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Enhancer buffering protects dosage-sensitive housekeeping genes during vulnerable developmental transitions

Chunyang Ni, Lucia IchinoiD, Tomek SwigutiD, Joanna WysockaiD

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In-depth summary

Claude Opus 5.5 · xhigh effort · 7 Nov 2026 · from the full text of version 2 (31 Aug 2026) · CC BY 4.0

TCOF1 dosage is limiting for transitional human neural crest cells; its enhancers buffer promoter output against MYC fluctuations rather than amplifying it, a pattern seen across 14 dosage-sensitive promoters.

Housekeeping genes keep stable expression across cell types, yet their haploinsufficiency causes tissue-specific congenital disease, with TCOF1 in Treacher Collins syndrome (TCS) as the test case. The paper argues that enhancers at such promoters, which show little amplification in earlier reporter assays, instead act as buffers that are revealed when promoter-regulating factors fluctuate.

Key results

  1. ShownTransitional cranial neural crest cells (tCNCC) are lost with modest TCOF1 reduction, and TCOF1 is far more limiting than POLR1A or POLR1B for tCNCC yield and rRNA synthesis.

    Degron titration: tCNCC fell dose-dependently at 82% and 60% TCOF1 (scRNA-seq). tCNCC yield halved at 57% TCOF1 vs 11% POLR1A (ED50). About 50% loss of POLR1A/B had no effect, while TCOF1 induced haploinsufficiency gave a mean difference of -84.3% (p = 1.5×10-3, n=3). · High vulnerability of transitional CNCC states…; TCOF1 but not POLR1 shows high dosage…; Figure legends

  2. ShownNeural crest cells are more sensitive than embryonic stem cells (ESC) to loss of rRNA synthesis, which may compound TCOF1 dosage sensitivity into tissue-selective disease.

    At matched 5-EU-measured rRNA synthesis reductions, CNCC viability (CellTiter-Glo) fell steeply while ESC tolerated substantial loss. p53-linked stress genes rose in CNCC by 6 h (~70% 5-EU signal), with no ESC response at 6 h. · CNCC are highly sensitive to perturbations in…; Figure legends

  3. ShownThree CNCC-stage enhancers (E1-E3) modestly support TCOF1 expression, and deleting them blocks the mesenchymal transition, phenocopying TCOF1 reduction.

    Homozygous E123 deletion lowered TCOF1 by 11% (tCNCC2), 30% (tCNCC3) and 10% (passaged CNCC) by RT-ddPCR, with no change in NE/tCNCC1. Two clones lost tCNCC2/3 and migrated less; transcriptome correlation with TCOF1 depletion rose across stages. · Developmental enhancers support stage-specific…; TCOF1 enhancers support CNCC developmental…; Figure legends

  4. ShownThe enhancers convert a linear TCOF1 promoter response to MYC into a threshold-protected one, without raising baseline output.

    Wild-type CNCC kept TCOF1 transcript stable under moderate MYC depletion; E123KO fell immediately. In reporters, TCOF1+E123 stayed buffered until MYC reached 25%, while the promoter alone declined linearly. E123 raised the SV40 promoter ~90 fold but left the TCOF1 promoter unchanged. · Enhancers buffer TCOF1 expression against…; Figure legends

  5. SuggestedThe buffering pattern held for 14 dosage-sensitive housekeeping (DSH) promoters: minimal enhancer amplification, but enhancer-dependent stability when MYC is lowered.

    DSH set chosen by pHaplo > 0.8 and expression CV < 0.06. TCOF1 E1 barely boosted them in CNCC luciferase reporters, unlike the SV40 promoter. At 55% MYC the promoters alone lost expression; with E1 expression stayed near complete (paired Wilcoxon, n=14 promoters). · Enhancer-mediated buffering represents a…; Figure legends

Methods, data and reproducibility

Human ESC-derived CNCC differentiation with endogenous degron tags (dTAGv-1) for graded protein dosage, read out by scRNA-seq, flow cytometry, 5-EU rRNA synthesis, ATAC-seq and RT-ddPCR. Enhancer function was tested by CRISPR deletion, a MYC degrader, and luciferase reporters, with dose-response models (linear, log-logistic) compared by BIC.

  • Day 8 CNCC differentiation scRNA-seq across genotypes and dTAGv-1 doses (Seurat, Harmony) · human (H9 hESC) · n = 12 samples merged; ~2000 cells captured per sample
  • Bulk ATAC-seq of sorted NE and tCNCC1-3 populations at day 8 · human (H9 hESC) · n = 2 independent differentiations (representative replicate shown)
  • Bulk RNA-seq of CNCC vs ESC under matched POLR1A titration · human (H9 hESC)
  • Luciferase reporter assays: 14 DSH promoters, TCOF1 promoter and enhancers E1-E3, with and without MYC degrader · human (CNCC) · n = 8 to 10 transfections per assay across 2 to 3 differentiations
  • Reanalysed MYC ChIP-seq from neuroblastoma cell lines (Upton et al. 2020) · human · GSE138315
Methods & tools
Seurat v4, Harmony, Monocle3 v0.2.0, DESeq2, DRAGEN, BWA 0.7.19, samtools 1.22.1, bedtools v2.31.1, salmon, dTAGv-1 degron (FKBP12 F36V), MYC degrader 1 (MCE HY-162318), pHaplo scores
Reagents & environment
Seurat v4 on R 4.6; Monocle3 v0.2.0; BWA 0.7.19-r1273; samtools 1.22.1; bedtools v2.31.1. No container or requirements file stated.
Reproducibility gaps
No code repository is stated. The data statement points to the text, supplement and/or GEO, with accessions of reanalysed public data in Table S2; no accession is printed for the new sequencing data.
Limitations (3)
  • Enhancer deletion reduced TCOF1 only subtly (11% to 30%), so TCOF1 transcription does not strictly depend on these enhancers. · stated by the authors
  • Generalisation rests on 14 promoters tested with a single TCOF1 enhancer (E1) in episomal luciferase reporters, and with MYC as the only perturbed promoter regulator. · noted in this summary
  • Everything is in one hESC line (H9) in vitro, and several scRNA-seq and rRNA-synthesis comparisons use only n=2 differentiations. The prenatal MYC-stabilisation idea is untested. · noted in this summary

Read this if you study enhancer function at housekeeping or haploinsufficient genes, or want degron dose-response data in human neural crest differentiation.

Citations
1

Abstract

Housekeeping genes maintain robust expression across cell types despite dynamic transcription factor fluctuations, yet their haploinsufficiency is associated with many tissue-specific developmental disorders. To understand this paradox, we focus on TCOF1, a broadly expressed regulator of rRNA synthesis, whose haploinsufficiency causes Treacher Collins syndrome (TCS). We show that transitional cranial neural crest cells (tCNCC) undergoing mesenchymal specification exhibit extreme dosage-sensitivity to TCOF1, but not to RNA polymerase I, explaining both cellular origins of TCS and prevalence of TCOF1 mutations in the disease. To maintain robust expression, TCOF1 deploys CNCC-specific enhancers that buffer against fluctuations in promoter-regulating factors, converting sensitive expression responses into threshold-protected outputs. Systematic promoter-enhancer coupling experiments demonstrate this principle generalizes across many dosage-sensitive housekeeping genes, whose promoters operate near saturation and only reveal their enhancer-dependency under suboptimal conditions. Thus, enhancers play a unique role at housekeeping promoters: rather than amplifying expression, they ensure its robustness during vulnerable developmental transitions.

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