BRCA1 haploinsufficiency for replication stress suppression in primary cells

BRCA1—a breast and ovarian cancer suppressor gene—promotes genome integrity. To study the functionality of BRCA1 in the heterozygous state, we established a collection of primary human BRCA1+/+ and BRCA1mut/+ mammary epithelial cells and fibroblasts. Here we report that all BRCA1mut/+ cells exhibited multiple normal BRCA1 functions, including the support of homologous recombination- type double-strand break repair (HR-DSBR), checkpoint functions, centrosome number control, spindle pole formation, Slug expression and satellite RNA suppression. In contrast, the same cells were defective in stalled replication fork repair and/or suppression of fork collapse, that is, replication stress. These defects were rescued by reconstituting BRCA1mut/+ cells with wt BRCA1. In addition, we observed ‘conditional’ haploinsufficiency for HR-DSBR in BRCA1mut/+ cells in the face of replication stress. Given the importance of replication stress in epithelial cancer development and of an HR defect in breast cancer pathogenesis, both defects are candidate contributors to tumorigenesis in BRCA1-deficient mammary tissue

Combined immunodeficiency and Epstein-Barr virus-induced B cell malignancy in humans with inherited CD70 deficiency

In this study, we describe four patients from two unrelated families of different ethnicities with a primary immunodeficiency, predominantly manifesting as susceptibility to Epstein-Barr virus (EBV)–related diseases. Three patients presented with EBV-associated Hodgkin’s lymphoma and hypogammaglobulinemia; one also had severe varicella infection. The fourth had viral encephalitis during infancy. Homozygous frameshift or in-frame deletions in CD70 in these patients abolished either CD70 surface expression or binding to its cognate receptor CD27. Blood lymphocyte numbers were normal, but the proportions of memory B cells and EBV-specific effector memory CD8+ T cells were reduced. Furthermore, although T cell proliferation was normal, in vitro–generated EBV-specific cytotoxic T cell activity was reduced because of CD70 deficiency. This reflected impaired activation by, rather than effects during killing of, EBV-transformed B cells. Notably, expression of 2B4 and NKG2D, receptors implicated in controlling EBV infection, on memory CD8+ T cells from CD70-deficient individuals was reduced, consistent with their impaired killing of EBV-infected cells. Thus, autosomal recessive CD70 deficiency is a novel cause of combined immunodeficiency and EBV-associated diseases, reminiscent of inherited CD27 deficiency. Overall, human CD70–CD27 interactions therefore play a nonredundant role in T and B cell–mediated immunity, especially for protection against EBV and humoral immunity

Role of Bre2 and Sdc1 in the regulation of histone lysine methylation by Set1 Complex

The Set1 complex, often called COMPASS (Complex associated with Set1 subunit), is a multi-protein complex containing the trithorax-related protein, Set1, which methylates histone H3 lysine-4 (H3K4) and additional substrates. The catalytic SET domain of Set1 requires a host of associated subunits to catalyze the transfer of methyl groups from S-adenosylmethionine to lysine residues of target substrates. Bre2 and Sdc1 are two subunits that promote H3K4 tri-methylation, a hallmark modification found at promoters of activatated RNA polymerase II genes. In collaboration with Sharon Dent\u27s group, we have previously demonstrated that Bre2 and Sdc1 are methylated by Set1 in vitro and in vivo. The significance of Bre2 methylation in regulating Set1 catalytic activity is demonstrated by changes in the global levels of H3K4 di-methylation upon blocking methylation of Bre2 at a specific lysine residue (K287). Indirect evidence suggests that demethylation of Bre2 and Sdc1 is possibly mediated by the H3K4 demethylase complex. The mechanism by which Bre2 and Sdc1 regulate Set1\u27s catalytic activity is not well understood, and represents the driving impetus for the current studies. In the present study we show that Sdc1 physically interacts with the C-terminus of Bre2, and that a Bre2/Sdc1 subcomplex can stably interact with the SET domain of Set1. We describe the development of an immobilized methyltransferase activity assay, and use this assay to demonstrate reconstitution of a Set1 complex functional for subunit methylation. The latter result suggests that Bre2/Sdc1 exchange between methyltransferase and demethylase complexes is a tenable hypothesis