WaveCNV: allele-specific copy number alterations in primary tumors and xenograft models from next-generation sequencing.

MOTIVATION: Copy number variations (CNVs) are a major source of genomic variability and are especially significant in cancer. Until recently microarray technologies have been used to characterize CNVs in genomes. However, advances in next-generation sequencing technology offer significant opportunities to deduce copy number directly from genome sequencing data. Unfortunately cancer genomes differ from normal genomes in several aspects that make them far less amenable to copy number detection. For example, cancer genomes are often aneuploid and an admixture of diploid/non-tumor cell fractions. Also patient-derived xenograft models can be laden with mouse contamination that strongly affects accurate assignment of copy number. Hence, there is a need to develop analytical tools that can take into account cancer-specific parameters for detecting CNVs directly from genome sequencing data. RESULTS: We have developed WaveCNV, a software package to identify copy number alterations by detecting breakpoints of CNVs using translation-invariant discrete wavelet transforms and assign digitized copy numbers to each event using next-generation sequencing data. We also assign alleles specifying the chromosomal ratio following duplication/loss. We verified copy number calls using both microarray (correlation coefficient 0.97) and quantitative polymerase chain reaction (correlation coefficient 0.94) and found them to be highly concordant. We demonstrate its utility in pancreatic primary and xenograft sequencing data. AVAILABILITY AND IMPLEMENTATION: Source code and executables are available at https://github.com/WaveCNV. The segmentation algorithm is implemented in MATLAB, and copy number assignment is implemented Perl. CONTACT: lakshmi.muthuswamy@gmail.com SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Differential recruitment of activating and inhibitory Fc gamma RII during phagocytosis.

Human myeloid cells express both activating and inhibitory receptors of the FcgammaRII family. FcgammaRIIA mediates processes associated with cell activation, including phagocytosis of IgG-opsonized particles, whereas coengagement of the inhibitory FcgammaRIIB downregulates such signaling. We analyzed the relative recruitment of these two receptors during phagocytosis of IgG-coated particles by ts20 Chinese hamster fibroblast cells cotransfected with both receptors carrying distinguishable fluorescent protein tags. We found that FcgammaRIIA is substantially enriched at sites of particle binding relative to its inhibitory counterpart, with a greater than 2-fold increase in the local ratio of activating to inhibitory receptor compared with that for the plasma membrane as a whole. Experiments with chimeric receptors revealed that the preferential enrichment of FcgammaRIIA results from differences between the extracellular domains of the receptors, and indicated that the lesser recruitment of FcgammaRIIB limits its ability to effectively inhibit FcgammaRIIA-mediated phagocytosis. Mutagenesis studies indicated that FcgammaRIIA residues leucine 132 and phenylalanine 160, which lie in IgG-binding regions of FcgammaRIIA and which differ in FcgammaRIIB, both contribute to the local relative enrichment of FcgammaRIIA by increasing its affinity for IgG1 relative to that of FcgammaRIIB. In human monocytes, engagement of approximately equal amounts of FcgammaRIIB was required to substantially inhibit FcgammaRIIA-mediated phagocytosis. These results demonstrate that differences in affinity for IgG between activating and inhibitory FcgammaR can result in substantial local changes in their relative concentrations during phagocytosis, with important functional consequences.

Breast cancer-associated gene 3 (BCA3) is a novel Rac1-interacting protein.

UNLABELLED: BCA3 was identified in a yeast two-hybrid screen as a novel Rac1-interacting partner in osteoclasts. BCA3 binds directly to Rac and, in vivo, binds GTP-Rac but not GDP-Rac. Perinuclear co-localization of BCA3 and Rac1 is observed in CSF-1-treated osteoclasts. Overexpression of BCA3 attenuates CSF-1-induced cell spreading. We conclude that BCA3 regulates CSF-1-dependent Rac activation. INTRODUCTION: Rac1, a ubiquitously expressed GTPase, is a mediator of colony-stimulating factor 1 (CSF-1)-dependent actin remodeling in osteoclasts. Because the role of Rac in osteoclasts has not been fully defined, we undertook a yeast two-hybrid screen to identify Rac-interacting partners in these cells. MATERIALS AND METHODS: A yeast two-hybrid screen was undertaken using a cDNA library prepared from osteoclast-like cells as prey and either native Rac1 or constitutively active Rac1 (Q61L) as bait. Radiolabeled breast cancer-associated gene 3 (BCA3) protein constructs were generated in vitro using rabbit reticulate lysates and used in vitro binding assays with Rac1. In vivo binding was assessed using myc-tagged Rac1(Q61L) and HA-tagged BCA3. PBD pull-down assays were used to determine if GTP-loaded Rac1 preferentially bound BCA3. Co-localization of Rac1 and BCA3 in osteoclasts was assessed using confocal immunofluorescence. The functional significance of the BCA3-Rac1 interaction was assessed by examining the effect of overexpressing BCA3 in RAW 264.7 cells on the subsequent spreading response to CSF-1. RESULTS: One of three positive clones from the wildtype Rac1 screen and all three positive clones from the Rac1(Q61L) screen encoded the same protein, BCA3. BCA3 expression in osteoclasts was confirmed by RT-PCR and immunocytochemistry. BCA3 bound directly to Rac1 in vitro. Deletional analysis indicated that amino acids 76-125 in BCA3 are important for its ability to bind Rac. In vivo association of the two proteins was shown by co-immunoprecipitation of BCA3 and Rac1. Only GTP-bound-Rac but not GDP-bound Rac could interact with BCA3 in vivo. Confocal immunocytochemistry showed perinuclear co-localization of BCA3 and Rac1 in CSF-1-treated neonatal rat osteoclasts but not in resting osteoclasts. Overexpression of BCA3 markedly attenuated the spreading response to CSF-1 in RAW 264.7 cells. CONCLUSIONS: These data establish that BCA3 is a novel Rac1-interacting protein and suggest that it may influence the ability of Rac1 to remodel the actin cytoskeleton.