Improved discovery of genetic interactions using CRISPRiSeq across multiple environments.

Large-scale genetic interaction (GI) screens in yeast have been invaluable for our understanding of molecular systems biology and for characterizing novel gene function. Owing in part to the high costs and long experiment times required, a preponderance of GI data has been generated in a single environmental condition. However, an unknown fraction of GIs may be specific to other conditions. Here, we developed a pooled-growth CRISPRi-based sequencing assay for GIs, CRISPRiSeq, which increases throughput such that GIs can be easily assayed across multiple growth conditions. We assayed the fitness of approximately 17,000 strains encompassing approximately 7700 pairwise interactions in five conditions and found that the additional conditions increased the number of GIs detected nearly threefold over the number detected in rich media alone. In addition, we found that condition-specific GIs are prevalent and improved the power to functionally classify genes. Finally, we found new links during respiratory growth between members of the Ras nutrient-sensing pathway and both the COG complex and a gene of unknown function. Our results highlight the potential of conditional GI screens to improve our understanding of cellular genetic networks.

iSeq: A New Double-Barcode Method for Detecting Dynamic Genetic Interactions in Yeast.

Systematic screens for genetic interactions are a cornerstone of both network and systems biology. However, most screens have been limited to characterizing interaction networks in a single environment. Moving beyond this static view of the cell requires a major technological advance to increase the throughput and ease of replication in these assays. Here, we introduce iSeq-a platform to build large double barcode libraries and rapidly assay genetic interactions across environments. We use iSeq in yeast to measure fitness in three conditions of nearly 400 clonal strains, representing 45 possible single or double gene deletions, including multiple replicate strains per genotype. We show that iSeq fitness and interaction scores are highly reproducible for the same clonal strain across replicate cultures. However, consistent with previous work, we find that replicates with the same putative genotype have highly variable genetic interaction scores. By whole-genome sequencing 102 of our strains, we find that segregating variation and de novo mutations, including aneuploidy, occur frequently during strain construction, and can have large effects on genetic interaction scores. Additionally, we uncover several new environment-dependent genetic interactions, suggesting that barcode-based genetic interaction assays have the potential to significantly expand our knowledge of genetic interaction networks.

The effect of microbial colonization on the host proteome varies by gastrointestinal location.

Endogenous intestinal microbiota have wide-ranging and largely uncharacterized effects on host physiology. Here, we used reverse-phase liquid chromatography-coupled tandem mass spectrometry to define the mouse intestinal proteome in the stomach, jejunum, ileum, cecum and proximal colon under three colonization states: germ-free (GF), monocolonized with Bacteroides thetaiotaomicron and conventionally raised (CR). Our analysis revealed distinct proteomic abundance profiles along the gastrointestinal (GI) tract. Unsupervised clustering showed that host protein abundance primarily depended on GI location rather than colonization state and specific proteins and functions that defined these locations were identified by random forest classifications. K-means clustering of protein abundance across locations revealed substantial differences in host protein production between CR mice relative to GF and monocolonized mice. Finally, comparison with fecal proteomic data sets suggested that the identities of stool proteins are not biased to any region of the GI tract, but are substantially impacted by the microbiota in the distal colon.

Postnatal Deletion of the Type II Transforming Growth Factor-ß Receptor in Smooth Muscle Cells Causes Severe Aortopathy in Mice.

OBJECTIVE: Prenatal deletion of the type II transforming growth factor-ß (TGF-ß) receptor (TBRII) prevents normal vascular morphogenesis and smooth muscle cell (SMC) differentiation, causing embryonic death. The role of TBRII in adult SMC is less well studied. Clarification of this role has important clinical implications because TBRII deletion should ablate TGF-ß signaling, and blockade of TGF-ß signaling is envisioned as a treatment for human aortopathies. We hypothesized that postnatal loss of SMC TBRII would cause aortopathy. APPROACH AND RESULTS: We generated mice with either of 2 tamoxifen-inducible SMC-specific Cre (SMC-CreER(T2)) alleles and homozygous floxed Tgfbr2 alleles. Mice were injected with tamoxifen, and their aortas examined 4 and 14 weeks later. Both SMC-CreER(T2) alleles efficiently and specifically rearranged a floxed reporter gene and efficiently rearranged a floxed Tgfbr2 allele, resulting in loss of aortic medial TBRII protein. Loss of SMC TBRII caused severe aortopathy, including hemorrhage, ulceration, dissection, dilation, accumulation of macrophage markers, elastolysis, abnormal proteoglycan accumulation, and aberrant SMC gene expression. All areas of the aorta were affected, with the most severe pathology in the ascending aorta. Cre-mediated loss of SMC TBRII in vitro ablated both canonical and noncanonical TGF-ß signaling and reproduced some of the gene expression abnormalities detected in vivo. CONCLUSIONS: SMC TBRII plays a critical role in maintaining postnatal aortic homeostasis. Loss of SMC TBRII disrupts TGF-ß signaling, acutely alters SMC gene expression, and rapidly results in severe and durable aortopathy. These results suggest that pharmacological blockade of TGF-ß signaling in humans could cause aortic disease rather than prevent it.

Transforming growth factor-ß signaling in myogenic cells regulates vascular morphogenesis, differentiation, and matrix synthesis.

OBJECTIVE: Transforming growth factor-ß (TGF-ß) signaling is required for normal vascular development. We aimed to discover the role of TGF-ß signaling in embryonic smooth muscle cells (SMCs). METHODS AND RESULTS: We bred mice with smooth muscle (SM) 22α-Cre and Tgfbr2(flox) alleles to generate embryos in which the type II TGF-ß receptor (TGFBR2; required for TGF-ß signaling) was deleted in SMCs. Embryos were harvested between embryonic day (E) 9.5 and E18.5 and examined grossly, microscopically, and by histochemical and RNA analyses. SM22α-Cre(+/0) Tgfbr2(flox/flox) (knockout [KO]) embryos died before E15.5 with defects that included cardiac outflow tract abnormalities, persistence of the right dorsal aorta, and dilation of the distal aorta. Histological analyses suggested normal expression of SMC differentiation markers in KO aortas; however, RNA analyses showed that SMC differentiation markers were increased in KO cardiac outflow vessels but decreased in the descending aorta. KO aortas had only rare mature elastin deposits and contained abnormal aggregates of extracellular matrix proteins. Expression of several matrix proteins was significantly decreased in KO descending aortas but not in cardiac outflow vessels. CONCLUSIONS: TGF-ß signaling in SMCs controls differentiation, matrix synthesis, and vascular morphogenesis. Effects of TGF-ß on SMC gene expression appear to differ depending on the location of SMCs in the aorta.

Overexpression of urokinase by plaque macrophages causes histological features of plaque rupture and increases vascular matrix metalloproteinase activity in aged apolipoprotein e-null mice.

BACKGROUND: The mechanisms of atherosclerotic plaque rupture are poorly understood. Urokinase-type plasminogen activator (uPA) is expressed at elevated levels by macrophages in advanced human plaques. Patients with evidence of increased plasminogen activation have an elevated risk of major cardiovascular events. We used atherosclerotic mice to test the hypothesis that increased macrophage uPA expression in advanced plaques would cause histological features similar to those in ruptured human plaques. METHODS AND RESULTS: Bone marrow from transgenic mice with increased macrophage uPA expression or nontransgenic controls (all apolipoprotein E-null [Apoe(-/-)]) was transplanted into 35-week-old Apoe(-/-) recipients, and innominate lesions and aortas were examined 8 to 13 weeks later. Donor macrophages accumulated in innominate lesions adjacent to plaque caps and in aortas, increasing uPA expression at both sites. Recipients of uPA-overexpressing macrophages had an increased prevalence of intraplaque hemorrhage (61% versus 13%; P=0.002) as well as increased lesion fibrin staining and fibrous cap disruption (P=0.06 for both). Transplantation of uPA-overexpressing macrophages increased aortic matrix metalloproteinase activity (40%; P=0.02). This increase was independent of matrix metalloproteinase-9. CONCLUSIONS: In advanced plaques of Apoe(-/-) mice, macrophage uPA overexpression causes intraplaque hemorrhage and fibrous cap disruption, features associated with human plaque rupture. uPA overexpression also increases vascular matrix metalloproteinase activity. These data provide a mechanism that connects macrophage uPA expression, matrix metalloproteinase activity, and plaque rupture features in mice. The data also suggest that elevated plaque plasminogen activator expression and plasminogen activation in humans may be causally linked to plaque rupture and cardiovascular events.

TGF-[beta]1 limits plaque growth, stabilizes plaque structure, and prevents aortic dilation in apolipoprotein E-null mice.

OBJECTIVE: Impairment of transforming growth factor (TGF)-beta1 signaling accelerates atherosclerosis in experimental mice. However, it is uncertain whether increased TGF-beta1 expression would retard atherosclerosis. The role of TGF-beta1 in aneurysm formation is also controversial. We tested whether overexpression of active TGF-beta1 in hyperlipidemic mice affects atherogenesis and aortic dilation. METHODS AND RESULTS: We generated apolipoprotein E-null mice with transgenes that allow regulated overexpression of active TGF-beta1 in their hearts. Compared to littermate controls, these mice had elevated cardiac and plasma TGF-beta1, less aortic root atherosclerosis (P< or =0.002), fewer lesions in the thoracic and abdominal aortae (P< or =0.01), less aortic root dilation (P<0.001), and fewer pseudoaneurysms (P=0.02). Mechanistic studies revealed no effect of TGF-beta1 overexpression on plasma lipids or cytokines, or on peripheral lymphoid organ cells. However, aortae of TGF-beta1-overexpressing mice had fewer T-lymphocytes, more collagen, less lipid, lower expression of inflammatory cytokines and matrix metalloproteinase-13, and higher expression of tissue inhibitor of metalloproteinase-2. CONCLUSIONS: When overexpressed in the heart and plasma, TGF-beta1 is an antiatherogenic, vasculoprotective cytokine that limits atherosclerosis and prevents aortic dilation. These actions are associated with significant changes in cellularity, collagen and lipid accumulation, and gene expression in the artery wall.