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BRD4 represses developmental and neuronal genes through interaction with PRC1.6

Fanny BouletiD, Manthan PateliD, Zeinab Shadman Zanjani, Nuria Andrés-SáncheziD, Anam IjaziD, Debosree PaliD, Pankaj Dubey, Aoife MurrayiD, Dean NižetićiD, Michael D. LeClaireiD, Karina L. BurschiD, Brian C. SmithiD, Pradeepa M. MadapuraiD

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Abstract

BRD4 is best known as a transcriptional co-activator, yet heterozygous loss-of-function variants cause craniofacial and neurodevelopmental abnormalities through unclear mechanisms. Using human embryonic stem cells and neural organoids as an in vitro model of human embryonic brain development, we show that BRD4 represses polycomb-regulated developmental and neuronal genes. BRD4 co-occupies bivalent promoters with PRC1.6 and directly interacts with PCGF6 and RING1B through its C-terminal domain. H3K23ac contributes to BRD4 recruitment through BD2, linking acetylated chromatin recognition to polycomb-associated repression. Acute BRD4 depletion alters PRC1.6 occupancy, reduces ubiquitination of H2AK119 and rapidly increases developmental gene transcription. In neural organoids, BRD4 BD2 mutations alter neuronal cell composition and increase expression and chromatin accessibility associated with neuronal transcription-factor programmes. These findings establish BRD4 as a context-dependent chromatin regulator that prevents premature activation of developmental genes and provide mechanistic insight into BRD4-associated neurodevelopmental disorders.

In-depth summary

Claude Sonnet 4.6 · xhigh effort · 8 Nov 2026 · from the full text of version 4 (5 Aug 2026) · CC BY 4.0

Acute BRD4 degradation and BD2 mutations de-repress bivalent developmental and neuronal genes in human stem cells and organoids, consistent with BRD4 recruitment by H3K14ac/H3K23ac and interaction with PRC1.6.

BRD4 is known as a general transcriptional activator, yet its loss causes specific neurodevelopmental defects, and its link to Polycomb-mediated repression was unclear. This work ties BRD4 to PRC1.6 and bivalent promoters as a repressive mechanism.

Key results

  1. ShownShort-term BRD4 loss quickly raises expression of neurodevelopmental and brain-specific genes in hESCs, and BRD4 is bound at their promoters, which suggests direct repression.

    C1 genes rose from 4 h and C2 genes at 8 and 20 h of ZxH, also with 20 h dTAGV-1 (adj-p<0.05 clustering). TT-seq after 1 h dTAG raised LMO2, OLIG2, NEUROG2, YBX2 and SYCE2, and lowered MYC. · BRD4 represses neurodevelopmental genes in stem…; BRD4 co-occupies and regulates PRC1.6 and…

  2. ShownBRD4 co-occupies PRC1.6-bound and bivalent promoters, and many PRC1.6 targets are up when BRD4 or PCGF6 is lost.

    1311 BRD4 peaks overlapped bivalent promoters (with EED and PolII-S5p) vs 19784 peaks at active elements. 454 genes were up in both PCGF6-KO and BRD4 degradation; 459 were down. Nearly 50% of bivalent genes rose at 8 h ZxH. · BRD4 is enriched at PRC-repressed and bivalent…; BRD4 co-occupies and regulates PRC1.6 and…

  3. ShownBRD4 physically associates with PRC1.6 subunits and EED, but not with canonical PRC1 (CBX7) or EZH2, in the assays tested.

    Public K562 IP-MS enriched RING1B, MGA, PCGF6, CBX3 and WDR5. Endogenous IP detected RING1B and PCGF6 but not CBX7. In HEK293T, GFP-BRD4-L co-precipitated E2F6, L3MBTL2 and EED, not EZH2. · BRD4 interacts with PRC1.6 and PRC2 proteins

  4. ShownThe second bromodomain of BRD4 binds H3K14ac/H3K23ac, a mark enriched at bivalent promoters, which provides a candidate recruitment route to PRC-marked chromatin.

    AlphaScreen: BD2, not BD1, bound the H3K14/K23ac peptide. A peptide pulldown with BD1-BD2 (E49-E460) was sensitive to iBET-BD2. PLA foci for BRD4-H3K23ac and BRD4-H3K27me3 fell after iBET-BD2 in hESCs. · BRD4 is recruited to PRC-marked regions through…

  5. SuggestedBD2 mutations upregulate bivalent and developmental genes and shift organoid composition toward diencephalic, optic vesicle and RPE cells. HOX and ZIC are upregulated, and the mut2 line shows matching motif accessibility gains in day 41 UNOs.

    Homozygous BD2mut1 hESCs: upregulated (n=1800) and downregulated (n=2502) genes overlapped significantly with 20 h dTAGV-1 degradation; 672 bivalent genes were up in neurons. Two heterozygous mutant UNOs (11746 cells day 41; 10537 day 63): fewer Diencephalic progenitor 1, more cluster 2, optic vesicle and RPE. · BRD4 BD2 mutations lead to upregulation of…; BRD4 mutations lead to upregulation of neuronal…; Upregulated transcription factor families lead…

Methods, data and reproducibility

Acute BRD4 degradation (PROTAC ZxH-3-26, dTAG) and CRISPR BD2 mutants in hESCs, neurons and unguided neural organoids, profiled by RNA-seq, TT-seq, CUT&Tag/CUT&RUN, IP-MS reanalysis, binding assays, scRNA-seq and scATAC-seq.

  • H9 hESC bulk RNA-seq after ZxH (4, 8, 20 h) or dTAGV-1 (20 h, BRD4-dTAG line) BRD4 degradation · human · n = at least 3 replicates (ZxH 4 h DMSO has 2) · GSE313810
  • BRD4 and interactor CUT&Tag and CUT&RUN in hESCs · human · n = at least two replicates · GSE313479
  • scRNA-seq of WT and two heterozygous BD2 mutant UNOs, days 41 and 63 · human · n = 11746 cells day 41; 10537 cells day 63
  • scATAC-seq of day 41 UNOs, WT vs BRD4 mut2 · human · n = 4236 WT and 4103 mut2 cells · GSE314451
  • Public PCGF6 signal data (PGP1 iPSC line, two merged replicates); PCGF6-KO hPSC RNA-seq reanalysed · human · GSE173690
  • Public scCUT&Tag of UNOs (H3K4me3, H3K27ac, H3K27me3) used to define bivalent genes (Zenk et al) · human · n = days 35, 60, 120
  • Public BRD4 IP-MS in K562 (Arnold et al 2021) and mouse ESC proximal interactomes of PRC components · human (K562); mouse
Methods & tools
ZxH-3-26 PROTAC, dTAG degron system, AlphaScreen, DESeq2, SEACR, bowtie2, deepTools, STAR, Seurat 5.3.0, Cluster similarity spectrum (CSS), ArchR, gimme maelstrom
Reagents & environment
Seurat 5.3.0; CellRanger with GRCh38 (2020-A) and CellRanger-ATAC with GRCh38 (2020-A-2.0.0); hg38 for CUT&Tag, CUT&RUN and RNA-seq; ArchR for scATAC-seq.
Reproducibility gaps
No code repository is given. scRNA-seq is only on a Broad portal reviewer-access link, with no accession. Plotted data are in Supplementary Data files.
Limitations (4)
  • The authors note that C1/C2 upregulation weakened by 20 h of degrader treatment, which they attribute possibly to drug decay or broad effects of long-term perturbation. · stated by the authors
  • Whether MAX/MGA recruit BRD4 to PRC1.6 targets, or the reverse, is left open by the authors. · stated by the authors
  • The organoid conclusions rest on two heterozygous mutant clones and one isogenic WT clone, with pooled-cell libraries. Biological replicate organoid batches are not described, so clone-level variability is not addressed. · noted in this summary
  • BRD4 requirement for PRC1.6 or EED recruitment is inferred from co-occupancy, co-IP and IP-MS. Loss of PRC1.6 or EED occupancy after BRD4 depletion was not shown. · noted in this summary

Read this if you study BET proteins, Polycomb repression, or how histone acetylation and bivalent chromatin shape neurodevelopmental disorders, using organoid single-cell data.

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