Cornelia-de Lange syndrome-associated mutations cause a DNA damage signalling and repair defect

Abstract

Cornelia de Lange Syndrome is a multisystem developmental disorder typically caused by mutations in 51 the gene encoding the cohesin loader NIPBL. The associated phenotype is generally assumed to be 52 the consequence of aberrant transcriptional regulation. Recently, we identified a residue substitution in 53 BRD4 associated with a Cornelia de Lange-like syndrome, that reduces BRD4 binding to acetylated 54 histones. Here we show that, although this mutation reduces BRD4-occupancy at enhancers in mouse 55 embryonic stem cells, it does not affect transcription. Rather it delays the cell cycle, increases DNA 56 damage signalling, and perturbs regulation of DNA repair in mutant cells. This uncovers a new role for 57 BRD4 in DNA repair pathway choice. Furthermore, we find evidence of a similar increase in DNA 58 damage signalling in cells derived from NIPBL-deficient individuals, suggesting that defective DNA 59 damage signalling and repair is also a feature of typical Cornelia de Lange Syndrome. 60 61 65 delayed growth 1 . CdLS is described as a 'cohesinopathy' 1 -most cases can be attributed to 66 heterozygous loss of function mutation in NIPBL encoding a protein involved in loading of the cohesin 67 complex onto chromatin 2 . Mutation in genes encoding cohesin complex proteins SMC1, SMC3 and 68 RAD21, or HDAC8 (SMC3 deacetylase), have also been identified in CdLS-like probands 2 . However 69 cells from CdLS patients have no obvious defects in sister chromatid cohesion 3 , and individuals with 70 mutations in SMC1, SMC3 and RAD21 are often considered 'atypical' in terms of facial appearance and 71 growth, and are less likely to have limb defects than those with NIPBL mutations 4 . 72Dysregulated gene expression has been proposed to be main mechanism underlying CDLS 5,6 . 73Mutations in genes encoding chromatin regulators unrelated to cohesin, such as ANKRD11, KMT2A, 74AFF4 and the bromodomain and extra-terminal domain (BET) protein BRD4, have been reported to 75 cause CdLS-like phenotypes 1 suggesting that chromatin dysregulation may play a role in CdLS as well. 76Additionally, increased sensitivity to DNA damage has been reported in CdLS patient cells 7 , but the 77 mechanism underlying this defect is unknown and its participation in the disease aetiology remains 78 unclear. 79Recently, we described de novo deletion and missense mutations in BRD4 associated with a clinical 80 phenotype overlapping CdLS 8 . BRD4 binds acetylated lysines residues in histones H3 and H4 through 81 its two N-terminal bromodomain domains (BD). BRD4 localises to promoters and enhancers of active 82 genes and is particularly enriched at super enhancers (SEs) 9,10 . BRD4 is a key regulator of transcription;; 83 through its C-terminal domain it recruits positive transcription elongation factor (P-TEFb) and the 84 Mediator complex to promoters and enhancers, whilst its extra-terminal domain confers transcriptional 85 activation through the recruitment of CHD4, JMJD6, and NSD3 11,12 . 86 3 The CdLS-associated BRD4 missense mutation is in the ...