BARD1 is necessary for ubiquitylation of nucleosomal histone H2A and for transcriptional regulation of estrogen metabolism genes

Abstract

Missense mutations that disrupt the RING domain of the tumor suppressor gene lead to increased risk of breast and ovarian cancer. The BRCA1 RING domain is a ubiquitin ligase, whose structure and function rely critically on forming a heterodimer with BARD1, which also harbors a RING domain. The function of the BARD1 RING domain is unknown. In families severely affected with breast cancer, we identified inherited BARD1 missense mutations Cys53Trp, Cys71Tyr, and Cys83Arg that alter three zinc-binding residues of the BARD1 RING domain. Each of these mutant BARD1 proteins retained the ability to form heterodimeric complexes with BRCA1 to make an active ubiquitin ligase, but the mutant BRCA1/BARD1 complexes were deficient in binding to nucleosomes and in ubiquitylating histone H2A. The BARD1 mutations also caused loss of transcriptional repression of BRCA1-regulated estrogen metabolism genes and ; breast epithelial cells edited to create heterozygous loss of showed significantly higher expression of and Reintroduction of wild-type into these cells restored and transcription to normal levels, but introduction of the cancer-predisposing RING mutants failed to do so. These results indicate that an intact BARD1 RING domain is critical to BRCA1/BARD1 binding to nucleosomes and hence to ubiquitylation of histone H2A and also critical to transcriptional repression of BRCA1-regulated genes active in estrogen metabolism.

Synergistic lethality between BRCA1 and H3K9me2 loss reflects satellite derepression

“…Mutations that compromise the BRCA1/BARD1 complex, a multifunctional E3 ubiquitin ligase implicated in the repair of DSB, represent the highest risk factor for hereditary forms of breast and ovarian cancer (King et al 2003). While this complex is best known to promote homologous recombination at DSB, BRCA1 is also implicated in interstrand cross-link repair and in replication fork stability, given that the complex associates both with stalled DNA and RNA polymerases (Schlacher et al 2012;Urban et al 2016;Stewart et al 2018). Moreover, BRCA1 was shown to recruit the RNA/DNA helicase senataxin to sites of paused RNA polymerase II, to ensure mRNA termination and prevent ensuing damage (Hatchi et al 2015).…”

Preparation and purification of mono-ubiquitinated proteins using Avi-tagged ubiquitin

“…Finally, mono-ubiquitination of nucleosomal histone H2A by BRCA1:BARD1, has also been shown to regulate DNA repair in the FA:BRCA pathway [4,16]. Using the Avi-ubiquitin system, we were able to purify mono-ubiquitinated histone H2A within a nucleosome ( Fig 3D).…”

“…Third, unhooking of the ICL creates a free DNA end that stimulates nucleosome mono-ubiquitination by BRCA1, which stimulates recombination and replication fork stabilization [15]. BRCA1, and its partner protein BARD1, interact through a RING-domain heterodimer to mono-ubiquitinate nucleosomes in concert with the UBCH5C E2-enzyme [4,16]. The BRCA1:BARD1 E3-complex specifically directs mono-ubiquitination to lysines 125, 127 and 129 of histone H2A both in vitro and in vivo [4], which is hypothesized to promote long-range resection of the DNA by SMAR-CAD-mediated chromatin unloading [17].…”

Literature Review of BARD1 as a Cancer Predisposing Gene with a Focus on Breast and Ovarian Cancers

“…One frameshift BARD1 variant was identified in a young age of onset BC case (29 years old at diagnosis) by multi-gene panel testing; this variant is predicted to result in premature termination of the protein and partial loss of the RING domain [46]. The BARD1 c.247A>G; p.Cys83Arg protein isoform retained the ability to interact with BRCA1 and retained E3 ubiquitin ligase activity, but the BARD1-BRCA1 complex was less efficient in binding to the nucleosomes and ubiquitylating histone H2A [117]. In contrast, the BARD1 c.253G>T; p.Val85Leu variant had no effect on the BARD1-BRCA1 interaction and homologous recombination DNA repair activity [118].…”