Critical Role for the Human Cytomegalovirus Major Immediate Early Proteins in Recruitment of RNA Polymerase II and H3K27Ac To an Enhancer-Like Element in Ori
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
REPRODUCED (1:1, qualitative). Fresh, independent re-run (SLURM «job» on «our HPC» n096, COMPLETED, 1:06:59) after the prior clean run (2209002) was auto-requeued and its «infra» workdir reclaimed. Ran the paper's described third-party pipeline (cutadapt -> Bowtie2 2.5.5 default -> samtools q10 uniquely-mapping -> HOMER 5.1 findPeaks + bedtools binned input-normalized coverage; combined hg19 + HCMV TB40r-GFP-IE-FKBP index) on the paper's OWN ChIP-seq (GSE171512 rep-1; the BRIEF's GSE185763 is a reused other-group dataset, the real data is GSE171512 / superseries GSE171522 / PRJNA719833). All 4 libraries aligned 95.4-98.3% with strong specific viral enrichment (Pol II ~8x, H3K27Ac concentrated into the oriLyt peak). C2 reproduces EXACTLY: the single dominant H3K27Ac peak sits at oriLyt and is the global maximum of viral H3K27Ac, dwarfing all other viral peaks (5.44x by HOMER tags, 8-12x by coverage height). C1 reproduces at the locus level (within-tol): HOMER calls only 4 viral Pol II peaks and the top-3 are precisely the 2.7-kb, 1.2-kb and oriLyt 4.9-kb RNAs; caveat is the 2.7-kb summit sits ~2.4 kb into the gene body rather than exactly at the 5' promoter. This run's H3K27Ac input was fully aligned (58.3M reads vs a truncated 10.2M previously), which sharpened background and INCREASED oriLyt dominance (5.44x vs prior 3.66x). NOT attempted (out of scope, wet-lab/manual): ChIP-qPCR fold-changes incl. the ~30-fold oriLyt-vs-RNA2.7 H3K27Ac, Shield-1 IE-depletion occupancy (Fig 5-8), and reused other-group browser snapshots. Tool versions differ from the paper (Bowtie2 2.5.5/HOMER 5.1 vs 2.2.9/4.11) but the qualitative localization/dominance claims are version-insensitive. All grades PROVISIONAL pending human audit.
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Assessment versions
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v1 current initial assessment Score 93assessed: 2026-06-20 ⛓ 17c46989b3f0
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Provenance — full disclosure
When this reproduction was carried out, which methodology version was used, and by whom — so the record can be audited and checked independently.
- Reproduced
- 2026-06-23
- Rubric version
- not recorded
- Assessed by
- —
- 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 the HCMV major immediate early (IE1-72 and IE2-86) proteins play a role, beyond direct activation of early gene transcription, in shaping the viral epigenome — specifically in recruiting RNA Polymerase II and H3K27Ac to an enhancer-like element at the OriLyt RNA4.9 promoter.
- ★ An enhancer-like element in OriLyt (RNA4.9 promoter) is extraordinarily enriched in H3K27Ac compared to other viral loci. finding
- ★ IE proteins are required for recruitment of Pol II and H3K27Ac to the OriLyt RNA4.9 enhancer-like element, in addition to activating gene expression. finding
- ★ A TB40/E-based virus (TB40r mGFP-IE-FKBP) with ddFKBP-tagged IE1/IE2 proteins allows Shield-1-dependent conditional expression of IE proteins without altering viral replication. method
- ★ The large difference in H3K27Ac signal between RNA4.9 and RNA2.7 promoters is not explained by a difference in nascent transcription level, as shown by PRO-Seq. finding
- ★ Depletion of IE proteins (Shield-1 withdrawal) reduces RNA2.7 and OriLyt RNA4.9 RNA expression without affecting host GAPDH RNA. finding
- ★ IE protein depletion effects on Pol II/H3K27Ac recruitment are specific to viral promoters and do not affect the host GAPDH promoter. finding
- Pol II ChIPseq/PRO-Seq/ChIP-qPCR occupancy is similar between RNA2.7 and RNA4.9 promoters despite the large disparity in H3K27Ac. finding
- H3K4me3 and H3K27Ac marks co-localize immediately downstream of the promoter-proximal Pol II peak at the RNA4.9 promoter in essential region 2 of OriLyt. finding
| Assay | System | Perturbation | Readout | Platform |
|---|---|---|---|---|
| ChIP-qPCR (Pol II and H3K27Ac) | HFF (human foreskin fibroblasts) | Shield-1 addition/withdrawal (IE protein depletion) with TB40r mGFP-IE-FKBP virus, MOI 3 | Pol II and H3K27Ac occupancy at RNA2.7, OriLyt RNA4.9, and GAPDH promoter-proximal regions | — |
| RT-qPCR | HFF | Shield-1 addition/withdrawal, TB40r mGFP-IE-FKBP virus, MOI 3 | OriLyt RNA4.9 and RNA2.7 RNA levels normalized to GAPDH | — |
| ChIPseq (Pol II and H3K27Ac) | MRC5 fibroblasts | TB40/Ewt-GFP infection, MOI 2, 24 hpi | Genome-wide Pol II and H3K27Ac occupancy across HCMV genome | — |
| RNA-seq | MRC5 fibroblasts | TB40/Ewt-GFP infection, MOI 2, 24 hpi | Abundance of viral transcripts | — |
| Western blot | HFF | Shield-1 addition/withdrawal, TB40r mGFP-IE-FKBP virus, 24 hpi | IE1-72F and IE2-86F protein expression, normalized to actin | iBright FL1500 Imaging System |
| PRO-Seq | HFF | TB40/E HCMV infection, MOI 3, 24 hpi | Nascent Pol II transcription (promoter-proximal pause and gene body) at RNA4.9 and RNA2.7 promoters | — |
| Viral DNA copy number / infectious virus yield assay | HFF | TB40r mGFP-IE-FKBP vs parental WT TB40/E infection, MOI 0.5 (DNA) and MOI 1.0 (infectivity) | Cell-associated viral genome copies and infectious virus titer over time | — |
| DFF ChIP-seq (Pol II and H3K4me3), reanalysis of published data | HFF | HCMV infection, 48 hpi (Spector et al. dataset) | Pol II and H3K4me3 occupancy around RNA2.7 and OriLyt RNA4.9 promoters | — |
- ▲ H3K27Ac ChIP-qPCR signal at OriLyt RNA4.9 is approximately 30-fold greater than at RNA2.7 ~30-fold
- ▼ Omitting Shield-1 (IE protein depletion) significantly reduced Pol II and H3K27Ac recruitment to RNA2.7 and OriLyt RNA4.9 promoter-proximal regions
- ▼ IE protein depletion decreased RNA2.7 and OriLyt RNA4.9 RNA levels without changing host GAPDH RNA level
- ▼ Full-length IE1-72 and IE2-86 protein expression was significantly reduced in the absence of Shield-1
- – RNA4.9 and RNA2.7 promoters produced similar amounts of Pol II nascent transcript by PRO-Seq at 24 hpi despite very different H3K27Ac levels
- – No difference in viral DNA copy number or infectious virus production over time between TB40r mGFP-IE-FKBP and parental WT TB40/E
- – Shield-1 withdrawal had no effect on Pol II or H3K27Ac binding at the host GAPDH promoter
- – Strong Pearson correlation between biological replicates of RNAseq, Pol II ChIPseq, and H3K27Ac ChIPseq
- fold_change ~30-fold (H3K27Ac ChIP-qPCR signal at OriLyt RNA4.9 vs RNA2.7)
- count 2 biological replicates (RNAseq, Pol II ChIPseq, H3K27Ac ChIPseq in MRC5 cells at 24 hpi)
- count 3 independent infections (Western blot of IE1-72F and IE2-86F expression)
- count triplicate infections (RT-qPCR and ChIP-qPCR of HFF infected with TB40r mGFP-IE-FKBP virus)
- other MOI 2 (MRC5 infection dose for RNAseq/ChIPseq experiments)
- other 24 hpi (Harvest time point for RNAseq/ChIPseq and ChIP-qPCR/RT-qPCR experiments)
- other 48 hpi (Time point of Spector et al. DFF ChIPseq comparison dataset)
- correlation strong correlation (Pearson) (Comparison of ChIPseq/RNAseq biological replicates)
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.
This study combined RNAseq and ChIP-seq (Pol II and H3K27Ac) in lytically HCMV-infected fibroblasts using two biological replicates, with replicate concordance assessed by Pearson correlation. Conditional IE-protein depletion via an FKBP degron system was evaluated at 24 hpi; effects on RNA expression were quantified by RT-qPCR and chromatin occupancy by ChIP-qPCR, each in triplicate, and described as statistically significant without naming the inferential test used. Results were primarily reported as fold-differences and representative genomic browser tracks, with Western blot band intensities quantified by imaging system.
| Test | Applied to | n | Assumptions |
|---|---|---|---|
| Pearson correlation | Validation of concordance between biological replicate pairs for RNAseq, Pol II ChIP-seq, and H3K27Ac ChIP-seq (Fig. 2A) | n=2 biological replicates per assay | not stated |
| RT-qPCR with GAPDH normalization (inferential test not named) | Comparison of Ori Lyt RNA4.9 and RNA2.7 expression ± Shield-1 (Fig. 7) | n=3 (triplicate infections) | not stated |
| ChIP-qPCR (inferential test not named) | Comparison of Pol II and H3K27Ac occupancy at viral and host promoters ± Shield-1 (Fig. 8) | n=3 (triplicate infections) | not stated |
| Western blot band intensity quantification via iBright FL1500 (inferential test not named) | Quantification of IE1-72F and IE2-86F protein levels ± Shield-1 (Fig. 6B) | n=3 independent infections | not stated |
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Replicate concordance for ChIP-seq was assessed with Pearson correlation↳ Could also: The ENCODE-recommended Irreproducible Discovery Rate (IDR) framework, or Spearman correlation, could also be used for ChIP-seq replicate QC — IDR directly quantifies the reproducibility of peak calls rather than genome-wide signal correlation, providing a principled threshold for retaining only consistently reproducible peaks; Spearman is more robust to outliers and non-linearity in signal distributions
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Differences in RT-qPCR and ChIP-qPCR signal between ± Shield-1 groups (n=3) were described as statistically significant without naming the inferential test↳ Could also: An explicit two-sample t-test (parametric) or Mann-Whitney U test (non-parametric) with reported test statistic and exact p-value could also be applied — Naming the test and reporting exact p-values allows readers to assess the inferential basis of significance claims and evaluate whether the chosen test's assumptions (e.g., normality) are appropriate for n=3
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Multiple qPCR comparisons were made across several primer sets and gene loci without a multiplicity adjustment↳ Could also: A Benjamini-Hochberg FDR correction or Bonferroni correction over the family of qPCR comparisons could also be applied — When multiple independent comparisons are made simultaneously, a correction procedure reduces the probability that any single finding is a false positive by chance; this is particularly informative for the ChIP-qPCR panel spanning several viral and host loci
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ChIP-seq data across conditions (± Shield-1) were compared by visual inspection of genome browser tracks and ChIP-qPCR↳ Could also: Spike-in normalization (e.g., with Drosophila chromatin or a defined exogenous control) combined with quantitative differential enrichment analysis (e.g., DiffBind or csaw) could also be used — Standard ChIP-seq library normalization assumes equal total signal between conditions; spike-in normalization enables quantitative between-sample comparisons of absolute chromatin occupancy, which is directly relevant when total histone acetylation levels may change upon IE depletion
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Dispersion around qPCR and Western blot measurements was not explicitly described in the text (error bar type not stated)↳ Could also: Reporting SD or 95% CI alongside mean values is also standard for n=3 experiments — At small n (3 per group), SD conveys the full observed spread, while 95% CI communicates uncertainty about the mean; either choice makes variability explicit and aids interpretation of effect magnitude relative to scatter
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RNAseq data were displayed as log2 read counts in a heatmap without a described differential expression analysis framework↳ Could also: A formal differential expression analysis using DESeq2 or edgeR (with size-factor normalization and Wald or likelihood-ratio tests) could also be applied to the n=2 replicate RNAseq data — Even with n=2 replicates, these frameworks provide regularized dispersion estimates and FDR-adjusted p-values that support statistical ranking of differentially expressed viral genes, complementing the descriptive heatmap visualization
What was reproduced
The exact results taken into scope, with each reported value next to the value our attempt produced.
scope.md — pmid-36645269
Paper: Forte et al. 2023, Microbiol Spectr 11:e03144-22. "Critical Role for the Human Cytomegalovirus Major Immediate Early Proteins in Recruitment of RNA Polymerase II and H3K27Ac To an Enhancer-Like Element in OriLyt." PMID 36645269 · PMCID PMC9927211 · DOI 10.1128/spectrum.03144-22
Code / data resolution
- "Code" repo = https://github.com/Mary-«our HPC»/HCMV-reference-genomes — this is
NOT analysis code. It ships HCMV reference genomes (FASTA + GTF) used to align
the deposited bigwig files in IGV. Per BRIEF rule P16, applying the paper's
described third-party pipeline to the paper's own data is an equally valid
reproduction. We do exactly that, using this repo's reference genome.
TB40r-GFP-IE-FKBP-final.fa(244,567 bp, contigTB40r-GFP-IE-FKBP) + its*.MH.gtfcarry the author-curated non-coding-RNA annotations (2.7-kb, 1.2-kb, 4.9-kb/oriLyt RNAs) we need to test the paper's localization claims.genome.fa(EF999921.1, plain TB40-BAC4) has only the standard US/UL/RL gene GTF and lacks the ncRNA names. We therefore align to the MH reference; the three ncRNAs all sit UPSTREAM of the IE-FKBP insertion, so their coordinates are unaffected by the degron-tag insertion (which is at the IE locus, far downstream).
- Paper's own data = GEO superseries GSE171522 (NOT GSE185763 — that is a different Price-lab paper, PMID 35579393, which this study merely reuses for two browser snapshots). The HCMV ChIP-seq is subseries GSE171512 "HCMV epigenome during early lytic infection" (MRC5 fibroblasts, TB40/E wt-GFP, MOI 2, 24 hpi).
Samples used (GSE171512 / PRJNA719833)
| Assay | Role | GSM | SRR | layout |
|---|---|---|---|---|
| Pol II | ChIP Rep1 | GSM5226481 | SRR14145989 | single |
| Pol II | Input Rep1 | GSM5226479 | SRR14145987 | single |
| H3K27Ac | ChIP Rep1 | GSM5226485 | SRR14145993 | paired |
| H3K27Ac | Input Rep1 | GSM5226483 | SRR14145991 | paired |
| (Rep2 available — SRR14145990/988/994/992 — used only as optional stretch.) |
Pipeline (paper Methods, "ChIP-seq and data analysis")
FastQC → cutadapt (trim Illumina 3′ adapters) → Bowtie2 v2.2.9 default
params, aligned to hg19 + HCMV (TB40-BAC4 EF999921.1 + GFP) → keep uniquely
mapping reads → HOMER v4.11 (findPeaks) to call Pol II and H3K27Ac peaks and
generate read-density tracks.
IN SCOPE (pipeline-derived; what we attempt)
- C1 (Pol II localization): major Pol II ChIP-seq peaks at the promoters of the 2.7-kb, 1.2-kb, and oriLyt 4.9-kb RNAs (Results §"ChIP-seq analyses of the viral genome…"; Fig 2/3). Test = are the largest viral Pol II peaks at these loci?
- C2 (H3K27Ac dominance): a single dominant H3K27Ac peak at oriLyt (RNA4.9 promoter) that "dwarfed all other peaks" on the viral genome (Results; Fig 2/3). Test = is the top viral H3K27Ac peak at oriLyt, and by what ratio does it exceed the next peak?
STRETCH / optional (80-20, may skip)
- Replicate Pearson correlation (Fig 2A) — needs both reps; reported only as a panel.
OUT OF SCOPE (wet-lab / manual / not pipeline-derived)
- ChIP-qPCR fold changes incl. "~30-fold" H3K27Ac oriLyt-vs-RNA2.7 (Fig 5–8) — qPCR.
- Shield-1 IE-depletion occupancy changes (Fig 8) — wet-lab.
- RNA4.9 expression / RNA-seq DE calls beyond coverage — not the central claim.
- The reused GSE185763 / GSE139114 browser snapshots — other groups' data.
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Reproduction footprint
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