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The identification of the breast cancer susceptibility genes BRCA1 and BRCA2 enhanced clinicians' ability to select high-risk individuals for aggressive surveillance and prevention, and led to the development of targeted therapies. However, BRCA1/2 mutations account for only 25% of familial breast cancer cases. To systematically identify rare, probably pathogenic variants in familial cases of breast cancer without BRCA1/2 mutations, we developed a list of 312 genes, and performed targeted DNA enrichment coupled to multiplex next-generation sequencing on 104 'BRCAx' patients and 101 geographically matched controls in Ireland. As expected, this strategy allowed us to identify mutations in several well-known high-susceptibility and moderate-susceptibility genes, including ATM (~ 5%), RAD50 (~ 3%), CHEK2 (~ 2%), TP53 (~ 1%), PALB2 (~ 1%), and MRE11A (~ 1%). However, we also identified novel pathogenic variants in 30 other genes, which, when taken together, potentially explain the etiology of the missing heritability in up to 35% of BRCAx patients. These included novel potential pathogenic mutations in MAP3K1, CASP8, RAD51B, ZNF217, CDKN2B-AS1, and ERBB2, including a splice site mutation, which we predict would generate a constitutively active HER2 protein. Taken together, this work extends our understanding of the genetics of familial breast cancer, and supports the need to implement hereditary multigene panel testing to more appropriately orientate clinical management.

SummaryThe chromatin remodeler CHD5 is expressed in neural tissue and is frequently deleted in aggressive neuroblastoma. Very little is known about the function of CHD5 in the nervous system or its mechanism of action. Here we report that depletion of Chd5 in the developing neocortex blocks neuronal differentiation and leads to an accumulation of undifferentiated progenitors. CHD5 binds a large cohort of genes and is required for facilitating the activation of neuronal genes. It also binds a cohort of Polycomb targets and is required for the maintenance of H3K27me3 on these genes. Interestingly, the chromodomains of CHD5 directly bind H3K27me3 and are required for neuronal differentiation. In the absence of CHD5, a subgroup of Polycomb-repressed genes becomes aberrantly expressed. These findings provide insights into the regulatory role of CHD5 during neurogenesis and suggest how inactivation of this candidate tumor suppressor might contribute to neuroblastoma.

Polycomb group proteins are repressive chromatin modifiers with essential roles in metazoan development, cellular differentiation and cell fate maintenance. How Polycomb proteins access active chromatin to confer transcriptional silencing during lineage transitions remains unclear. Here we show that the Polycomb repressive complex 2 (PRC2) component PHF19 binds trimethylated histone H3 Lys36 (H3K36me3), a mark of active chromatin, via its Tudor domain. PHF19 associates with the H3K36me3 demethylase NO66, and it is required to recruit the PRC2 complex and NO66 to stem cell genes during differentiation, leading to PRC2-mediated trimethylation of histone H3 Lys27 (H3K27), loss of H3K36me3 and transcriptional silencing. We propose a model whereby PHF19 functions during mouse embryonic stem cell differentiation to transiently bind the H3K36me3 mark via its Tudor domain, forming essential contact points that allow recruitment of PRC2 and H3K36me3 demethylase activity to active gene loci during their transition to a Polycomb-repressed state.

Fluorescence microscopy is an important and fundamental tool for biomedical research. Optical microscopy is almost non-invasive and allows highly spatially resolved images of organisms, cells, macromolecular complexes, and biomolecules to be obtained. Generally speaking, the architecture of the observed structures is not significantly modified and the environmental conditions can be kept very close to physiological reality. The development of fluorescence microscopy was revolutionized with the invention of laser scanning confocal microscopy (LSCM). With its unique three-dimensional representation and analysis capabilities, this technology gives us a more real view of the world.This chapter introduces the reader to the methodology of setting up basic experiments for use with a laser scanning confocal microscope. There are practical guidelines about sample preparation for both fixed and living specimens, as well as examples of some of the applications of confocal microscopy.

The chromatin remodeler CHD5 is expressed in neural tissues and is frequently deleted in aggressive neuroblastoma. Very little is known about the normal function of CHD5, its mechanism of action or how its deletion may contribute to neuroblastoma. Here we report that depletion of CHD5 in the developing murine neocortex blocks neuronal differentiation leading to an accumulation of undifferentiated progenitors. Consistent with this, ChIP-SEQ analysis of CHD5 shows it binds a large cohort of neuronal genes and is required for facilitating their activation during differentiation. However, CHD5 also binds a cohort of Polycomb target genes and is required for the maintenance of H3K27me3 on their promoters and their sustained repression during differentiation. Finally, we show that the chromodomains of CHD5 directly bind H3K27me3 and are essential for the function of CHD5 during neuronal differentiation. These findings provide new insights into the essential role of CHD5 during neurogenesis and show how the inactivation of this candidate tumor suppressor might contribute to the progression of neuroblastoma via a defect in differentiation. This abstract is also presented as Poster A05. Citation Format: Christopher M. Egan, Ulrika Nyman, Julie Skotte, Gundula Streubel, Siobhán Turner, David J. O'Connell, Vilma Rraklli, Michael J. Dolan, Naomi Chadderton, Hansen Klaus, Farrar Gwyneth Jane, Kristian Helin, Johan Holmberg, Adrian P. Bracken. CHD5 is required for neurogenesis and has a dual role in facilitating gene expression and Polycomb gene repression. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Jun 19-22, 2013; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2013;73(13 Suppl):Abstract nr PR12.