Enhancers act to regulate cell type specific gene expression by facilitating the transcription of target genes. In mammalian cells active or primed enhancers are commonly marked by monomethylation of Histone H3 at lysine 4 (H3K4me1) in a cell-type specific manner. Whether and how this histone modification regulates enhancer-dependent transcription programs in mammals is unclear. In this study, we conducted SILAC Mass-spec experiments with mono-nucleosomes and identified multiple H3K4me1 associated proteins, including many involved in chromatin remodeling. We demonstrate that H3K4me1 augments the association of the chromatin remodeling complex BAF to enhancers in vivo and that in vitro, H3K4me1 nucleosomes are more efficiently remodeled by the BAF complex. Crystal structures of BAF component BAF45c reveal that monomethylation, but not trimethylation, is accommodated by BAF45c’s H3K4 binding site. Our results suggest that H3K4me1 plays an active role at enhancers by facilitating the binding of the BAF complex and possibly other chromatin regulators.

The relevance of many BRCA2 variants of uncertain significance (VUS) to breast cancer has not been determined due to limited genetic information from families carrying these alterations. Here we classified six new variants as pathogenic or non-pathogenic by analysis of genetic information from families carrying 65 individual BRCA2 DBD missense mutations using a multifactorial likelihood model of cancer causality. Next, we evaluated the utility of a homology directed DNA break repair (HDR) functional assay as a method for inferring the clinical relevance of VUS in the DNA binding domain (DBD) of BRCA2 using 18 established non-pathogenic missense variants and all 13 established pathogenic missense mutations from the BRCA2 DBD. Compared to the known status of these variants based on the multifactorial likelihood model, the sensitivity of the HDR assay for pathogenic mutations was estimated at 100% (95%CI: 75.3%-100%), and specificity was estimated at 100% (95%CI: 81.5%-100%). A statistical classifier for predicting the probability of pathogenicity of BRCA2 DBD variants was developed using these functional results. When applied to 33 additional VUS, the classifier identified eight with >99% probability of non-pathogenicity and 18 with ≥ 99% probability of pathogenicity. Thus, in the absence of genetic evidence a cell based HDR assay can provide a probability of pathogenicity for all VUS in the BRCA2 DBD, suggesting that the assay can be used in combination with other information to determine the cancer relevance of BRCA2 VUS.