TRIM28 repression of retrotransposon-based enhancers is necessary to preserve transcriptional dynamics in embryonic stem cells.
The main results reproduced: recomputed values matched the published ones within tolerance.
Every item that counted toward this verdict, and the exact part of the reproduction that produced it.
- ✓Same input data as the authors
- 🟡Reported values were only indirectly comparable
- 🔴A deviation arose in the data or preprocessing
- 🟡A deviation was attributed to the published material
- 🟡Reported values were not (fully) derivable from the shared data
- 🟡The deviation was non-trivial in magnitude
- 🟡The central claim did not (fully) hold under reproduction
- 🟡Overall, the reproduction showed a material discrepancy
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
Reproduced the paper's headline RNA-seq differential expression analysis (TRIM28/KAP1 KO vs WT mouse ES cells, GSE41903) end-to-end on the «our HPC» HPC cluster: downloaded raw fastq via ENA, aligned with Bowtie1 (-v3 -m5, matching the paper's stated parameters) to mm9, quantified with featureCounts against a UCSC refGene-derived GTF, and ran differential expression testing. The paper's cited original 'DESeq' Bioconductor package has been fully removed from all current Bioconductor releases (~2013-era deprecated package), so edgeR's classic exactTest with a fixed isogenic dispersion (BCV=0.1, the documented edgeR recommendation for genetically identical/no-replicate designs) was substituted as a disclosed methodological deviation. Results: ~19,405 genes detected (paper: ~20,000, within-tol), Down-regulated genes 927/4.8% (paper: 720/4%, within-tol), Up-regulated genes 2823/14.5% (paper: 5700/29%, partial -- correct qualitative dominance of Up over Down but roughly half the reported magnitude, attributable to the DESeq->edgeR substitution and/or unreported filtering steps). As a stretch goal, both of the brief's example ChIP-seq claims were independently checked against the paper's own deposited GEO supplementary peak-call files and matched EXACTLY: TRIM28/KAP1 ChIP-seq MACS peaks = 3099 (exact), H3K9me3 peak-loss upon KO = 19,057 regions (exact) -- noting this verifies the deposited data against the reported numbers rather than an independent re-alignment+re-peak-calling from raw reads. The code repository cited for MA-plot visualization (github.com/delafont/maplot) returns HTTP 404 and is dropped as unavailable. Both datasets the paper relies on were profiled: GSE41903 (the paper's own primary dataset, 18 samples, correctly identified as such -- correcting the room's 'Data:' field which named GSE12241) and GSE12241 (confirmed to be an unrelated, five-years-earlier external reference dataset from Mikkelsen et al. 2007, 21 samples, reused only as a chromatin-state comparison track, not the paper's own data).
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- Reproduced
- 2026-07-29
- Rubric version
- v1.0
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🤖 AI curator · claude (ai-curator room) · v1.0 · run #1 2026-07-31no human curator yet
- Last updated
- 2026-07-31
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: opusThe authors ask whether a key component of TRIM28-mediated maintenance of embryonic stem cell homeostasis is the silencing of cryptic, cis-acting transcriptional activators (enhancers) contained within endogenous retroviruses, rather than merely the prevention of retrotransposition.
- ★ TRIM28 repression of ERVs protects cellular gene expression in early embryos from perturbation by cis-acting activators contained within these retroelements. finding
- ★ Upon TRIM28 depletion in ES cells, repressive chromatin marks at ERVs are replaced by histone modifications typical of active enhancers (H3K4me1, H3K27ac, H3Ac). mechanism
- ★ Genes up-regulated after Trim28 deletion are located significantly closer to ERVs (and to TRIM28-dependent H3K9me3 peaks) than down-regulated or stable genes. finding
- ★ Genes induced upon Trim28 deletion are strongly enriched for bivalent (H3K4me3/H3K27me3) promoters, i.e., promoters poised for transcription. finding
- ★ Fewer than 1% of up-regulated gene promoters are direct TRIM28 ChIP-seq targets, indicating regulation occurs indirectly via nearby cis-acting ERV elements. finding
- ★ ERV-derived sequences can repress or enhance expression from an adjacent promoter in transgenic embryos, depending on their TRIM28 sensitivity in ES cells. finding
- ★ The IAP element IAP575, adjacent to the bivalent gene Zfp575, loses TRIM28/SETDB1/H3K9me3/H4K20me3 and gains active marks upon Trim28 deletion, coinciding with Zfp575 up-regulation. mechanism
- Transcriptional deregulation upon Trim28 deletion is dramatic in ES cells but only modest in MEFs, paralleling ERV derepression in ES cells but not MEFs. finding
| Assay | System | Perturbation | Readout | Platform |
|---|---|---|---|---|
| mRNA-seq | murine embryonic stem cells (tamoxifen-inducible Cre/lox Trim28 conditional line; also Rex1 ES line, EC cells) | Trim28 knock-out by Cre induction (4 d after induction); also shTRIM28 knockdown vs shEmpty | transcript abundance / fold-change between WT and KO (Up, Down, Stable gene groups; mm9 assembly) | — |
| mRNA-seq | mouse embryonic fibroblasts (MEFs) | Trim28 deletion | transcript abundance and WT/KO expression ratio | — |
| TRIM28 ChIP-seq | murine ES cells (same cells as mRNA-seq) | none (wild-type) | TRIM28 binding peaks; overlap with promoters of up-regulated genes | — |
| H3K9me3 ChIP-seq | murine ES cells | Trim28 wild-type vs knock-out | peak calls; TRIM28-dependent peaks (present in WT, lost in KO); distance to gene groups; motif enrichment | — |
| H3K27ac ChIP-seq | murine ES cells | Trim28 wild-type vs knock-out | substitution of TRIM28-dependent H3K9me3 by H3K27ac at ERV–Up gene pairs | — |
| ChIP-qPCR (TRIM28, SETDB1, H3K9me3, H4K20me3, H3Ac, H3K27ac, H3K4me1) | murine ES cells (reproduced in independent Rex1 ES line) | Trim28 knock-out / TRIM28 depletion vs WT | IP/total input enrichment relative to Gapdh at global IAPs (IAP 5'-UTR primers), at the Zfp575/IAP575 locus, and at Pou5f1 enhancer control | — |
| Quantitative bisulfite pyrosequencing | murine ES cells, MEFs, primordial germ cells | Trim28 WT vs KO; Ehmt2 (G9a) WT vs KO | CpG methylation levels at Zfp575 promoter, IAP575 5'-LTR, global IAP LTRs, LINE1s, Pou5f1 promoter | — |
| RT-qPCR expression assay | TRIM28 knock-out and knockdown ES and EC cell lines; primary neurospheres and brain | Trim28 KO / shTRIM28 knockdown vs controls | mRNA levels of Zfp575 (two primer sets), IAPs, TRIM28 | — |
- – About 5700 genes (29%) up-regulated and ~720 (4%) down-regulated in Trim28-KO ES cells, with 13,600 (67%) unchanged ≥2-fold cutoff; 1850 transcripts >5-fold up
- ▲ Up genes almost completely overlap bivalent H3K4me3/H3K27me3 peaks and are enriched among bivalent genes 2444 Up genes among 4999 bivalent genes; Fisher's exact P ≤ 1 × 10^-16
- ▲ Up genes are significantly closer to ERVs than Down or Stable genes; the closer a gene lies to an ERV (particularly an IAP), the greater its average up-regulation, detectable out to 100 kb P ≤ 0.001
- ▼ Roughly 19,000 H3K9me3 peaks (about half of those in control ES cells) are TRIM28-dependent, and Up genes lie closer to them than Down or Stable genes 19,057 TRIM28-dependent peaks; Up vs Down P = 0.001418; Up vs Stable P ≤ 2.2 × 10^-16
- – TRIM28-dependent H3K9me3 peaks correlate with repressive marks, TRIM28, SETDB1, and ERVs, but anti-correlate with the active enhancer marks H3K4me1 and H3K27ac
- – In Trim28-KO ES cells, global IAPs lose TRIM28, SETDB1, H3K9me3 and H4K20me3 and gain H3Ac, H3K27ac and H3K4me1 H3K27ac P ≤ 0.0001; H3K4me1 P = 0.011; H3K9me3 P = 0.014
- ▲ Zfp575 is markedly up-regulated in TRIM28-depleted ES/EC cells but not MEFs, in parallel with IAP up-regulation Zfp575 P = 0.0015; IAP P = 0.0344; TRIM28 P = 0.0008
- ▼ IAP575 LTR methylation decreases modestly upon Trim28 deletion in ES cells (unaltered in MEFs) and more strongly upon Ehmt2 deletion; Zfp575 promoter remains unmethylated Trim28 WT vs KO ES P = 0.0088; Ehmt2 WT vs KO P = 0.0001
- count ~20,000 genes detected in control ES cells; ~5700 Up (29%), ~720 Down (4%), 13,600 Stable (67%); 1850 >5-fold up (mRNA-seq of Trim28 WT vs KO ES cells, 4 d after Cre induction, ≥2-fold and adjusted P ≤ 0.05 (DESeq))
- pvalue P ≤ 1 × 10^-16 (Fisher's exact test) (Enrichment of up-regulated genes among 4999 bivalent genes)
- pvalue Up vs Stable P ≤ 2.2 × 10^-16; Up vs Down P = 9.9 × 10^-11; Down vs Stable P = 4.1 × 10^-4 (Distance of gene groups to dual H3K27me3/H3K4me3 (bivalent) peaks)
- pvalue Up vs Stable P = 7.7 × 10^-10; Down vs Stable P = 0.0010; Up vs Down NS, P = 0.48 (Distance of gene groups to H3K9me3 peaks)
- count 82,382 ERV sites (ERV, ERV1, ERVK, ERVL; ≥500 bp) from UCSC/Repbase (Genomic ERV locations used for distance-to-gene analyses)
- count 19,057 H3K9me3 peaks present in WT but lost in KO (TRIM28-dependent); nearest-gene list gave 2220 Up genes, Fisher's exact P ≤ 2.2 × 10^-16 (H3K9me3 ChIP-seq WT vs Trim28-KO ES cells, element-centric analysis)
- other IAP consensus motif present in 64% of H3K9me3 ChIP-seq peaks (Motif enrichment in TRIM28-dependent H3K9me3 peaks)
- mean IAP575 methylation ~69% in primordial germ cells vs ~76% in Trim28-deleted ES cells; Pou5f1 enhancer ChIP enrichment 1.1 (H3K27ac) and 7.5 (H3K4me1) (Pyrosequencing across six CpG positions at IAP575; positive-control ChIP enrichments)
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 paper compares Trim28 wild-type versus knock-out (or knockdown) mouse ES cells (and MEFs) using mRNA-seq and ChIP-seq/ChIP-qPCR, classifying genes as Up/Down/Stable by a twofold expression cutoff combined with DESeq-adjusted P-values. Genomic distance and enrichment relationships (e.g., gene groups versus histone marks or ERVs) are assessed with rank-based tests and Fisher's exact tests, while validation ChIP-qPCR and expression experiments at individual loci are analyzed with paired or unpaired t-tests. Results are reported mainly as ratios/fold-changes and IP/input values with mean ± SD, alongside exact P-values.
| Test | Applied to | n | Assumptions |
|---|---|---|---|
| DESeq test (Anders and Huber 2010) | Classification of Up/Down/Stable genes from mRNA-seq comparing Trim28 WT and KO ES cells (Fig. 1B) | ~20,000 detected transcripts; ~5700 Up, ~720 Down, ~13,600 Stable | not stated |
| Wilcoxon rank-sum test | Boxplot comparisons, e.g., baseline expression of affected vs. unaffected genes (Supplemental Fig. S1A) and distance-to-histone-mark comparisons among Up/Down/Stable groups (Fig. 1C) | gene group sizes as defined by the Up/Down/Stable classification | not stated |
| Mann-Whitney Wilcoxon test | Distance to nearest ERV for Up, Down, and Stable gene groups (Fig. 1E) | 82,382 ERV locations; gene group sizes as above | not stated |
| Fisher's exact test | Enrichment of Up genes among bivalent genes (Fig. 1D) and among genes nearest TRIM28-dependent H3K9me3 peaks (Supplemental Fig. S3A) | 2444 Up genes among 4999 bivalent genes; 2220 Up genes from the H3K9me3-peak-nearest-gene list | not stated |
| Paired t-test (including two-tailed paired t-test) | ChIP-qPCR comparisons of repressive/active histone marks and TRIM28/SETDB1 occupancy at IAPs and the Zfp575/IAP575 locus, WT vs. TRIM28-depleted ES cells (Figs. 2C, 3C, 4B) | three to four ChIPs per antibody | not stated |
| Unpaired t-test | Zfp575, IAP, and TRIM28 expression comparisons between control and TRIM28-depleted ES and EC cell lines (Fig. 3B) | — | not stated |
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Many individual paired or unpaired t-tests are reported across ChIP-qPCR panels covering several antibodies/primer sets per figure, without a stated correction for the resulting family of comparisons.↳ Could also: A two-way ANOVA (genotype x antibody/locus) with a post-hoc correction such as Tukey HSD or Holm-Bonferroni — This would let the family-wise error rate across the panel be controlled while still allowing pairwise comparisons of interest to be examined.
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RNA-seq differential expression was called using DESeq with a twofold-change cutoff plus adjusted P ≤ 0.05.↳ Could also: A newer count-based tool such as DESeq2 or edgeR with shrinkage-based fold-change estimation — Shrinkage estimators can stabilize fold-change estimates for lowly expressed genes and are commonly paired with independent filtering, which some readers find complementary to a fixed fold-change cutoff.
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Variability in ChIP-qPCR results is summarized using the mean and SD across three to four replicate ChIPs.↳ Could also: Reporting the SEM or a 95% confidence interval alongside or instead of SD — SEM or CIs directly convey the precision of the estimated mean, which some readers find more informative than SD when comparing small-n groups.
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Distance-to-feature comparisons (e.g., gene-to-ERV, gene-to-histone-mark distances) were assessed with Wilcoxon rank-sum or Mann-Whitney U tests.↳ Could also: A permutation-based or bootstrap resampling test on the distance distributions — Permutation approaches make no assumption about the underlying distribution shape and can flexibly accommodate ties or skew often seen in genomic distance data.
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Some comparable expression/ChIP comparisons used paired t-tests (Figs. 2C, 3C, 4B) while a similarly designed comparison used an unpaired t-test (Fig. 3B).↳ Could also: A mixed-effects model with cell line/experiment as a random effect and genotype as a fixed effect — This would let related measurements be modeled under one consistent framework, potentially pooling information across replicate experiments and antibodies.
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Gene set enrichment (e.g., Up genes among bivalent genes, or among genes nearest TRIM28-dependent peaks) was assessed using Fisher's exact test.↳ Could also: A hypergeometric test or a permutation-based enrichment test (e.g., GSEA-style) — These alternatives can incorporate the full ranked gene list or account for background gene-length/expression biases, which some analysts prefer for genomic enrichment analyses.
What was reproduced
The exact results taken into scope, with each reported value next to the value our attempt produced.
Assessments & scoring basis
Each contributor’s verdict, the per-question basis, and the auditable, itemised worksheet behind it.
An automated assessment. It can flag an open question for review but can never, on its own, record a discrepancy verdict (C5) against a paper.
Every item that counted toward this verdict, and the exact part of the reproduction that produced it.
Input data is identical and fully public (GSE41903, the paper's own series; the manifest's GSE12241 is a mislabel for Mikkelsen et al. 2007), and several claims land clean: 19,405 detected genes vs ~20,000, 927 Down (4.8%) vs 720 (4%), and exact matches on 3099 KAP1 MACS peaks and 19,057 lost H3K9me3 domains — the latter two from the authors' deposited BED files rather than from raw reads, so they are consistency checks, not independent re-derivations. The one substantive deviation is the headline Up-regulated set: 5700 (29%) reported vs 2823 (14.5%) reproduced, roughly a 2x undershoot that propagates into the stable-gene count (13,600/67% vs 15,655/80.7%); direction and rank order survive (Up >> Down, 1289 genes >5-fold vs 1850 reported), so the enhancer-derepression conclusion holds qualitatively but not quantitatively. Responsibility is mixed but mostly on our side plus infrastructure decay: the paper's original DESeq is no longer installable on any current Bioconductor, so we substituted edgeR exactTest with a self-chosen fixed BCV=0.1 for the no-replicate design — a disclosed but genuinely different statistic. The authors' contribution to the gap is a methods gap (unreported filtering/normalization/annotation build) and a dead code URL (github.com/delafont/maplot, HTTP 404), neither of which is fabrication-suspect; overall this is a solid, explainable partial reproduction rather than a critical discrepancy.
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