Person: Sack, Laura
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Sack
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Laura
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Sack, Laura
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Publication Gain-of-Function Genetic Screens Using Barcoded Libraries of Human Open Reading Frames Identify Regulators of Proliferation and Cancer Drivers(2015-05-17) Sack, Laura; Thomas, Sheila; Sicinski, Piotr; Toker, Alex; Lees, JacquelineThe identification of genetic events that cause tumorigenesis is a key goal of cancer research. While recent sequencing efforts have provided unprecedented illumination of cancer genomes, the inherent genomic instability of tumors results in vast numbers of somatic mutations and copy number alterations (CNAs), hampering identification of causative driver events. Functional genetic screens provide a complementary approach to genomics in the search for cancer drivers. While many studies have focused on loss-of-function studies in cancer cells using RNAi technology, we present a platform for gain-of-function screening using sequence-verified human open reading frames (ORFs). We have paired genome-scale ORF collections with unique DNA barcodes of uniform length, facilitating quantitative readout by next generation sequencing. ORFs are expressed from an inducible promoter, allowing precise control of screening conditions. We have used our libraries to identify regulators of proliferation in non-transformed human mammary epithelial cells (HMECs), seeking to uncover early driving events in tumorigenesis. We rediscovered many known oncogenes and observe significant enrichment of our pro-proliferative gene set within regions of recurrent focal amplification in human cancers, indicating the efficacy of our method for cancer gene driver discovery. We have identified a novel family of pro-proliferative genes, the keratin associated proteins, which strongly promote proliferation in HMECs and may contribute to human cancers. Additionally, we report several common sources of error that contribute to noise in pooled genetic screens in mammalian cells. We have observed that library plasmid DNA present in viral supernatants can contaminate screen samples resulting in inaccurate reference measurements of the abundance of library elements. A further artifact of this contamination is a perceived bias towards enrichment of library elements with low GC content. Additionally, libraries containing multiple unique elements carried on a single retroviral genome are subject to recombination during reverse transcription. Our inducible ORF libraries circumvent these problems, allowing for highly accurate, reproducible screen results. As CNAs are considered to be key initiating events in tumorigenesis, and many oncogenes are activated by amplification, we believe that modeling increased gene expression in untransformed human cells will provide critical functional data facilitating the discovery of bona fide cancer drivers.Publication Leaf palmate venation and vascular redundancy confer tolerance of hydraulic disruption(Proceedings of the National Academy of Sciences, 2008) Sack, Laura; Dietrich, E. M.; Streeter, Chris; Sanchez-Gomez, D.; Holbrook, NoelLeaf venation is a showcase of plant diversity, ranging from the grid-like network in grasses, to a wide variety of dendritic systems in other angiosperms. A principal function of the venation is to deliver water; however, a hydraulic significance has never been demonstrated for contrasting major venation architectures, including the most basic dichotomy, ‘‘pinnate’’ and ‘‘palmate’’ systems. We hypothesized that vascular redundancy confers tolerance of vein breakage such as would occur during mechanical or insect damage. We subjected leaves of woody angiosperms of contrasting venation architecture to severing treatments in vivo, and, after wounds healed, made detailed measurements of physiological performance relative to control leaves. When the midrib was severed near the leaf base, the pinnately veined leaves declined strongly in leaf hydraulic conductance, stomatal conductance, and photosynthetic rate, whereas palmately veined leaves were minimally affected. Across all of the species examined, a higher density of primary veins predicted tolerance of midrib damage. This benefit for palmate venation is consistent with its repeated evolution and its biogeographic and habitat distribution. All leaves tested showed complete tolerance of damage to second- and higherorder veins, demonstrating that the parallel flow paths provided by the redundant, reticulate minor vein network protect the leaf from the impact of hydraulic disruption. These findings point to a hydraulic explanation for the diversification of low-order vein architecture and the commonness of reticulate, hierarchical leaf venation. These structures suggest roles for both economic constraints and risk tolerance in shaping leaf morphology during 130 million years of flowering plant evolution.Publication Sources of Error in Mammalian Genetic Screens(Genetics Society of America, 2016) Sack, Laura; Davoli, Teresa; Xu, Qikai; Li, Mamie Z.; Elledge, StephenGenetic screens are invaluable tools for dissection of biological phenomena. Optimization of such screens to enhance discovery of candidate genes and minimize false positives is thus a critical aim. Here, we report several sources of error common to pooled genetic screening techniques used in mammalian cell culture systems, and demonstrate methods to eliminate these errors. We find that reverse transcriptase-mediated recombination during retroviral replication can lead to uncoupling of molecular tags, such as DNA barcodes (BCs), from their associated library elements, leading to chimeric proviral genomes in which BCs are paired to incorrect ORFs, shRNAs, etc. This effect depends on the length of homologous sequence between unique elements, and can be minimized with careful vector design. Furthermore, we report that residual plasmid DNA from viral packaging procedures can contaminate transduced cells. These plasmids serve as additional copies of the PCR template during library amplification, resulting in substantial inaccuracies in measurement of initial reference populations for screen normalization. The overabundance of template in some samples causes an imbalance between PCR cycles of contaminated and uncontaminated samples, which results in a systematic artifactual depletion of GC-rich library elements. Elimination of contaminating plasmid DNA using the bacterial endonuclease Benzonase can restore faithful measurements of template abundance and minimize GC bias.