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Cited by 775Epigenetics and DNA MethylationGenomics and Chromatin DynamicsCancer-related gene regulation

Intragenic DNA methylation prevents spurious transcription initiation

Open access
Neri, Francesco, Rapelli, Stefania, Krepelova, Anna, Incarnato, Danny, Parlato, Caterina, Basile, Giulia, Maldotti, Mara, Anselmi, Francesca +1 more·2017·Nature
DOI 10.1038/nature21373
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Abstract

In mammals, DNA methylation occurs mainly at CpG dinucleotides. Methylation of the promoter suppresses gene expression, but the functional role of gene-body DNA methylation in highly expressed genes has yet to be clarified. Here we show that, in mouse embryonic stem cells, Dnmt3b-dependent intragenic DNA methylation protects the gene body from spurious RNA polymerase II entry and cryptic transcription initiation. Using different genome-wide approaches, we demonstrate that this Dnmt3b function is dependent on its enzymatic activity and recruitment to the gene body by H3K36me3. Furthermore, the spurious transcripts can either be degraded by the RNA exosome complex or capped, polyadenylated, and delivered to the ribosome to produce aberrant proteins. Elongating RNA polymerase II therefore triggers an epigenetic crosstalk mechanism that involves SetD2, H3K36me3, Dnmt3b and DNA methylation to ensure the fidelity of gene transcription initiation, with implications for intragenic hypomethylation in cancer.

AI snapshot

Written from the paper’s full text.

TL;DR

Dnmt3b-mediated DNA methylation within gene bodies, recruited via H3K36me3, suppresses spurious intragenic RNA polymerase II entry and cryptic transcription initiation in mouse embryonic stem cells. The resulting aberrant transcripts can be degraded by the RNA exosome or capped, polyadenylated, exported, and ribosome-associated, with possible relevance to intragenic hypomethylation in cancer.

Key findings

  • Dnmt3b knockout reduced gene-body 5-methylcytosine, especially on exons and introns, and increased intragenic transcription: 1,445 genes (18% of genes with RPKM > 1) showed an intermediate-exon/first-exon log2 ratio above 1.
  • DECAP-seq identified 2,627 high-expression intragenic transcription start sites (RPM > 6) specific to Dnmt3b−/− cells, versus 780 shared and 936 WT-specific sites; Dnmt3b−/−-specific sites accounted for 2.76% of mapped reads, while shared sites accounted for 5.22%.
  • SetD2 silencing reduced H3K36me3 and Dnmt3b gene-body binding and produced 3,560 high-confidence intragenic TSSs, of which 2,759 were specific to the knockdown and more than 2,000 overlapped with Dnmt3b−/− sites.
  • Rescue experiments showed that wild-type Dnmt3b, but not the catalytically inactive V725G mutant or H3K36me3-binding mutants S277P and VW-RR, reduced the intermediate/first-exon ratio; cryptic RNAs were partly degraded by the RNA exosome, partly polyadenylated, and showed increased ribosome occupancy in intronic regions.

Methods

  • Mouse E14 embryonic stem cells were used, with two independent Dnmt3b knockout lines generated by TALEN targeting of exon 17; SetD2, Dis3, and Rrp6 were silenced by shRNA, and Dnmt3b rescue used wild-type or mutant constructs.
  • Genome-wide assays included ChIP-seq for Dnmt3b, RNA Pol II, H3K36me3, and other marks; whole-genome bisulfite sequencing; total, polyA+, and cytoplasmic RNA-seq; CAPIP-seq; DECAP-seq for single-base TSS mapping; and ART-seq ribosome profiling.
  • Analyses mapped reads to an E14-specific mm9 variant and used TopHat/Cuffdiff, Bowtie, BSMAP, MACS, and custom scripts, with significance assessed by Wilcoxon rank-sum, chi-square, hypergeometric, and correlation tests.

Limitations

  • The paper has no dedicated limitations section; it notes that alternative splicing and repetitive-element activation events in Dnmt3b−/− cells were sporadic, with only 4 of 2,563 genes showing intragenic alternative-promoter reactivation.
  • The cancer relevance is discussed as an implication rather than tested directly, and the production of aberrant proteins from cryptic transcripts is suggested by ribosome profiling rather than demonstrated.
  • Evident from the text: the work is restricted to mouse ESCs and relies heavily on knockout/knockdown and overexpression systems, so the findings may not generalize to other cell types or in vivo tumor settings.

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

Integrity signals

Registry

Crossref · 10.1038/nature21373

Cites

55 of 55 references verified0 citation errors · 0 not checkable

Statistics

Nothing to recompute.

Claims the paper makes

0 of 14 cited sources checked · 0 supported · 0 flagged

  • Findingown data

    In DRB-treated cells, Dnmt3b-/- cells show a significant increase of RNA Pol II binding on intragenic regions compared with WT, especially on introns and exons of q3-q4 genes, using both pan and Ser5 antibodies.

    “In contrast, we observed a significant increase of RNA Pol II binding on intragenic regions when comparing Dnmt3b -/-to WT cells treated with DRB.This was especially true on introns and exons of q3-q4 genes using both antibodies (Fig. 2c, d and Extended Data Fig. 4f)”
    Quote in the paperSubstantial claim
  • Findingown data

    Dnmt3b-/- cells have a significantly higher ratio of intermediate exon RPKM to first exon RPKM starting from the second exon.

    “Dnmt3b -/- cells displayed a significantly higher ratio starting from the second exon (Fig. 2a).”
    Quote in the paperSubstantial claim
  • Findingown data

    Untreated Dnmt3b-/- cells show no significant changes in H3K36me3 and RNA Pol II genomic profiles compared with WT cells.

    “We did not observe any significant changes in H3K36me3 and RNA Pol II genomic profiles when comparing untreated WT to Dnmt3b -/-cells (Fig. 2c, d and Extended Data Fig. 4d-f).”
    Quote in the paperSubstantial claim
  • Findingown data

    In Dnmt3b-/- cells, 1445 genes, representing 18% of genes with RPKM greater than 1, had a log2 ratio of intermediate exons versus first exon greater than 1.

    “1445 genes (18% of total genes having RPKM > 1) had a log2 ratio of intermediate exons versus first exon > 1 (Fig. 2b).”
    Quote in the paperSubstantial claim
  • Findingown data

    Dnmt3b loss does not cause general genome-wide reactivation of alternative promoters.

    “Even though some alternative promoters' reactivation events take place in Dnmt3b -/-cells, the Dnmt3b loss did not show evidence of a general reactivation of these events at genome-wide level (Extended Data Fig. 3).”
    Quote in the paperSubstantial claim
  • May combine two claims. 32
  • Classified as probably not a substantial claim. 11
  • The quote only partly states this claim. 1

  • May combine two claims.

    Findingown data

    DECAP-seq identified 2627 highly expressed TSSs with RPM greater than 6 specific to Dnmt3b-/- cells, 780 TSSs common to both cell lines, and 936 TSSs specific to WT cells.

    “This analysis identified 2627 highly expressed TSSs (RPM > 6) specific of the Dnmt3b -/-cells, 780 TSSs common in both the cell lines and 936 specific of WT cells (Fig. 3c).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Endogenous Dnmt3b binds intragenically, especially on genes in the third and fourth expression quartiles, and this binding correlates with H3K36me3.

    “ChIP-seq analysis revealed the intragenic binding of the endogenous Dnmt3b especially on the genes belonging to the third and fourth quartiles (q3-q4) of gene expression and correlated with H3K36me3 histone modification (Fig. 1a-c and Extended Data Fig 1g-q).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Dnmt3b knockout in mouse ESCs causes a global reduction of genomic DNA methylation, with a significant decrease of 5mC on exons and introns.

    “Whole genome bisulfite sequencing (WGBS) in WT or Dnmt3b -/-ESCs revealed a global reduction of genomic DNA methylation, with a significant decrease of the level of 5methylcytosine (5mC) on exons and introns (Fig. 1d and Extended Data Fig. 1r).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Dnmt3b binding and DNA methylation loss in Dnmt3b-/- cells correlate with H3K36me3 abundance.

    “Genome splitting in deciles of H3K36me3 occupancy showed that both Dnmt3b binding and DNA methylation loss in Dnmt3b -/-cells markedly correlate with the abundance of H3K36me3 (Fig. 1e).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Intragenic TSSs in Dnmt3b-/- cells show loss of pyrimidine enrichment at position -1 and reduced purine enrichment at position +1 compared with canonical TSSs.

    “Our data confirm this observation on canonical TSSs, while on intragenic TSSs we observed the loss of the pyrimidine enrichment at position -1 and a reduced enrichment of the purine at position +1 (Fig. 3f).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Intragenic TSSs identified in Dnmt3b-/- cells lie in regions with higher H3K36me3 and Dnmt3b binding, loss of methylation upon Dnmt3b depletion, and lower nucleosome occupancy than random intragenic regions.

    “The intragenic TSSs identified in Dnmt3b -/-cells were within genomic regions with significantly higher H3K36me3 and Dnmt3b binding, showed loss of methylation upon Dnmt3b depletion, and lower nucleosome occupancy with respect to randomly chosen intragenic regions (Fig. 3e).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Almost all highly expressed TSSs identified in Dnmt3b-/- cells are present in PolyA+ and cytosolic DECAP-seq fractions, but at significantly lower expression levels.

    “Interestingly, almost all the highly expressed TSSs identified in Dnmt3b -/-cells can be found in the PolyA+ and cytosolic DECAP-seq fractions (Fig. 5c), even though, with a significantly lower level of expression with consequent reduction of the percentage of the total mapped reads (Fig. 5d, e)”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    mRNAs with fast decay are mainly linked to cell cycle and transcription, whereas slow-decay mRNAs are more linked to metabolism and translation.

    “The analysis revealed that fast decay mRNAs are mainly involved in cell cycle and transcription biological processes, while slow decay mRNAs are more related to metabolism and translation.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    WT and catalytically inactive V725G Dnmt3b proteins show similar intragenic binding profiles, but only WT increases DNA methylation levels.

    “Both the Dnmt3b proteins showed similar intragenic binding profiles (Fig. 4g, h and Extended Data Fig. 9b, c), although only the cells expressing the WT protein showed an increase of DNA methylation levels (Extended Data Fig. 9d, e).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    In Dnmt3b-/- cells, few events of alternative splicing and activation of repetitive elements occur, and these regulations were sporadic in the model.

    “We tested these hypotheses and, in agreement with the literature, we found few events of alternative splicing and activation of repetitive elements actually occurring in Dnmt3b -/-cells (data not shown). However, these regulations were sporadic in our model.”
    Quote in the paperNot a substantial claim0 of 3 sources checked
    • cites [32] Maunakea, A. K., Chepelev, I., Cui, K. & Zhao, K. Intragenic DNA methylation modulates alternative splicing by recruiting MeCP2 to promote exon recognition. Cell Res. 23, 1256–1269 (2013) source

      Not checked in the free run.

    • cites [33] Yearim, A. et al. HP1 is involved in regulating the global impact of DNA methylation on alternative splicing. Cell Reports 10, 1122–1134 (2015) source

      Not checked in the free run.

    • cites [34] Jones, P. A. Functions of DNA methylation: islands, start sites, gene bodies and beyond. Nat. Rev. Genet. 13, 484–492 (2012) source

      Not checked in the free run.

  • May combine two claims.

    Findingown data

    SetD2-silenced cells have 3560 high-confidence intragenic TSSs with RPM greater than 6, of which 2759 are specific to SetD2 knockdown and more than 2000 are shared with Dnmt3b-/- TSSs.

    “This analysis revealed 3560 high confident (RPM > 6) intragenic TSSs in SetD2 silenced cells, of which 2759 were specific of the SetD2 knockdown and more than 2000 were in common with the TSSs identified in Dnmt3b -/-cells (Extended Data Fig. 8l-o and 9a).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The percentage of total mapped reads specific for Dnmt3b-/- TSSs was 2.76%, while the percentage for common TSSs was 5.22%.

    “The percentage of the total mapped reads specific for Dnmt3b -/-TSSs was 2.76%, while the percentage of common TSSs was 5.22%”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    SetD2 knockdown drastically reduces H3K36me3 in both WT and Dnmt3b-/- cells and causes loss of Dnmt3b intragenic binding in WT cells.

    “SetD2 knockdown resulted in a drastic reduction of H3K36me3 both in WT and Dnmt3b -/-cells and loss of Dnmt3b intragenic binding in WT cells (Fig. 4a-c and Extended Data Fig. 8a-f).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Both Dnmt3b mutants show impaired Dnmt3b binding and DNA methylation relative to rescue with the WT enzyme.

    “Specific impairment of Dnmt3b binding and DNA methylation is observed in both the mutants compared to the rescue by using WT Dnmt3b enzyme.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Dnmt3b WT, but not the V725G mutant, significantly raises global DNA 5mC (p = 0.003) according to dot-blot quantification from three biological replicates.

    “Dot intensity quantification from three biological replicates revealed that Dnmt3b WT (but not the V725G mutant) significantly (p=0.003)increases global DNA 5mC.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Total RNA-seq showed a significant reduction of the intermediates/first exon ratio in cells expressing WT Dnmt3b, but not in those expressing the Dnmt3b V725G mutant.

    “Total RNA-seq (Extended Data Fig. 9f, g) revealed a significant reduction of the intermediates/first exon ratio in the cells expressing the WT Dnmt3b enzyme, but not in those expressing Dnmt3bV725G mutant (Fig. 4i, j).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Dnmt3b mutants unable to bind H3K36me3, S277P and VW-RR, show lower intragenic binding, reduced DNA methylation activity, and fail to reduce the intermediates/first exon ratio.

    “These Dnmt3b mutants showed lower intragenic binding with reduced DNA methylation activity (Extended Data Fig. 9i, j) and were unable to reduce the intermediates/first exon ratio (Extended Data Fig. 10a-d).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Silencing Dis3 and Rrp6 components of the RNA exosome complex does not change the total number of intragenic TSSs but significantly increases their expression level.

    “Silencing of the Dis3 and Rrp6 component of the RNA exosome complex did not show changes in the total number of intragenic TSSs (Extended Data Fig. 10e-g), but a significant increase of their expression level (Extended Data Fig. 10h-k).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationown data

    DNA methylation within gene bodies that depends on Dnmt3b protects the gene body against spurious entry by RNA Pol II and against cryptic transcription initiation.

    “Here, we show that the Dnmt3b-dependent intragenic DNA methylation protects the gene body from RNA Polymerase II spurious entry and cryptic transcription initiation.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    Spurious transcripts can either be degraded by the RNA exosome complex or be capped, polyadenylated, and delivered to ribosomes to produce aberrant proteins.

    “Furthermore, the spurious transcripts can be either degraded by the RNA exosome complex or capped, polyadenylated, and delivered to the ribosome to produce aberrant proteins.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    The ability of Dnmt3b to prevent spurious transcription requires both its catalytic activity and its recruitment to gene bodies by H3K36me3.

    “Using different genome-wide approaches, we demonstrate that this Dnmt3b function is dependent on its enzymatic activity and recruitment on the gene body by H3K36me3.”
    Quote in the paperSubstantial claim

  • Classified as probably not a substantial claim.

    Premisecitation

    Dnmt3b preferentially binds to gene bodies through interaction with the histone modification H3K36me3.

    “Recent studies have reported that Dnmt3b binds preferentially to the gene bodies by interacting with the histone modification H3K36me3 10,11 .”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites [10] Baubec, T. et al. Genomic profiling of DNA methyltransferases reveals a role for DNMT3B in genic methylation. Nature 520, 243–247 (2015) source

      Not checked in the free run.

    • cites [11] Morselli, M. et al. In vivo targeting of de novo DNA methylation by histone modifications in yeast and mouse. eLife 4, e06205 (2015) source

      Not checked in the free run.

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

    Premisecitation

    Global DNA hypomethylation, especially on intragenic regions, is a general feature of most tumours.

    “Indeed, the global DNA hypomethylation, especially occurring on intragenic regions, is a general feature of most tumours [35][36][37] .”
    Quote in the paperNot a substantial claim0 of 3 sources checked
    • cites [35] Jones, P. A. & Baylin, S. B. The fundamental role of epigenetic events in cancer. Nat. Rev. Genet. 3, 415–428 (2002) source

      Not checked in the free run.

    • cites [36] Gaudet, F. et al. Induction of tumors in mice by genomic hypomethylation. Science 300, 489–492 (2003) source

      Not checked in the free run.

    • cites [37] Feinberg, A. P. & Vogelstein, B. Hypomethylation distinguishes genes of some human cancers from their normal counterparts. Nature 301, 89–92 (1983) source

      Not checked in the free run.

  • May combine two claims.

    Interpretationown data

    A fraction of spurious transcripts is degraded, and not all cryptic transcripts undergo polyadenylation and nuclear export.

    “confirming that a fraction of the spurious transcripts is actually degraded and indicating that not all cryptic transcripts undergo polyadenylation and nuclear export.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationreasoning

    Partial internal RNAs generated by loss of transcription initiation fidelity could impact multiple biological processes and favour tumour cell heterogeneity and neoplastic predisposition.

    “Partial internal RNAs generated by the loss of transcription initiation fidelity, could impact on multiple biological processes impairing molecular mechanisms such as regulation of the gene expression, miRNA targeting, truncated proteins generation and others, thus favouring (stochastic) tumour cell heterogeneity and neoplastic predisposition.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Study design and conductown data

    The intragenic transcription initiation result was confirmed on technical and biological replicates and by RT-qPCR on a subset of genes.

    “Analysis performed on technical and biological replicates, and validation by RT-qPCR on a subset of genes confirmed this result (Extended Data Fig. 2c-e).”
    Quote in the paperSubstantial claim

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

    Study design and conductown data

    Comparative methylation analyses used only CpGs covered at least 5x in both WT and Dnmt3b-/- cells.

    “Methylation calling was performed using the methratio.pyscript provided with the BSMAP tool and comparative analyses were performed by using only CpG covered at least 5x in both wt and Dnmt3b -/-cells.”
    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 conductown data

    Both RppH-treated and untreated samples had a double peak near 130 bp from adapter dimers, but only the RppH-treated sample showed the higher enrichment corresponding to decapped RNA fragments.

    “RppH treated and untreated samples showed a double peak around 130bp corresponding to the dimers of adapter, but only the RppH treated sample showed a higher enrichment (in the red box) corresponding to the decapped RNA fragments.”
    Quote in the paperNot a substantial claim

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

    Premisecitation

    Loss or mutation of SETD2 and the consequent loss of H3K36me3 is a key event in promoting cancer growth and malignancy.

    “Moreover, several recent studies reported the loss/mutation of SETD2 and the consequent loss of H3K36me3 histone mark as a key event in promoting cancer growth and malignancy [38][39][40] .”
    Quote in the paperNot a substantial claim0 of 3 sources checked
    • cites [38] Kanu, N. et al. SETD2 loss-of-function promotes renal cancer branched evolution through replication stress and impaired DNA repair. Oncogene 34, 5699–5708 (2015) source

      Not checked in the free run.

    • cites [39] Fontebasso, A. M. et al. Mutations in SETD2 and genes affecting histone H3K36 methylation target hemispheric high-grade gliomas. Acta Neuropathol. 125, 659–669 (2013) source

      Not checked in the free run.

    • cites [40] Duns, G. et al. Histone methyltransferase gene SETD2 is a novel tumor suppressor gene in clear cell renal cell carcinoma. Cancer Res. 70, 4287–4291 (2010) source

      Not checked in the free run.

  • May combine two claims.

    Interpretationown data

    Only reexpression of WT Dnmt3b rescues the loss of spurious intragenic transcription initiation in Dnmt3b-/- cells, demonstrating that Dnmt3b-dependent DNA methylation prevents cryptic intragenic transcription.

    “Thus, only the reexpression of the WT Dnmt3b was able to rescue the loss of spurious intragenic transcription initiation in Dnmt3b -/-cells demonstrating that Dnmt3bdependent DNA methylation is responsible for preventing the occurrence of cryptic intragenic transcription.”
    Quote in the paperSubstantial claim

  • Classified as probably not a substantial claim.

    Study design and conductown data

    Two independent Dnmt3b knockout ESC lines were generated to exclude experimental artefacts from different genetic backgrounds or prolonged culturing.

    “we generated two independent Dnmt3b knockout cell lines from mouse embryonic stem cell (ESC) line E14 to exclude experimental artefacts due to different genetic backgrounds, or prolonged cell culturing (Extended Data Fig. 1a-f).”
    Quote in the paperSubstantial claim

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

    Study design and conductcitation

    The gene ontology result matches earlier observations in mouse embryonic stem cells, supporting the validity of the experiment.

    “This result is in agreement with that previously observed in mESCs 54,55 , thus suggesting the bona fide of the experiment.”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [54] Sharova, L. V. et al. Database for mRNA half-life of 19 977 genes obtained by DNA microarray analysis of pluripotent and differentiating mouse embryonic stem cells. DNA Res. 16, 45–58 (2009) source

      Not checked in the free run.

    • cites [55] Schwanhäusser, B. et al. Global quantification of mammalian gene expression control. Nature 473, 337–342 (2011) source

      Not checked in the free run.

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

    Study design and conductnone given

    The cells used were mycoplasma free.

    “The cells were mycoplasma free.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims. The quote only partly states this claim.

    Study design and conductown data

    The ChIP-seq results were confirmed by ChIP-qPCR on selected genomic regions and by co-immunoprecipitation of Dnmt3b with H3K36me3 in wild-type and Dnmt3b -/- embryonic stem cells.

    “The ChIP-seq data were validated by ChIP-qPCR, using several biological replicates, on target genomic regions and by not cross-linked co-immunoprecipitation experiments between Dnmt3b and H3K36me3 in WT or Dnmt3b -/-ESCs (Extended Data Fig.1o, p).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationown data

    Dnmt3b-dependent DNA methylation on the gene body prevents aberrant transcription initiation events and is necessary to guarantee mRNA transcription initiation fidelity.

    “We now demonstrate that Dnmt3b-dependent DNA methylation on the gene body is responsible for the prevention of aberrant transcription initiation events necessary to guarantee the mRNA transcription initiation fidelity (Extended Data Fig. 12a).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    Mock embryonic stem cells correlated more strongly with Dnmt3b mutant-transfected cells (r = 0.99) than with Dnmt3b WT-transfected cells (r = 0.95), suggesting that DNA methylation activity is the main driver of transcriptome changes caused by Dnmt3b.

    “Of note, Mock ESCs showed higher correlation with Dnmt3b mutant transfected ESCs (r = 0.99) with respect that Dnmt3b WT transfected ESCs (r = 0.95), thus suggesting that DNA methylation enzymatic activity is the major driver of the Dnmt3b-dependent transcriptome alterations.”
    Quote in the paperSubstantial claim

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

    Study design and conductown data

    DECAP-seq replicates showed high overlap in the number of intragenic TSSs and read distribution.

    “The number of intragenic TSSs and the reads distribution was similar in both the DECAP-seq replicates showing high overlap (Extended Data Fig. 6k-m)”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Study design and conductown data

    The Dnmt3b ChIP-seq antibody immunoprecipitated endogenous Dnmt3b cross-linked to chromatin and showed no background signal in Dnmt3b-/- cells.

    “To investigate the distribution of the endogenous Dnmt3b we tested different antibodies and found one that was able to immunoprecipitate the endogenous Dnmt3b cross-linked to chromatin, which showed no background signal in Dnmt3b -/-(Extended Data Fig. 1g-i”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Study design and conductown data

    DECAP-seq is cap- and strand-specific, as indicated by a sharp TSS peak appearing only on the relevant DNA strand.

    “A pronounced sharp peak (red arrow) is present on the TSS only on the respective gene strand, thus reflecting both the cap-and strandnessspecificity of the method.”
    Quote in the paperSubstantial claim

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

    Premisecitation

    RppH in Thermopol buffer can remove the 5' pyrophosphate and cap from RNA, leaving a 5' monophosphate.

    “in Thermopol buffer is able to mediate decapping and pyrophosphate removal from the 5´ end of RNA to leave a 5´ monophosphate RNA (5'-P).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites S1

      Not checked in the free run.

40 of 40

  • The paper relies on this without support.

    The function of gene body DNA methylation in highly expressed genes has not yet been clarified.

    “while the functional role of gene body DNA methylation in highly expressed genes has yet to be clarified.”
    “the function of gene body DNA methylation has not yet been clarified.”

  • The paper relies on this without support.

    Silencing SetD2 in mammalian cells causes cryptic transcription initiation in a large fraction of active genes.

    “Silencing of SetD2 in mammalian cells leads to cryptic transcription initiation of a large fraction of active genes.”

  • The paper relies on this without support.

    Silencing Dis3 and Rrp6 impairs RNA exosome degradation of cryptic transcripts.

    “Silencing of the Dis3 and Rrp6 component of the RNA exosome complex did not show changes in the total number of intragenic TSSs (Extended Data Fig. 10e-g), but a significant increase of their expression level (Extended Data Fig. 10h-k).”
3 of 3

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?

  • Loss of DNMT3B in mouse embryonic stem cells results in spurious initiation of alternative promoters.

    “loss of DNA methyltransferase 3B (DNMT3B) in mouse embryonic stem (ES) cells was shown to result in spurious initiation of alternative promoters21.”

    From Alternative promoters in CpG depleted regions are prevalently associated with epigenetic misregulation of liver cancer transcriptomes (2023) · cites this paper as [21]

    The Analysis of intragenic spurious transcription section reports increased cryptic initiation within gene bodies in Dnmt3b-knockout embryonic stem cells. It also says some alternative promoters were reactivated, but found no general reactivation; Extended Data Figure 3 identifies only 4 of 2,563 genes with such reactivation. The claim conflates the broader spurious-initiation finding with the much narrower alternative-promoter finding. (LLM, conf=0.87)

    Experimental — accuracy not yet measured

  • SETD2 deposits H3K36me3 after Pol2 passage, which in turn recruits DNMT3B to maintain a repressive chromatin state.

    “The methyltransferase SETD2 deposits H3K36me355 after Pol2 passage56, which in turn recruits DNMT3B21 to maintain a repressive chromatin state57.”

    From Alternative promoters in CpG depleted regions are prevalently associated with epigenetic misregulation of liver cancer transcriptomes (2023) · cites this paper as [21]

  • The gene body coverage of H3K36me3 coincides with the DNMT3B coverage.

    “As H3K36me3 recruits DNMT3B21, the gene body coverage of H3K36me3 (Supplementary Fig. 6b) coincides with the DNMT3B coverage”

    From Alternative promoters in CpG depleted regions are prevalently associated with epigenetic misregulation of liver cancer transcriptomes (2023) · cites this paper as [21]

  • Genome-wide perturbation strategies are associated with pleiotropic phenotypes and inevitably confound interpretation of results.

    “Although discoveries regarding 5mC function and regulation have been made with genome-wide perturbation strategies, these methods are associated with pleiotropic phenotypes and inevitably confound the interpretation of results (35).”

    From Dynamic DNA methylation: In the right place at the right time (2018) · cites this paper as [35]

  • Genes in the A compartment had lower methylation at TSS but higher methylation at gene bodies than genes in the B compartment.

    “We observed that genes in the A compartment had lower methylation at transcription starting site (TSS) but higher methylation at gene bodies than genes in the B compartment (Extended Data Fig. 11b), consistent with the distinct roles of DNA methylations at different regions47.”

    From Subtype-specific 3D genome alteration in acute myeloid leukaemia (2022) · cites this paper as [47]

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Authors
Neri, F., Rapelli, S., Krepelova, A., Incarnato, D., Parlato, C., Basile, G., Maldotti, M., Anselmi, F., & Oliviero, S.
Affiliations
  • Italian institute for Genomic Medicine (IT)
  • University of Turin (IT)
Corresponding author
  • Neri, Francesco
  • Oliviero, Salvatore
ORCID
Venue
Nature
Publisher
Springer Science and Business Media LLC
Published
2017-02-22
Volume
543
Issue
7643
Pages
72-77
ISSN
  • 0028-0836 (ISSN-L)
  • 1476-4687
DOI
10.1038/nature21373
OpenAlex
W2589386142
Semantic Scholar
1e92f02718723d76aae589d66f60a1d9a01188d5
Cited by
739
References
55
Citations by year
2026: 54 · 2025: 53 · 2024: 79 · 2023: 92 · 2022: 97 · 2021: 92 · 2020: 75 · 2019: 89 · 2018: 63 · 2017: 43 · 2012: 1
FWCI
30.15
Type
journal article
Language
en
Open access
Licence
Locations
  • Nature (publishedVersion)
  • PubMed (publishedVersion)
  • Institutional Research Information System University of Turin (University of Turin) (submittedVersion, other-oa)
  • RePEc: Research Papers in Economics (submittedVersion)
  • University of Groningen research database (University of Groningen / Centre for Information Technology) (submittedVersion)
Funders
  • Associazione Italiana per la Ricerca sul Cancro (IG-2014)
  • Institute of Molecular and Cell Biology
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Epigenetics and DNA Methylation
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Genomics and Chromatin Dynamics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Cancer-related gene regulation
Keywords
gene body methylation, DNMT3B, RNA polymerase II, H3K36me3, SETD2, RNA exosome, epigenetic crosstalk, mouse embryonic stem cells, CpG methylation
MeSH terms
Mouse Embryonic Stem Cells, DNA Methyltransferase 3B, Animals, Cell Line, DNA, DNA (Cytosine-5-)-Methyltransferases, Genes, Histones, Lysine, Neoplasms, Histone-Lysine N-Methyltransferase, Ribosomes, RNA Caps, RNA Polymerase II, RNA, Messenger, DNA Methylation, RNA Stability, Transcription Initiation Site, Polyadenylation, Epigenesis, Genetic, Mice, Transcription Initiation, Genetic

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