BRD4 interacts with NIPBL and BRD4 is mutated in a Cornelia de Lange-like syndrome

Abstract

We found that the clinical phenotype associated with BRD4 haploinsufficiency overlapped with that of Cornelia de Lange syndrome (CdLS), which is most often caused by mutation of NIPBL. More typical CdLS was observed with a de novo BRD4 missense variant, which retained the ability to coimmunoprecipitate with NIPBL, but bound poorly to acetylated histones. BRD4 and NIPBL displayed correlated binding at super-enhancers and appeared to co-regulate developmental gene expression.

Figure 1. BRD4 mutations in CdLS and CdLS-like disorders.

A. Pedigree drawing of proband with CdLS-like disorder associated with a de novo 1.04 Mb microdeletion of 19p (red bar), the location of which is shown on the Log₂ ratio plot of the array-based comparative genomic hybridization. Below on the left is a key to the symbols using in the pedigree images. To the right of this is the RefSeq gene content of the deleted region with the location of BRD4 indicated in orange. The genes colored green are known disease genes associated with phenotypes that are not consistent with the clinical presentation in this case. Details of each is given in the supplementary notes. B. Pedigree drawings and facial photographs of probands with intragenic mutations of BRD4, with cartoon of BRD4 protein indicating the position of each of the variants in relation to the first bromodomain (BD1), the second bromodomain (BD2) and the N-terminal extra terminal domain (NET). The position of an inherited p.His304Tyr variant (orange text) reported in a single family with inherited cataracts is indicated and discussed in supplementary notes.

Figure 2. Binding of BRD4 wild-type and BRD4 p.Tyr430Cys variant to histone and non-histone proteins.

A. Specificity of binding to acetylated histone tail peptides of wild-type BRD4 Bromodomain 1 (BRD4 BD1 WT), wild-type BRD4 Bromodomain 2 (BRD4 BD2 WT), and BRD4 p.Tyr430Cys mutant BD2 (BRD4 BD2 Y430C). B. Cropped immunoblots of endogenous BRD4 IPs and rabbit normal IgG (control) from Brd4 wild-type and Brd4^Y430C/Y430C mESCs. Input =1% of mESC nuclear extract. Antibodies detect BRD4, H3K9ac, H3K27ac, H4K8ac and H3. C. Heatmap of the label-free mass spectrometry quantitative output values (average of triplicates) assigned to each protein following IP from BRD4 wild-type (WT) and BRD4-Y430C (MUT) mESC using IgG only control or Abcam/Bethyl antibody against BRD4. D. A plot of the log Andromeda scores assigned to the 90 proteins which are absent in the IgG controls and present in both cell lines using both BRD4 antibodies. Horizontal scatter aids visibility of each open circle and has no data correlate. E. Cropped immunoblot of reciprocal IP using BRD4 and NIPBL antibodies in Brd4 wild-type and Brd4^Y430C/Y430C mESCs. Antibodies detect BRD4, NIPBL and SOX2. F. Percentage (%) input bound for BRD4 ChIP-qPCR across genomic regions in WT and BRD4 Y430C mutant MEFs (error bars = standard error of the mean from n=2 biological replicates). G. Forest plot of the log2 odds ratio with confidence intervals (CI) of different functional genomic categories within intersecting regions from BRD4 and NIPBL mESC ChIP. H. UCSC Genome Browser graphic showing colocalisation of BRD4 and NIPBL ChIPseq peaks over the super enhancer (blue bar) at Klf4 locus. H3K27ac, H3K4me1, H3K122ac and super enhancer tracks are previously published.