine two claims.

    Findingown data

    Genes located in A-to-B switching regions showed decreased expression, and genes in B-to-A switching regions showed increased expression.

    “Genes located in the A-to-B or B-to-A switching regions showed decreased or increased expression, respectively (Fig. 1e).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    On average, comparing AML samples with HSPC controls, there was 3.2% A-to-B compartment switch and 2.9% B-to-A switch.

    “On average, there was 3.2% A-to-B compartment switch and 2.9% B-to-A switch comparing AML samples with HPSC controls (Fig. 1d).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    WT1 gene showed B-to-A switch and expression activation exclusively in samples with TET2 and/or FLT3-ITD mutations, accompanied by gain of ATAC-seq and H3K27ac peaks.

    “For example, the WT1 gene showed B-to-A switch and expression activation exclusively in samples with TET2 and/or FLT3-ITD mutations, accompanied by the gain of ATAC-seq and H3K27ac peaks (Extended Data Fig. 2a-b).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Multiple gained chromatin loops appeared in these AML samples across the lost CTCF binding sites, including a loop linking WDR66 promoter with distal regions.

    “Correspondingly, we observed multiple gained chromatin loops appeared in these AML samples across the lost CTCF binding sites, including a loop linking WDR66 promoter with distal regions.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    In four AML samples (424, 018, 546, 472), lost binding of CTCF was observed, with motif hypermethylated.

    “For example, we observed in four AML samples (424, 018, 546, and 472) a lost binding of CTCF, the motif of which was hypermethylated in those samples (Bar plot in Extended Data Fig. 11g).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Variable methylation levels were observed globally in AML samples, and methylation at CpG islands was significantly higher in AML compared with normal PBMC and HSPC.

    “We observed variable methylation levels globally in AML samples (Extended Data Fig. 11a) and significantly higher methylation at CpG islands in AML compared with normal PBMC and HSPC (Fig. 6a), consistent with previous findings46.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [46] Akalin, A. et al. Base-pair resolution DNA methylation sequencing reveals profoundly divergent epigenetic landscapes in acute myeloid leukemia. PLoS Genet. 8, e1002781 (2012). source

      Not checked in the free run.

  • May combine two claims.

    Findingown data

    220 genes located in AML-specific loops were significantly upregulated in AML and 88 were downregulated.

    “As shown in Fig. 2e, 220 genes were significantly upregulated in AML and 88 genes were downregulated.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    261 cancer-related genes had promoters located in neo-loop anchors, 44 of which were recurrent across different samples.

    “Overall, we found 261 cancer-related genes whose promoters were located in the neo-loop anchors, 44 of which were recurrent across different samples.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    MYCN formed a subtype-specific interaction with a cluster of co-occurring enhancers ~650Kb downstream, observed only in AML samples with TET2/FLT3-ITD, CEBPA or STAG2/KRAS/NRAS mutations.

    “For example, MYCN formed a subtype-specific interaction with a cluster of co-occurring enhancers ~650Kb downstream, and this loop was only observed in AML samples with TET2/FLT3-ITD, CEBPA or STAG2/KRAS/NRAS mutations (Fig. 2d).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Across all samples, 42.4% of AML-specific loops were between gene promoter and enhancers (P-E), and 11.2% were between promoter and silencers (P-S).

    “Across all samples, 42.4% of AML-specific loops were between gene promoter and enhancers (P-E), and 11.2% are between promoter and silencers (P-S) (Fig. 2g, Supplementary Table 2).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    5.7% of all neo-loops in this study lead to silencer hijacking, while 17.2% introduce enhancer-hijacking.

    “Overall, 5.7% of all the neo-loops in this study lead to silencer hijacking, while 17.2% of the neo-loops introduce enhancer-hijacking (Fig. 5e).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    A fusion between chr7 and chr11 was present in AML 270 but not in HSPC, and a neo-loop connected CDK5 gene to several enhancers on chr11.

    “A representative example was shown in Extended Data Fig. 9c, where there was a fusion between chr7 and chr11 in AML 270, but not in HSPC. This neo-loop connected the CDK5 gene (located on chr7) to several enhancers on chr11. In contrast, there were no such inter-chromosomal Hi-C signals in HSPC.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Loss of CTCF binding sites in AML samples was potentially due to hypermethylation, with an average of 76 such sites per AML sample.

    “We also identified the loss of CTCF binding sites in AML samples that were potentially due to hypermethylation (Extended Data Fig. 11d). There are in average 76 such sites in each AML samples.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    CBL gene was linked to enhancers on chr7 in sample 270, but in THP-1 cells, it was linked with enhancers on chr9.

    “Similarly, the CBL gene was linked to enhancers on chr7 in sample 270, but in THP-1 cells, it was linked with enhancers on chr9 (Extended Data Fig. 9d);”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    AML-specific loops showed subtype-specific patterns and contained known AML proto-oncogenes (MYCN, WT1, ERG, MEIS1, RUNX1) in loop anchors.

    “The AML-specific loops also showed subtype-specific patterns and contained many known AML proto-oncogenes (such as MYCN, WT1, ERG, MEIS1, and RUNX1) in the loop anchors (Fig. 2c).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    P-S loops account for 9.2% of all loops, with an average size of 169 kb.

    “P-S loops account for 9.2% of all loops, with an average size of 169 kb (Extended Data Fig. 6a and 6b).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    On average, each AML sample has 1.7 large deletions (>1 Mb), 2.1 inversions (>1 Mb), and 13.2 inter-chromosomal translocations.

    “On average, each AML sample has 1.7 large deletions (>1 Mb), 2.1 inversions (>1 Mb), and 13.2 inter-chromosomal translocations (Supplementary Table 5).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    MYC gene was looped with a cluster of enhancers on chr14 in sample 1360 due to t(8;14), but was looped with a different set of enhancers on chr11 in HL-60 cells due to t(8;11).

    “For example, the MYC gene (located on chr8) was looped with a cluster of enhancers on chr14 in sample 1360 due to t(8;14), but was looped with a different set of enhancers on chr11 in HL-60 cells due to t(8;11) (Fig. 4b).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Stable A compartments were slightly enriched for CpGi, while stable B compartment regions were depleted of CpGi.

    “The stable A compartments were slightly enriched for CpGi, while the stable B compartment regions were depleted of CpGi (Extended Data Fig. 13e).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    865 out of 905 (95.5%) TKD-induced A-to-B reversion regions overlapped with A-to-B revision regions induced by 5-AZA treatment, and overlap was 79% for B-to-A reversion regions.

    “865 out of the 905 (95.5%) TKD-induced A-to-B reversion regions overlapped with the A-to-B revision regions induced by 5-AZA treatment, and the overlap is 79% for the B-to-A reversion regions (Fig. 6f).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    HMA exposure partially restored chromatin structure, including reverting switched compartment and dissociating AML-specific loops.

    “We demonstrated that the HMA exposure partially restored the chromatin structure, including reverting switched compartment and dissociating AML-specific loops.”
    Quote in the paperSubstantial claim

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

    Findingown data

    In HSPC 213, there are no visible inter-chromosomal interactions at the region shown.

    “We also showed the inter-chromosomal Hi-C map in the HSPC 213, where there is no visible inter-chromosomal interactions.”
    Quote in the paperNot a substantial claim

  • The quote only partly states this claim.

    Findingown data

    5-AZA treatment at 0.5μM for 12 days slowed down cell proliferation.

    “The treatment slowed down the cell proliferation (Extended Data Fig. 12a).”
    Quote in the paperNot a substantial claim

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

    Findingown data

    The diamond-shaped region between the two dashed lines contained the interactions across the lost CTCF sites.

    “The diamond-shaped region between the two dashed lines resided the interactions across the lost CTCF sites.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    The loops at the MEIS1 and ERG gene regions are absent in all three PBMC samples and less frequent in the four HSPC samples.

    “The loops are absent in all three PBMC samples (only one is plotted) and less frequent in the four HSPC samples (only one is plotted).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    AML sample 018 has the same P-S loop for RTTN but with less interacting signals and almost no H3K27me3 signals at the loop anchor.

    “AML sample 018 has the same loop but with less interacting signals and almost no H3K27me3 signals at the loop anchor.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    After 5-AZA treatment, A/A and B/B interactions were decreased, while A/B interactions were increased.

    “the results showed that after 5-AZA treatment A/A and B/B interactions were decreased, while the interactions between A/B compartment were increased (Fig. 6i).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    CpGi originally in A compartment (A-to-A and A-to-B) were hypomethylated, while CpGi originally in B compartment (B-to-B and B-to-A) were hypermethylated.

    “We noticed that the CpGi originally in A compartment (A-to-A and A-to-B) were hypomethylated, while CpGi originally in B compartment (B-to-B and B-to-A) were hypermethylated.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    5-AZA treatment dramatically decreased DNA methylation levels for all four regions (CpGi, shores, shelves, open sea), with the biggest changes in open sea, as well as on CTCF binding motifs.

    “5-AZA treatment dramatically decreased DNA methylation levels for all four regions, with the biggest changes in open sea (Extended Data Fig. 13c), as well as on CTCF binding motifs (Extended Data Fig. 13d).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    Disruption of CTCF site might lead to loss of insulation and change the local chromatin interaction profile.

    “We observed increased interactions across the lost CTCF binding sites, suggesting that disruption of CTCF site might lead to loss of insulation and change the local chromatin interaction profile.”
    Quote in the paperSubstantial claim

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

    Study design and conductnone given

    SV calls from Delly and Speedseq were merged and only SVs detected by both methods were kept to reduce false positives.

    “SV calls from Delly and Speedseq were merged and only SVs detected by both methods were kept to reduce false positives.”
    Quote in the paperSubstantial claim

  • Classified as probably not a substantial claim.

    Study design and conductnone given

    Human blood samples were obtained with consent from AML patients or healthy donors under the Penn State Hershey IRB-approved protocol STUDY00005272.

    “Human blood samples were obtained with consent from AML patients or healthy donors under the Penn State Hershey IRB-approved protocol STUDY00005272.”
    Quote in the paperSubstantial claim

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

    Study design and conductown data

    The quantity and molecular size of the HiChIP library was confirmed by Qubit HS DNA assay and Tapestation 2200 system.

    “The quantity and molecular size of the library was confirmed by Qubit HS DNA assay (ThermoFisher) and Tapestation 2200 system(Agilent).”
    Quote in the paperNot a substantial claim

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

    Study design and conductnone given

    In two separate replicates of experiments, lentiviruses of sgRNA plasmids were transfected into Kasumi-1 cells.

    “In two separate replicates of experiments, lentiviruses of sgRNA plasmids were then transfected into 2 million of the treated Kasumi-1 cells with an MOI<0.3.”
    Quote in the paperNot a substantial claim

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

    Study design and conductnone given

    Detected SVs were compared against the DGV database to reduce germline SV calls.

    “Furthermore, the detected SVs were compared against the DGV database (version 2016-05-15) to reduce germline SV calls.”
    Quote in the paperNot a substantial claim

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

    Study design and conductnone given

    This final set of point mutations was used to confirm the clinical molecular diagnosis of AML samples, and both results were used for optimizing the subtyping of the AML samples.

    “This final set of point mutations was further used to confirm the clinical molecular diagnosis of our AML samples, and both results were used for optimizing the subtyping of the AML samples.”
    Quote in the paperNot a substantial claim

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

    Study design and conductown data

    The qPCR for RTTN RNA expression used CRISPR-processed Kasumi-1 cells without the deletion as the control.

    “The qPCR was performed for RTTN RNA expression with the primer listed in Supplementary Table 11, using CRPSIR-processed Kasumi-1 cells without the deletion as the control.”
    Quote in the paperNot a substantial claim

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

    Study design and conductnone given

    All insertions were verified by Sanger sequencing.

    “All insertions have been verified by Sanger sequencing.”
    Quote in the paperNot a substantial claim

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

    Study design and conductnone given

    Each cloning was verified by Sanger sequencing.

    “Each cloning was verified by Sanger sequencing.”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim.

    Study design and conductnone given

    Myeloblasts accounted for more than 90% of purified PBMC for most samples.

    “Myeloblasts accounted for more than 90% of purified PBMC for most samples”
    Quote in the paperSubstantial claim

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

    Study design and conductown data

    Two biological replications were applied to each of CRISPR interference, CRISPR deletion, and reporter assay with similar results.

    “Two biological replications were applied to each of CRISPR interference, CRISPR deletion, and reporter assay with similar results.”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim.

    Study design and conductreasoning

    Firefly luciferase activity was normalized to renilla luciferase to control for cell number and transfection efficiency.

    “To control for cell number and transfection efficiency, firefly luciferase activity was normalized to renilla luciferase.”
    Quote in the paperSubstantial claim

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

    Study design and conductreasoning

    Read alignments were shifted +4bp on the '+' strand and −5bp on the '-' strand to account for Tn5 insertion before peak calling.

    “Read alignments were shifted +4bp on “+” strand and −5bp on “-” strand to account for Tn5 insertion before peak calling.”
    Quote in the paperNot a substantial claim

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

    Study design and conductown data

    The Western Blots for yH2AX, Caspase-3, GAPDH, and β-actin under 5-AZA treatment were repeated twice with similar results.

    “The Western Blots for yH2AX, Caspase-3, GAPDH, and β-actin under 5-AZA treatment were repeated for twice with similar results (Extended Data Fig. 12c-d).”
    Quote in the paperNot a substantial claim

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

    Premisenone given

    Reads mapped to the same MboI restriction fragment are not informative to chromatin interactions and are removed for downstream analysis.

    “Reads mapped to the same MboI restriction fragment are not informative to chromatin interactions and thus are removed for downstream analysis.”
    Quote in the paperSubstantial claim

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

    Study design and conductnone given

    Each biological replicate contains three technical replicates.

    “Each biological replicate contains three technical replicates.”
    Quote in the paperNot a substantial claim

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

    Study design and conductown data

    The Western Blots for DNMT3A, DNMT3B, DNMT1, and GAPDH in the DNMT TKD experiment were repeated three times with similar results.

    “The Western Blots for DNMT3A, DNMT3B, DNMT1, and GAPDH in the DNMT TKD experiment were repeated for three times with similar results (Fig. 7d).”
    Quote in the paperNot a substantial claim

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

    Premisecitation

    Peakachu reports a probability score associated with each loop that demarcates confidence of loop calling and is positively associated with loop intensity.

    “Peakachu reports a probability score associated with each loop which demarcates confidence of loop calling. The probability score is also positively associated with the loop intensity, making it convenient for differential loop analysis.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [27] Salameh, T. J. et al. A supervised learning framework for chromatin loop detection in genome-wide contact maps. Nat. Commun. 11, 3428 (2020). source

      Not checked in the free run.

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

    Study design and conductreasoning

    Raw stripe calls were filtered to retain stripes larger than 300kb to ensure specificity and avoid calls less distinguishable from loop clusters or background noise.

    “To ensure specificity, we filter raw calls to retain stripes larger than 300kb (eight of 40Kb binds) to avoid calls that are less distinguishable from loop clusters or background noise.”
    Quote in the paperNot a substantial claim

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

    Study design and conductnone given

    A minimal quality score of 20 was used to reduce false positive point mutation calls.

    “and minimal quality score of 20 was used to reduce false positive calls.”
    Quote in the paperNot a substantial claim

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

    Study design and conductreasoning

    Point mutations that overlap with dbSNP150 mutations were removed to minimize the number of germline calls.

    “To minimize the number of germline calls, point mutations that overlap with dbSNP150 mutations were removed.”
    Quote in the paperNot a substantial claim

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

    Study design and conductnone given

    Measurements were taken in triplicates.

    “measurements were taken in triplicates.”
    Quote in the paperNot a substantial claim

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

    Premisecitation

    Peakachu detects loops as CTCF or H3K27ac loops depending on a pre-trained CTCF model or H3K27ac model.

    “Peakachu detects loops as CTCF or H3K27ac loops depending on a pre-trained CTCF model or H3K27ac model.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [27] Salameh, T. J. et al. A supervised learning framework for chromatin loop detection in genome-wide contact maps. Nat. Commun. 11, 3428 (2020). source

      Not checked in the free run.

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

    Study design and conductown data

    The CRISPRi screening library passed QC by parameters: skew ratio = 2.53, perfect match = 91.5%, undetected guides = 0, and coverage = ~5,500 per sgRNA.

    “The library passed QC by the following parameters: skew ratio = 2.53 (criterial <10); perfect match = 91.5 (>70%); undetected guides = 0 (<0.5%); Coverage = ~5,500 per sgRNA (>100).”
    Quote in the paperNot a substantial claim

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

    Study design and conductnone given

    Peaks in the ENCODE hg38 blacklist regions were filtered out.

    “Peaks in the ENCODE hg38 blacklist regions (http://mitra.stanford.edu/kundaje/akundaje/release/blacklists/hg38-human/hg38.blacklist.bed.gz) were filtered out.”
    Quote in the paperNot a substantial claim

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

    Study design and conductnone given

    Cell proliferation was independently performed with three technical replicates every 24 hours.

    “Cell proliferation was independently performed with three technical replicates every 24 hours.”
    Quote in the paperNot a substantial claim

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

    Study design and conductreasoning

    Loops were first detected using both the CTCF and H3K27ac models, and only those detected by both models were reported to reduce false positive loop calls.

    “In this work, loops were first detected using both models and only those detected by both models were reported to reduce the false positive loop calls.”
    Quote in the paperSubstantial claim
58 of 58

  • The paper relies on this without support.

    AML is characterized by a global aberrant DNA methylation profile.

    “AML is also characterized by a global aberrant DNA methylation profile.”

  • The paper relies on this without support.

    Genome-wide enhancer hijacking in AML has not been studied.

    “However, genome-wide enhancer hijacking in AML have not been studied.”
2 of 2

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?

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.

  • 5-AZA treatment concentration differs by one million-fold

    Minor

    The results describe a 12-day treatment at 0.5 μM, but two Methods sections specify 0.5 M for the same treatment. These concentrations differ by a factor of one million, leaving the reported treatment dose inconsistent.

  • Enhancer-validation result names a different histone mark from its figure caption

    Minor

    The results call the depleted signal H3K37ac, whereas the caption for the cited validation data identifies H3K27ac at the targeted loci.

2 of 2

Cites

Flagged first, then by citations

Research works printed but not linked: 1

  1. Spracklin, G. et al. Heterochromatin diversity modulates genome compartmentalization and loop extrusion barriers. Preprint at bioRxiv https://doi.org/10.1101/2021.08.05.455340 (2021). · DOI

Cited by

Sorted by citations

Related

Authors
Xu, J., Song, F., Lyu, H., Kobayashi, M., Zhang, B., Zhao, Z., Hou, Y., Wang, X., Luan, Y., Jia, B., Stasiak, L., Wong, J. H.-Y., Wang, Q., Jin, Q., Jin, Q., Fu, Y., Yang, H., Hardison, R. C., Dovat, S., Platanias, L. C., Diao, Y., Yang, Y., Yamada, T., Viny, A. D., Levine, R. L., Claxton, D., Broach, J. R., Zheng, H., & Yue, F.
Affiliations
  • Northwestern University (US)
  • Penn State Cancer Institute (US)
  • Pennsylvania State University (US)
  • Duke University (US)
  • Columbia University Irving Medical Center (US)
  • Memorial Sloan Kettering Cancer Center (US)
Corresponding author
  • Zheng, Hong
  • Yue, Feng
ORCID
Venue
Nature
Publisher
Springer Science and Business Media LLC
Published
2022-10-26
Volume
611
Issue
7935
Pages
387-398
ISSN
  • 0028-0836 (ISSN-L)
  • 1476-4687
DOI
10.1038/s41586-022-05365-x
PMC
PMC10060167
OpenAlex
W4307659092
Semantic Scholar
132618fe8c2b6c087d9eae40298458947550ac3b
Cited by
208
References
78
Citations by year
2026: 38 · 2025: 61 · 2024: 68 · 2023: 40 · 2022: 1
FWCI
31.82
Type
journal article
Language
en
Open access
Licence
Locations
  • Nature (publishedVersion)
  • PubMed (publishedVersion)
  • PubMed Central (acceptedVersion)
  • RePEc: Research Papers in Economics (submittedVersion)
Funders
  • Damon Runyon Cancer Research Foundation (DRG 117-15, P30 CA008748)
  • Memorial Sloan-Kettering Cancer Center (CA008748, P30 CA008748, NIH P30CA008748)
  • Edward P. Evans Foundation
  • National Institutes of Health (CA008748, NIH P30 CA008748)
  • National Institute on Drug Abuse (U01DA053691)
  • National Human Genome Research Institute (R01HG011207, R01HG009906)
  • National Cancer Institute (NIH P30 CA008748, R01CA213912, CA008748, U54CA143869, U01CA200060, R01CA216421, K08CA215317, P30CA008748, R35CA197594, P30CA014236)
  • National Institute of General Medical Sciences (R35GM124820)
  • National Institute of Diabetes and Digestive and Kidney Diseases (R24DK106766)
Topics
  • Health Sciences › Medicine › Hematology › Acute Myeloid Leukemia Research
  • 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
Keywords
acute myeloid leukemia, 3D genome organization, chromatin looping, A/B compartments, topologically associating domains, enhancer hijacking, CTCF binding, DNA methylation, DNMT1, DNMT3A, DNMT3B, hypomethylating agents, leukemia subtypes, CRISPR interference, CRISPR screen, cancer therapy targets
MeSH terms
CCCTC-Binding Factor, Chromatin, DNA (Cytosine-5-)-Methyltransferases, Enhancer Elements, Genetic, Humans, Promoter Regions, Genetic, Sulfites, Reproducibility of Results, Leukemia, Myeloid, Acute, Genome, Human, Gene Expression Regulation, Leukemic, Sequence Analysis, DNA Methylation, Gene Silencing, CRISPR-Cas Systems

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.

What counts as a claim ›How we measure confidence ›How we check this ›

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