Dynamic interaction of MYC enhancer RNA with YEATS2 protein regulates MYC gene transcription in pancreatic cancer.
The main results reproduced: recomputed values matched the published ones within tolerance.
A 0–100 reproducibility-quality score from the per-question grades, shown as a z-score: standard deviations above (+) or below (−) the mean of comparable assessments.
▸Reproduction agent’s raw note
Described well enough to reproduce 1:1. Fresh, independent re-run from raw GSE288088 FASTQ on «our HPC» SLURM («job», 2026-06-23) of the fully-specified pipeline: FastQC 0.11.7 -> Trim Galore 0.6.10 -> Bowtie2 (default, hg38 GRCh38_noalt_as, unique reads) -> HOMER makeTagDirectory/findPeaks -style groseq/coverage. C1 (headline eRNA count) is an EXACT integer match: 19 eRNAs in the 260 kb MYC super-enhancer (chr8:127,163,754-127,428,551) at both per-condition pools, matching the paper. C2 (TNFa induction, Fig 1B) reproduces qualitatively at all 4 loci (24h>0h). V (validation) shows strong matched-strand concordance (|r|=0.89-0.99) with the authors' deposited BigWigs. Alignment rates (96.04/95.97/96.00/95.69%) and unique-read counts are identical to an earlier 2026-06-20 run, confirming the pipeline is deterministic; that earlier genuine run had been lost to a false-positive infrastructure quarantine (its «infra» workdir reclaimed). NOT attempted: external overlay ChIP/ATAC datasets (out of scope) and all wet-lab/experimental results (not pipeline-derived). Grades are provisional and must be human-checked.
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Assessment versions
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v1 current initial assessment Score 90assessed: 2026-06-20 ⛓ 2e06c8a79def
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- Reproduced
- 2026-06-23
- Rubric version
- not recorded
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- Last updated
- 2026-08-05
Provisional, curator- or AI-assessed, and independently checkable. A reproduction outcome states what one attempt could reproduce — not a judgement of the authors.
Deep full-text extraction
Model: sonnetThe paper tests whether a MYC-associated enhancer RNA (eRNA), induced under chronic inflammatory (TNF-α) conditions, regulates MYC gene transcription in pancreatic cancer cells through interaction with the histone reader protein YEATS2.
- ★ MYC eRNAs (notably MYC-490-kb) are transcribed from the MYC super-enhancer and are upregulated by chronic TNF-α stimulation specifically in pancreatic cancer cells, not normal pancreatic epithelial cells finding
- ★ MYC-490-kb eRNA levels are elevated in chronic pancreatitis patient samples and in pancreatic cancer tissue versus adjacent normal tissue finding
- ★ MYC-490-kb eRNA positively regulates MYC mRNA transcription (knockdown reduces, overexpression increases MYC mRNA) in a dose-dependent manner finding
- ★ MYC-490-kb eRNA interacts with YEATS2 protein in a TNF-α-dependent, cancer-cell-specific manner finding
- ★ YEATS2 is required for MYC gene transcription, as its knockdown reduces MYC mRNA levels finding
- ★ TNF-α induces tyrosine dephosphorylation of the YEATS domain (involving residue Y313), which increases MYC eRNA binding to YEATS2 mechanism
- ★ MYC gene amplification is associated with reduced overall survival in PDAC patients (TCGA PAAD cohort) finding
- ★ MYC eRNA augments recruitment of the YEATS2-containing ATAC-HAT complex to the MYC promoter/enhancer, driving MYC expression mechanism
| Assay | System | Perturbation | Readout | Platform |
|---|---|---|---|---|
| Survival analysis (TCGA PAAD cohort) | PDAC patient cohort, n=176 | none (MYC amplification status) | overall survival | TCGA database |
| ChIP-seq (H3K27ac, H3K4me1) and ATAC-seq | PDAC cell line (published datasets) | none | active enhancer marks / open chromatin at MYC super-enhancer | — |
| Nascent RNA sequencing | MIAPaCa-2 cells | TNF-α (24h) | enhancer-directed transcription/eRNA levels from MYC super-enhancer | — |
| RT-PCR/qPCR | MIAPaCa-2, AsPC-1, HPNE (and HCT-116) cells | TNF-α (24h) | MYC-490-kb, MYC-425-kb eRNA and MYC mRNA levels | — |
| qPCR on patient samples | Chronic pancreatitis patients (n=7) and pancreatic cancer patient tissue (n=5) vs normal/adjacent normal | none (disease state) | MYC-490-kb eRNA and MYC mRNA levels | — |
| Click-iT nascent RNA capture with nuclear/cytosolic fractionation | MIAPaCa-2 and HPNE cells | TNF-α | subcellular localization of nascent MYC-490 eRNA | Click-iT kit; Western blot (β-Actin, Lamin) |
| shRNA knockdown | MIAPaCa-2 cells (MYC-490-kb eRNA KD and YEATS2 KD) | shRNA knockdown | MYC mRNA/eRNA and YEATS2 levels | — |
| Plasmid overexpression | MIAPaCa-2 and HPNE cells | MYC-490-kb / MYC-425-kb eRNA overexpression (dose titration) | MYC mRNA levels | — |
| In-silico RNA-protein interaction prediction | MYC-490-kb eRNA vs histone modifier proteins | none | binding propensity/docking score | RPIseq, HDOCK web server |
| UV-RIP (RNA immunoprecipitation) | MIAPaCa-2, AsPC-1, HPNE cells | TNF-α stimulation | YEATS2-associated MYC-490 eRNA (and BRD4 association) | — |
| Western blot / immunoprecipitation (phospho-tyrosine, 4G10 antibody) | MIAPaCa-2, AsPC-1, HPNE cells; HEK293T-expressed YEATS domain, Y313/Y313F mutants | TNF-α; in vitro phosphatase assay | tyrosine phosphorylation of YEATS2/YEATS domain and correlation with eRNA binding | — |
- ▼ MYC-amplified PDAC patients show significantly lower overall survival than unaltered patients P=0.018, n=176
- ▲ MYC-490-kb and MYC-425-kb eRNA and MYC mRNA levels increase after TNF-α treatment in MIAPaCa-2 and AsPC-1 cells but not HPNE P=0.0068/0.0419/0.0483 (MIAPaCa-2); P=0.0005/0.0015/0.0008 (AsPC-1)
- ▲ MYC-490-kb eRNA is elevated in chronic pancreatitis patient samples vs normal P=0.0167, n=7 vs 7
- ▲ MYC-490-kb eRNA and MYC mRNA are upregulated in pancreatic cancer tissue compared to adjacent normal tissue n=5
- ▼ shRNA knockdown of MYC-490-kb eRNA reduces MYC mRNA expression ~30% reduction
- ▲ Overexpression of MYC-490-kb eRNA (peaking at 200 ng DNA) significantly increases MYC mRNA in MIAPaCa-2 cells but not HPNE cells P=0.0025 (upper), P=0.0051 (lower panel)
- ▲ UV-RIP shows increased YEATS2-MYC-490-kb eRNA association under TNF-α in MIAPaCa-2 and AsPC-1, but not HPNE; no increase seen with BRD4 or MYC-425-kb eRNA P=0.0001/0.0002 (MIAPaCa-2); P=0.0012/0.002 (AsPC-1)
- ▼ shRNA knockdown of YEATS2 significantly reduces cMYC mRNA expression P=0.0017 to <0.0001 across 4 shRNAs
- pvalue P=0.018 (survival difference by MYC amplification status, TCGA PAAD)
- pvalue P=0.0068, P=0.0419, P=0.0483 (MYC-490, MYC-425 eRNA and MYC mRNA induction by TNF-α in MIAPaCa-2)
- pvalue P=0.0005, P=0.0015, P=0.0008 (MYC-490, MYC-425 eRNA and MYC mRNA induction by TNF-α in AsPC-1)
- pvalue P=0.0167 (MYC-490-kb eRNA elevated in chronic pancreatitis patients vs normal)
- fold_change ~30% reduction (MYC mRNA decrease after MYC-490-kb eRNA knockdown)
- count n=5 (pancreatic cancer patient tissue samples vs adjacent normal analyzed for MYC-490 eRNA/mRNA)
- pvalue P=0.0001 (Input), P=0.0002 (IP) (UV-RIP YEATS2-MYC-490 eRNA association in TNF-treated MIAPaCa-2)
- pvalue P=0.0026 (Input), P=0.027 (IP) (in vitro MYC eRNA binding assay correlating tyrosine dephosphorylation with YEATS domain binding)
Statistical methods review
Model: sonnetA neutral, descriptive read of the statistical approach — what was done, and (for shared learning, not as criticism) what could also have been done.
The study combined TCGA public cohort survival analysis (n=176), in vitro cell-line assays in two pancreatic cancer lines and one normal epithelial line, and patient tissue comparisons (chronic pancreatitis n=7/group; pancreatic cancer n=5 vs. adjacent normal). Primary statistical inference across all quantitative assays (RT-PCR/qPCR) relied on repeated application of unpaired two-tailed Student's t-tests on n=3 independent experiments. Results were reported as mean ± SD with exact p-values; no multiplicity correction was described.
| Test | Applied to | n | Assumptions |
|---|---|---|---|
| Survival analysis — test not explicitly named (log-rank is the standard method for this output type) | Fig 1A — overall survival by MYC amplification status in TCGA PAAD cohort | 176 patients | not stated |
| Unpaired two-tailed Student's t-test | Fig 1C–E — RT-PCR of MYC eRNA and MYC mRNA in MIAPaCa-2, AsPC-1, HPNE at TNF-α 0 h vs 24 h | n=3 independent experiments per cell line | not stated |
| Unpaired two-tailed Student's t-test | Fig 1F — MYC-490-kb eRNA in chronic pancreatitis vs. normal patient samples | n=7 normal, n=7 chronic pancreatitis patients | not stated |
| Unpaired two-tailed Student's t-test (as inferred from study-wide pattern; pairing not stated) | Fig 1G — MYC-490-kb eRNA and MYC mRNA in pancreatic cancer vs. adjacent normal tissue | n=5 patients | not stated |
| Unpaired two-tailed Student's t-test | Figs 2A–D — Click-iT nascent RNA fractionation, shRNA knockdown, eRNA overexpression in MIAPaCa-2 and HPNE | n=3 independent experiments | not stated |
| Unpaired two-tailed Student's t-test | Figs 3A–B, 3E, 3J, 3L — UV-RIP quantification, YEATS2 KD effect on cMYC, in vitro eRNA binding with phospho-mutants | n=3 independent experiments | not stated |
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Numerous unpaired t-tests were applied across multiple figures, cell lines, and shRNA constructs without any correction for multiple comparisons↳ Could also: One-way or two-way ANOVA (depending on factorial structure) followed by a post-hoc test such as Tukey HSD or Dunnett's vs. control, or application of Benjamini-Hochberg FDR correction across the full set of t-tests — When many pairwise tests are performed, an omnibus test or explicit correction controls the family-wise error rate and reduces the probability of spurious findings accumulating across the experiment set
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Pancreatic cancer tissue (n=5) was compared to matched adjacent normal tissue from the same patients using an unpaired test framework↳ Could also: A paired t-test, or Wilcoxon signed-rank test given the very small n, that treats each patient's tumor/normal pair as a matched unit — Paired analysis removes between-patient variability and typically increases statistical power, more accurately reflecting the within-patient contrast between tumor and normal tissue
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Parametric unpaired t-tests were applied to cell-line data from n=3 independent experiments↳ Could also: Non-parametric Mann-Whitney U test, or supplementing bar graphs with plots of individual data points — With n=3, normality cannot be formally assessed; non-parametric alternatives require no distributional assumption, and displaying individual data points alongside summary statistics preserves the full distribution for readers
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The survival analysis (Fig 1A) reports P=0.018 but does not name the underlying statistical test↳ Could also: Explicitly state the log-rank test and report a hazard ratio with 95% confidence interval — Naming the test and providing a hazard ratio with CI conveys both the analytical basis and the magnitude and precision of the survival difference, improving interpretability and reproducibility
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Effect magnitude was conveyed only through p-values and visual inspection of bar chart heights↳ Could also: Report standardized effect sizes (e.g., Cohen's d) or fold changes with 95% confidence intervals alongside p-values — Effect size metrics separate statistical significance from biological magnitude, helping readers assess practical relevance and enabling future meta-analyses or power calculations
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Dispersion was reported as SD across all quantitative figures with n=3↳ Could also: Report 95% confidence intervals or show individual data points, approaches increasingly recommended for small-n experimental data — For n=3, the SD describes sample spread but CIs communicate precision of the mean estimate; individual data points additionally reveal the actual distribution and any outliers, consistent with current reporting standards in molecular biology journals
What was reproduced
The exact results taken into scope, with each reported value next to the value our attempt produced.
Scope — pmid-40216980
Paper: Roy et al. 2025, EMBO Rep — "Dynamic interaction of MYC enhancer RNA with YEATS2 protein regulates MYC gene transcription in pancreatic cancer." DOI 10.1038/s44319-025-00446-0 · PMCID PMC12117045.
Data: GEO GSE288088 (BioProject PRJNA1216178) — ex vivo Nascent RNA-seq (5-EU Click-iT capture, nuclear fraction) of MIA PaCa-2 cells, TNFα 0 h vs 24 h, 2 biological replicates each. 4 paired-end (2×100 bp) NovaSeq 6000 runs:
- SRR32136284 → GSM8758825 — 0 h rep1 (~42.0 M spots)
- SRR32136283 → GSM8758826 — 0 h rep2 (~47.2 M spots)
- SRR32136282 → GSM8758827 — 24 h rep1 (~33.9 M spots)
- SRR32136281 → GSM8758828 — 24 h rep2 (~36.0 M spots) Deposited processed data = strand-specific BigWig coverage tracks (rep1 only): 0hneg/0hpos (GSM8758825), 24hneg/24hpos (GSM8758827).
Code link in record: github.com/FelixKrueger/TrimGalore (Trim Galore v0.6.10,
tag commit 4edff97). This is a third-party tool, not author code — valid per
brief rule P16. The full pipeline is third-party tools chained per the Methods.
Pipeline (fully specified in Methods "Bioinformatic processing" + GEO data_processing)
- FastQC v0.11.7 — QC of raw reads.
- Trim Galore v0.6.10 — adapter trimming.
- Bowtie2 (Langmead & Salzberg 2012), default parameters, align to hg38; retain only uniquely aligned reads.
- HOMER
makeTagDirectoryfrom uniquely-aligned SAM/BAM. - HOMER
findPeaks -style groseq— call nascent transcripts (eRNAs). - HOMER
makeBigWig.pl -strand— strand-specific coverage BigWigs (UCSC viz).
IN SCOPE (pipeline-derived, attempted)
- C1 — transcript/eRNA call. "MYC-490-kb and MYC-425-kb eRNA are part of a group of 19 eRNAs expressed from a 260 kb region (chr8:127,163,754–127,428,551) of the MYC super-enhancer" (Results, ¶ "MYC eRNAs are expressed from super-enhancer regions"). → reproduce by HOMER findPeaks -style groseq, count transcripts in that interval. PRIMARY pinnable claim.
- C2 — TNFα induction (Fig 1B). Nascent RNA-seq signal at MYC-490 / MYC-425 eRNA and MYC loci is higher at 24 h than 0 h TNFα. → quantify coverage over the SE region / MYC gene, 0 h vs 24 h; check direction (increase).
- V — track concordance. Correlate our reproduced strand BigWigs vs the authors' deposited BigWigs over the MYC locus (chr8:127–128 Mb). Validates the alignment step independent of the claim text.
OUT OF SCOPE (not attempted — reason)
- ChIP-seq/ATAC-seq re-analysis of external datasets (GSE64557 H3K27ac/H3K4me1,
GSE164974 ATAC) shown as overlay tracks in Fig 1B — external data, supportive
context, not the GSE288088 result; HOMER
-style histone/-style dnaseruns are optional 20%. Could be added; skipped under 80/20. - All wet-lab / qPCR / RT-PCR / UV-RIP / Co-IP / ChIP-qPCR / phosphatase / MTT / wound-healing / survival / in-silico docking (RPISeq/HDOCK) results (Figs 1C–G, 2–5, EV1–4) — manual/experimental, not pipeline-derived.
Heavy compute
All alignment/HOMER runs on «our HPC» SLURM (account=kubisch_std, partition=std). Data + index + intermediates on «infra»; only small derived values returned to «host».
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Reproduction footprint
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