The tumour suppressor L(3)mbt inhibits neuroepithelial proliferation and acts on insulator elements.
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 directly comparable
- ✓No relevant deviation in data/preprocessing
- ✓No authors-side cause for any deviation
- ✓Reported values are derivable from the shared data
- ✓Any deviation was negligible
- ✓The central claim held under reproduction
- ✓Overall, the reproduction was clean
- Every checked point held up.
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
Richter, Bhogaraju et al., Nat Cell Biol 2011 (PMID 21857667). L(3)mbt ChIP-seq in Drosophila, peaks called with the authors' own tool pypeak (GEO GSE29206 / SRA SRP006763). REPRODUCED end-to-end. (1) Deposited final files contain EXACTLY the reported 3314 (0.5% FDR) and 4572 (3% FDR) L(3)mbt binding sites; the 0.5% set is a strict subset of the 3% set. (2) Full from-scratch reprocessing on «our HPC» (4 SRA runs -> bowtie1.3.1/dm3 uniquely-mapped -> pypeak IP-vs-control and swapped decoy) regenerates the deposited intermediates almost exactly: 18544 IP peaks vs 18529 deposited, 46618 control/decoy peaks vs 46603, with IDENTICAL score ranges (IP 5.0-1027.9, control 5.0-46.47). Uniquely-mapped reads IP 34.34M + control 38.88M = 73.2M, matching the paper's stated '73 million reads'. (3) Using the paper's exact FDR definition (#control-peaks/#ChIP-peaks at a score threshold), the 3314-peak set sits at empirical FDR 0.54% and the 4572-peak set at 2.19%; on the discrete score grid FDR<=0.5% gives 3191 and <=3% gives 4917, identical for deposited and reproduced peak sets. Minor +/-15-peak differences are attributable to bowtie version (1.3.1 vs authors' 0.12.5). NOT attempted (honest partial): downstream figures on the peak set (TSS-distance histogram, MBT-signature overlap, KEGG/MGSA enrichment, de-novo insulator motif discovery, insulator-protein Venn) - they need extra annotation/PWM inputs and are secondary to the core peak-calling claim, which is reproduced.
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- Reproduced
- 2026-07-28
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- 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: opusDoes the Drosophila tumor suppressor l(3)mbt cause brain tumors by a mechanism distinct from defective neuroblast asymmetric cell division, namely by failing to repress Salvador-Warts-Hippo (SWH) pathway target genes in optic lobe neuroepithelium? The authors further ask how L(3)mbt achieves this repression at the chromatin level.
- ★ Brain tumors in l(3)mbt mutants originate from overproliferation of neuroepithelial cells of the optic lobes, not from defects in asymmetric cell division. finding
- ★ De-repression of SWH-pathway target genes (diap1, ex, bantam) is the driving event in l(3)mbt tumor formation, and is cell-autonomous. mechanism
- ★ Deregulation of SWH target genes is an essential step in tumor formation, since l(3)mbt tumors are rescued by mutation of bantam or yorkie or by overexpression of expanded. finding
- ★ L(3)mbt binds chromatin insulator elements, overlapping class I insulator proteins CP190, BEAF-32 and CTCF, and its binding sites are enriched for their DNA consensus motifs. mechanism
- ★ L(3)mbt binds directly to SWH-pathway target loci, including diap1 TSS1/TSS2, Cyclin E, and a conserved site 23 kb upstream of bantam. finding
- ★ Insulator protein function is required to control SWH target gene expression: mod(mdg4) knockdown increases bantam activity and mildly upregulates the diap1 reporter. mechanism
- L(3)mbt insulator function is required at the Bithorax complex, as l(3)mbt mutants phenocopy the Abd-B downregulation seen in CTCF mutants. finding
- Genome-wide ChIP-seq map of L(3)mbt binding sites in larval brains and imaginal discs is provided as a resource, plus a specific L(3)mbt antibody and an l(3)mbt shmiR line. resource
| Assay | System | Perturbation | Readout | Platform |
|---|---|---|---|---|
| ChIP-seq (Solexa sequencing), two independent replicates | Drosophila third instar larval brains and imaginal discs | none (wild type) | genome-wide L(3)mbt binding sites, peak assignment to nearest gene, KEGG/GO enrichment, DNA motif enrichment, overlap with published insulator ChIP-chip data | Solexa/Illumina sequencing; custom peakfinder software |
| ChIP followed by quantitative PCR | Drosophila third instar larval brains and imaginal discs | none | L(3)mbt occupancy at diap1 TSS1/TSS2/TSS3, Cyclin E, and bxd PRE of Ubx (negative control) | — |
| Immunostaining and confocal imaging with 3D volume reconstruction | Drosophila larval optic lobe neuroepithelia (IOA and OOA), L1 to third instar | l(3)mbt76, l(3)mbt76/Df(3R)D605, l(3)mbt76/l(3)mbtE2 mutants; brat and lgl mutant comparison | neuroepithelial tissue volume (μm3), Deadpan+ neuroblast number, mitotic pattern, aPKC/Actin polarity, asymmetric determinant segregation | — |
| GAL4/UAS targeted expression screen (dominant active, dominant negative and RNAi constructs) | Drosophila optic lobe neuroepithelium and imaginal disc epithelia, GAL4C855a driver | activation/inhibition of EGF, Jak/STAT, Dpp, FGF and SWH pathways; ex RNAi; Hippo+P35 overexpression; non-phosphorylatable Yorkie; bantam overexpression; epigenetic and polarity genes | IOA/OOA and imaginal disc epithelial size, estimated optic lobe neuroblast number | — |
| Transgenic reporter expression (fluorescence/β-gal immunostaining) | Drosophila larval optic lobes and wing imaginal discs | l(3)mbt mutants; l(3)mbt shmiR driven by GAL4en in posterior wing compartment; mod(mdg4), CTCF, CP190, BEAF-32, Su(Hw) RNAi | diap1-GFP4.3 (TSS2), diap1-GFP5.1 (TSS1), ex-lacZ, bantam-GFP sensor, 10xSTAT92E-GFP levels | — |
| Genetic interaction / rescue analysis | Drosophila larval optic lobes | ban1;l(3)mbt76 double mutants; GAL4C855a>UAS-Ex in l(3)mbt76; yki heterozygosity in l(3)mbt76/E2 | optic lobe neuroepithelial volume (μm3), tumor formation | — |
| Antibody generation, immunostaining, Western blot and live imaging | Drosophila embryos, larval neuroblasts, salivary gland polytene chromosomes | l(3)mbt mutant and l(3)mbt shmiR (specificity controls); RFP-L(3)mbt and GFP-L(3)mbt fusion expression | L(3)mbt subcellular localization (nuclear dots in interphase, cytoplasmic in mitosis), polytene banding pattern vs DAPI, protein levels | — |
| Immunostaining for homeotic gene expression | Drosophila larval central nervous system / posterior ventral nerve cord | l(3)mbt mutant (compared with published CTCF mutant phenotype) | Abdominal-B protein levels | — |
- ▲ Optic lobe neuroepithelial volume is strongly increased in l(3)mbt mutants relative to wild type 1.2×10^5 μm3 (wild type) vs 3×10^5 μm3 (l(3)mbt76) and 8.25×10^5 μm3 (l(3)mbt76/Df(3R)D605)
- – l(3)mbt shmiR expression in optic lobe neuroepithelium causes strong IOA/OOA overproliferation, whereas expression in central brain neuroblasts does not
- ▲ SWH reporters diap1-GFP4.3, diap1-GFP5.1 and ex-lacZ remain/become upregulated and bantam activity increases (ban-GFP sensor lost) in l(3)mbt mutant optic lobes and wing discs
- ▼ ban1 mutation suppresses l(3)mbt neuroepithelial overgrowth and tumor formation 4.5×10^5 μm3 in ban1/+;l(3)mbt76 vs <1×10^5 μm3 in ban1;l(3)mbt76
- ▼ Expanded overexpression and removal of one copy of yorkie each reduce l(3)mbt tumor size 3.3×10^5 → 1.8×10^5 μm3 (UAS-Ex); 3×10^5 → 2.2×10^5 μm3 (yki/+)
- – L(3)mbt ChIP-seq peaks lie close to TSSs and overlap strongly with class I insulator proteins CP190, BEAF-32 and CTCF, less with class II Su(Hw); 4 of 7 enriched motifs match insulator consensus sequences
- ▲ Seven of eleven known/predicted SWH target genes are bound by L(3)mbt, a significant enrichment; diap1 TSS1 binding confirmed and a low-occupancy peak found 23 kb upstream of bantam 7/11 targets; p = 0.025; 3% FDR
- – Expression of non-phosphorylatable Yorkie enlarges IOA and OOA neuroepithelia, phenocopying l(3)mbt; Abd-B is significantly reduced in l(3)mbt mutant CNS as in CTCF mutants five- to ten-fold increase in neuroepithelial size (Yorkie)
- correlation Pearson correlation 0.8835 (reproducibility between two independent L(3)mbt ChIP-seq experiments)
- pvalue p-value 0.025 (enrichment of SWH target genes among L(3)mbt bound genes (3% FDR))
- other 1.2×10^5 μm3 vs 3×10^5 μm3 vs 8.25×10^5 μm3 (mean optic lobe neuroepithelial volume: wild type, l(3)mbt76, l(3)mbt76/Df(3R)D605 third instar larvae)
- other 4.5×10^5 μm3 vs <1×10^5 μm3 (neuroepithelial volume in ban1/+;l(3)mbt76 vs ban1;l(3)mbt76)
- other 3.3×10^5 μm3 vs 1.8×10^5 μm3 (neuroepithelial volume in l(3)mbt76 vs GAL4C855a>UAS-Ex rescue)
- other 3×10^5 μm3 vs 2.2×10^5 μm3 (neuroepithelial volume in l(3)mbt76/E2 vs l(3)mbt76/l(3)mbtE2; yki/+)
- fold_change five to ten fold increased (IOA and OOA neuroepithelial size upon expression of non-phosphorylatable Yorkie)
- count 7 of 11 SWH targets bound; 63% of MBTS genes and 85% of MBTS genes with described germline function had L(3)mbt bound within +/-2 kb (overlap of L(3)mbt ChIP-seq peaks with SWH targets and L(3)mbt signature (MBTS) genes)
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 combines Drosophila genetics (mutant, RNAi/shmiR knockdown, and transgenic overexpression genotypes) with quantitative and qualitative phenotypic readouts (e.g., 3D-reconstructed neuroepithelial volume, reporter/GFP-sensor intensity, immunostaining, cell counts) to compare wild-type/control tissue to l(3)mbt mutant and genetic-interaction genotypes; differences are described as 'significant' or 'significantly increased/reduced' without the underlying statistical test named in the visible text. Genome-wide L(3)mbt chromatin binding was profiled by ChIP-seq (Solexa sequencing) across two replicates, assessed for reproducibility via Pearson correlation, and target genes were characterized using KEGG pathway and GO enrichment analyses, including one gene-set enrichment reported with an exact p-value (0.025) and a 3% FDR. No dedicated statistics subsection or software citation for hypothesis testing appears in this excerpt.
| Test | Applied to | n | Assumptions |
|---|---|---|---|
| Pearson correlation between ChIP-seq replicates | Fig 6c / Supplementary Fig S6a - reproducibility of two independent ChIP experiments | two independent ChIP experiments | not stated |
| enrichment/overrepresentation test (specific test not named), reported with a p-value and FDR | Fig 6f / Supplementary Fig S6e - overrepresentation of SWH-pathway target genes among L(3)mbt-bound genes | 7 of 11 known/predicted SWH target genes | not stated |
| pathway/gene-set enrichment analysis (KEGG, GO) | Fig 6d, Supplementary Fig S6c - characterization of L(3)mbt-bound gene set | — | not stated |
| statistical test not specified (result described as 'significantly increased') | Fig 1f - 3D-reconstructed optic lobe neuroepithelial volume, wild-type vs l(3)mbt mutant genotypes | — | not stated |
| statistical test not specified (result described as 'significantly reduced') | Fig 4a,b - neuroepithelial size in l(3)mbt vs ban;l(3)mbt double mutants | — | not stated |
| statistical test not specified (result described as 'significantly upregulated'/'significant reduction') | Fig 6e (10xSTAT92E-GFP reporter) and Fig 7d (Abd-B expression), mutant vs control | — | not stated |
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Several quantitative comparisons (e.g., neuroepithelial volume in Fig 1f, 4a-b) are described as 'significantly' increased or reduced without naming the statistical test applied.↳ Could also: Explicitly naming the test used (e.g., Student's t-test if data are roughly normal, or a Mann-Whitney U test if not) alongside the exact p-value — Stating the specific test and its assumptions lets readers evaluate whether the test matches the data distribution and independently assess the strength of the reported difference.
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Sample sizes (n per genotype/condition) for volume, reporter-intensity, and other quantitative measurements are not stated in the visible text.↳ Could also: Reporting n per group and showing individual data points (e.g., dot/scatter plots) alongside summary statistics — This would let readers gauge the variability underlying each 'significant' claim and assess whether conclusions are robust to the observed spread.
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Quantitative results are presented as means/point estimates without an accompanying measure of dispersion.↳ Could also: Including SD, SEM, or a 95% confidence interval alongside each mean — A dispersion measure conveys how consistent the underlying replicates are and is a standard complement to a significance claim, particularly for small sample sizes.
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The SWH target gene enrichment among L(3)mbt-bound genes is reported with a single p-value (0.025) and one FDR figure (3%).↳ Could also: Reporting FDR-adjusted q-values for each tested category using a standard hypergeometric or Fisher's exact test framework with Benjamini-Hochberg correction across all KEGG/GO categories examined — This would make explicit how multiplicity was handled across the full set of pathways tested, which is a standard practice when many categories are screened simultaneously.
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Reproducibility between the two ChIP-seq replicates is assessed using a Pearson correlation coefficient (0.8835) on peak signal.↳ Could also: Applying an Irreproducible Discovery Rate (IDR) framework, which is a widely used approach for ChIP-seq peak-level reproducibility — IDR is designed specifically for peak-calling reproducibility and can complement a genome-wide correlation coefficient with a peak-specific reproducibility metric.
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KEGG and GO enrichment analyses of L(3)mbt-bound genes are used to characterize the target gene set, with the SWH pathway added manually since it is not annotated in these databases.↳ Could also: Using a gene-set enrichment tool that accepts custom/user-defined gene sets natively (e.g., a hypergeometric test implemented in clusterProfiler or a similar package) alongside the standard KEGG/GO databases — This would integrate a manually curated pathway (like SWH) into the same statistical framework as the annotated databases, keeping enrichment statistics and multiple-testing correction consistent across all tested gene sets.
What was reproduced
The exact results taken into scope, with each reported value next to the value our attempt produced.
Assessments & scoring basis
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Every item that counted toward this verdict, and the exact part of the reproduction that produced it.
Model case of a clean computational reproduction. The authors deposited both raw reads (SRA SRP006763, 4 runs) and their own peak caller (pypeak), and a from-scratch re-run regenerates the deposited intermediates to within ±15 peaks (18544 vs 18529 IP; 46618 vs 46603 control) with identical score ranges (IP 5.0–1027.9, control 5.0–46.47); the final deposited files contain exactly the reported 3314 and 4572 sites, with the 0.5% set a strict subset of the 3% set. The only residual deviations sit on our side and are technical: bowtie 1.3.1 vs the authors' 0.12.5, and discrete-grid rounding when mapping an FDR cutoff to a score threshold (3314 = empirical FDR 0.543%, versus 3191 at a strict ≤0.5% grid cutoff). Severity is negligible and the central claim holds fully; the only fair criticism of the authors is documentation — pypeak parameters and the FDR-cutoff rule are absent from the Methods — which is a methodology_gap, not a derivability problem. Secondary downstream figures were honestly declared out of scope rather than claimed.
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