A Scalable Genome-Editing-Based Approach for Mapping Multiprotein Complexes in Human Cells

SummaryConventional affinity purification followed by mass spectrometry (AP-MS) analysis is a broadly applicable method used to decipher molecular interaction networks and infer protein function. However, it is sensitive to perturbations induced by ectopically overexpressed target proteins and does not reflect multilevel physiological regulation in response to diverse stimuli. Here, we developed an interface between genome editing and proteomics to isolate native protein complexes produced from their natural genomic contexts. We used CRISPR/Cas9 and TAL effector nucleases (TALENs) to tag endogenous genes and purified several DNA repair and chromatin-modifying holoenzymes to near homogeneity. We uncovered subunits and interactions among well-characterized complexes and report the isolation of MCM8/9, highlighting the efficiency and robustness of the approach. These methods improve and simplify both small- and large-scale explorations of protein interactions as well as the study of biochemical activities and structure-function relationships

Should We Abandon the t-Test in the Analysis of Gene Expression Microarray Data: A Comparison of Variance Modeling Strategies

High-throughput post-genomic studies are now routinely and promisingly investigated in biological and biomedical research. The main statistical approach to select genes differentially expressed between two groups is to apply a t-test, which is subject of criticism in the literature. Numerous alternatives have been developed based on different and innovative variance modeling strategies. However, a critical issue is that selecting a different test usually leads to a different gene list. In this context and given the current tendency to apply the t-test, identifying the most efficient approach in practice remains crucial. To provide elements to answer, we conduct a comparison of eight tests representative of variance modeling strategies in gene expression data: Welch's t-test, ANOVA [1], Wilcoxon's test, SAM [2], RVM [3], limma [4], VarMixt [5] and SMVar [6]. Our comparison process relies on four steps (gene list analysis, simulations, spike-in data and re-sampling) to formulate comprehensive and robust conclusions about test performance, in terms of statistical power, false-positive rate, execution time and ease of use. Our results raise concerns about the ability of some methods to control the expected number of false positives at a desirable level. Besides, two tests (limma and VarMixt) show significant improvement compared to the t-test, in particular to deal with small sample sizes. In addition limma presents several practical advantages, so we advocate its application to analyze gene expression data

The Fanconi anemia group C protein interacts with uncoordinated 5A and delays apoptosis.

The Fanconi anemia group C protein (FANCC) is one of the several proteins that comprise the Fanconi anemia (FA) network involved in genomic surveillance. FANCC is mainly cytoplasmic and has many functions, including apoptosis suppression through caspase-mediated proteolytic processing. Here, we examined the role of FANCC proteolytic fragments by identifying their binding partners. We performed a yeast two-hybrid screen with caspase-mediated FANCC cleavage products and identified the dependence receptor uncoordinated-5A (UNC5A) protein. Here, we show that FANCC physically interacts with UNC5A, a pro-apoptotic dependence receptor. FANCC interaction occurs through the UNC5A intracellular domain, specifically via its death domain. FANCC modulates cell sensitivity to UNC5A-mediated apoptosis; we observed reduced UNC5A-mediated apoptosis in the presence of FANCC and increased apoptosis in FANCC-depleted cells. Our results show that FANCC interferes with UNC5A's functions in apoptosis and suggest that FANCC may participate in developmental processes through association with the dependence receptor UNC5A

FANCC interacts with UNC5A^ΔDD.

<p>Immunoprecipitations (IPs) were performed in HEK293T cells transiently transfected with Xpress-tagged death-domain deletion mutant UNC5A (UNC5A^ΔDD) and HA-tagged FANCC (A) or in B HA-tagged FANCC harboring the L554P mutation (HA-FANCC^L554P). IPs were performed using anti-Xpress, anti-HA or control mouse serum (IgG). Western immunoblotting (IB) was performed with the indicated antibodies. WCE: whole cell extract from transfected cells. * indicates non-specific or IgG bands. Numbers indicate molecular weight</p

Yeast-2-hybrid assays between FANCC^1-306 and the identified clones.

<p>+ indicates growth on selective media.</p

FANCC interacts with UNC5A.

<p>Immunoprecipitations (IPs) were performed in HEK293T cells transiently transfected with Myc-tagged rUNC5A (A), HA-tagged UNC5A^ICD (B–C), with (A–B) or without (C) full-length FANCC. In D and E, HEK293T cells were cotransfected with HA-tagged UNC5A^ICD and EGFP-tagged FANCC^1-306 (D) or EGFP-tagged FANCC^307-558) (E). IPs were performed using anti-FANCC, anti-Myc, anti-HA, anti-GFP or control mouse serum (IgG). Western immunoblotting (IB) was performed with the indicated antibodies. WCE: whole cell extract from transfected cells, C: control untransfected cells. * indicates non-specific or IgG bands. Numbers indicate molecular weight.</p

FANCC co-localizes with UNC5A in the cytoplasm.

<p>Representative immunofluorescence experiment performed in HEK293T cells double-stained with anti-UNC5A (red) and anti-FANCC (green) antibodies. Cells were visualized via confocal fluorescence microscopy using a Nikon E800 microscope equipped with a C1 confocal system at 100X magnification.</p

Schematic representation of FANCC interaction with UNC5A and possible role in apoptosis.

<p>Based on interactions studies, full length FANCC interacts with UNC5A via different parts of the intracellular domain. Upon apoptosis, FANCC and UNC5A are cleaved by a caspase. Cleaved FANCC fragments interact with UNC5A and interfere with the apoptosis signal (survival). In absence of FANCC or mutations in FANCC, UNC5A triggers apoptosis.</p

FANCC directly interacts with UNC5A in yeasts.

<p>Yeast two-hybrid assays were performed with various UNC5A constructs that included the Y2H clone coding for the C-terminal death domain (DEATH) and a part of intron 14 (shown in blue), UNC5A intracellular domain (UNC5A^ICD), the UNC5A C-terminus (UNC5A^DD) and the death-domain deletion mutant (UNC5A^ΔDD). The yeast strain AH109 was co-transformed with UNC5A constructs along with FANCC constructs as indicated. The plus sign (+) indicate a positive interaction. Negative controls were performed using empty vectors (Empty), and positive controls were performed using pGBKT7/p53 and pGADT7/SV40 T-antigen from Clontech (data not shown). Yeast two-hybrid assays were performed at least three times (independent transformation) with 3 to 4 clones each.</p