Faculty Bibliography

The yeast pheromone response pathway serves as a valuable model of eukaryotic mitogen activated protein kinase (MAPK) pathways and transcription of their downstream targets. Here we describe application of a screening method combining two technologies, fluorescence-activated cell sorting (FACS) with Barcode analysis by Sequencing (Bar-Seq). Using this screening method, and pFUS1-GFP as a reporter for MAPK pathway activation, we readily identified mutants in known mating pathway components. In this study, we also include a comprehensive analysis of the FUS1 induction properties of known mating pathway mutants by flow cytometry, featuring single cell analysis of each mutant population. We also characterized a new source of false positives resulting from the design of this screen. Additionally, we identified a deletion mutant, sub1Delta, with increased basal expression of pFUS1-GFP. Here, in the first ChIP-Seq of Sub1, our data shows that Sub1 binds to the promoters of about half the genes in the genome (tripling the 991 loci previously reported), including the promoters of several pheromone-inducible genes, some of which show increase upon pheromone induction. Here we also present the first RNA-Seq of a sub1Delta mutant; the majority of genes have no change in RNA but of the small subset that do, most of these show decreased expression, consistent with biochemical studies implicating Sub1 as a positive transcriptional regulator. The RNA-Seq data also show that certain pheromone-inducible genes induce less in the sub1Delta mutant relative to the wild-type, supporting a role of Sub1 in regulation of mating pathway genes. The sub1Delta mutant has increased basal levels of a small subset of other genes besides FUS1, including IMD2 and FIG1, a gene encoding an integral membrane protein necessary for efficient mating.

Retrotransposons are mutagenic units able to move within the genome. Despite many defenses deployed by the host to suppress potentially harmful activities of retrotransposons, these genetic units have found ways to meld with normal cellular functions through processes of exaptation and domestication. The same host mechanisms targeting transposon mobility allow for expansion and rewiring of gene regulatory networks on an evolutionary time scale. Recent works demonstrating retrotransposon activity during development, cell differentiation and neurogenesis shed new light on unexpected activities of transposable elements. Moreover, new technological advances illuminated subtler nuances of the complex relationship between retrotransposons and the host genome, clarifying the role of retroelements in evolution, development and impact on human disease.

LINE-1 (L1) retrotransposons are catalysts of evolution and disease whose sequences comprise a significant proportion of the human genome. L1 ribonucleoprotein particles incorporate a combination of permissive host factors that are essential to their lifecycle as well as repressive factors that constitute defenses against L1's mutagenic activity. We previously characterized host proteins associated with human L1 retrotransposons, as expressed in cell culture, using a combination of techniques including metabolic labeling and affinity proteomics. To build on these analyses, we have executed a suite of quantitative proteomic comparisons, yielding interactomic maps of affinity isolated L1s. These studies have revealed the presence of at least two populations of putative transposition intermediates that may exhibit distinctive intracellular localizations. We report the proteins partitioning within these distinct L1 populations and associated in vitro activities. Our observations provide a basis for the classification of L1 interactors into physical and functional modules and have enabled the development of in vitro systems to study L1 activity

Combinatorial assembly of DNA elements is an efficient method for building large-scale synthetic pathways from standardized, reusable components. These methods are particularly useful because they enable assembly of multiple DNA fragments in one reaction, at the cost of requiring that each fragment satisfy design constraints. We developed BioPartsBuilder as a biologist-friendly web tool to design biological parts that are compatible with DNA combinatorial assembly methods, such as Golden Gate and related methods. It retrieves biological sequences, enforces compliance with assembly design standards, and provides a fabrication plan for each fragment. AVAILABILITY: BioPartsBuilder is accessible at http://public.biopartsbuilder.org and an Amazon Web Services image is available from the AWS Market Place (AMI ID: ami-508acf38). Source code is released under the MIT license, and available for download at https://github.com/baderzone/biopartsbuilder. CONTACT: joel.bader@jhu.edu.

Synthetic chromosome rearrangement and modification by loxP-mediated evolution (SCRaMbLE) generates combinatorial genomic diversity through rearrangements at designed recombinase sites. We applied SCRaMbLE to yeast synthetic chromosome arm synIXR (43 recombinase sites), and then use a computational pipeline to infer or unscramble the sequence of recombinations that created the observed genomes. Deep sequencing of 64 synIXR SCRaMbLE strains revealed 156 deletions, 89 inversions, 94 duplications, and 55 additional complex rearrangements; several duplications are consistent with a double rolling circle mechanism. Every SCRaMbLE strain was unique, validating the capability of SCRaMbLE to explore a diverse space of genomes. Rearrangements occurred exclusively at designed loxPsym sites, with no significant evidence for ectopic rearrangements or mutations involving synthetic regions, the 99% non-synthetic nuclear genome, or the mitochondrial genome. Deletion frequencies identified genes required for viability or fast growth. Replacement of 3' UTR by non-UTR sequence had surprisingly little effect on fitness. SCRaMbLE generates genome diversity in designated regions, reveals fitness constraints, and should scale to simultaneous evolution of multiple synthetic chromosomes.

BACKGROUND: Gliomas are the most common primary brain tumors in adults. We sought to understand the roles of endogenous transposable elements in these malignancies by identifying evidence of somatic retrotransposition in glioblastomas (GBM). We performed transposon insertion profiling of the active subfamily of Long INterspersed Element-1 (LINE-1) elements by deep sequencing (TIPseq) on genomic DNA of low passage oncosphere cell lines derived from 7 primary GBM biopsies, 3 secondary GBM tissue samples, and matched normal intravenous blood samples from the same individuals. RESULTS: We found and PCR validated one somatically acquired tumor-specific insertion in a case of secondary GBM. No LINE-1 insertions present in primary GBM oncosphere cultures were missing from corresponding blood samples. However, several copies of the element (11) were found in genomic DNA from blood and not in the oncosphere cultures. SNP 6.0 microarray analysis revealed deletions or loss of heterozygosity in the tumor genomes over the intervals corresponding to these LINE-1 insertions. CONCLUSIONS: These findings indicate that LINE-1 retrotransposon can act as an infrequent insertional mutagen in secondary GBM, but that retrotransposition is uncommon in these central nervous system tumors as compared to other neoplasias.

BACKGROUND: The National Cancer Institute-60 (NCI-60) cell lines are among the most widely used models of human cancer. They provide a platform to integrate DNA sequence information, epigenetic data, RNA and protein expression, and pharmacologic susceptibilities in studies of cancer cell biology. Genome-wide studies of the complete panel have included exome sequencing, karyotyping, and copy number analyses but have not targeted repetitive sequences. Interspersed repeats derived from mobile DNAs are a significant source of heritable genetic variation, and insertions of active elements can occur somatically in malignancy. METHOD: We used Transposon Insertion Profiling by microarray (TIP-chip) to map Long INterspersed Element-1 (LINE-1, L1) and Alu Short INterspersed Element (SINE) insertions in cancer genes in NCI-60 cells. We focused this discovery effort on annotated Cancer Gene Index loci. RESULTS: We catalogued a total of 749 and 2,100 loci corresponding to candidate LINE-1 and Alu insertion sites, respectively. As expected, these numbers encompass previously known insertions, polymorphisms shared in unrelated tumor cell lines, as well as unique, potentially tumor-specific insertions. We also conducted association analyses relating individual insertions to a variety of cellular phenotypes. CONCLUSIONS: These data provide a resource for investigators with interests in specific cancer gene loci or mobile element insertion effects more broadly. Our data underscore that significant genetic variation in cancer genomes is owed to LINE-1 and Alu retrotransposons. Our findings also indicate that as large numbers of cancer genomes become available, it will be possible to associate individual transposable element insertion variants with molecular and phenotypic features of these malignancies.

BACKGROUND: The immunoprecipitation (IP) assay is a valuable molecular biology tool applied across a breadth of fields. The standard assay couples IP to immunoblotting (IP/IB), a procedure severely limited as it is not easily scaled for high-throughput analysis. RESULTS: Here we describe and characterize a new methodology for fast and reliable evaluation of an immunoprecipitation reaction. FLIP (FLuorescence IP) relies on the expression of the target protein as a chromophore-tagged protein and couples IP with the measurement of fluorescent signal coating agarose beads. We show here that FLIP displays similar sensitivity to the standard IP/IB procedure but is amenable to high-throughput analysis. We applied FLIP to the screening of mouse monoclonal antibodies of unknown behavior in IP procedures. The parallel analysis of the considered antibodies using FLIP and IP/western shows good correlation between the two procedures. We also show application of FLIP using unpurified antibodies (hybridoma supernatant) and we developed a publicly available tool for the easy analysis and quantification of FLIP signals. CONCLUSIONS: Altogether, our characterizations of this new methodology show that FLIP is an appealing and reliable tool for any application of high-throughput IP.

The LINE-1 retrotransposon (L1) encodes two proteins, ORF1p and ORF2p, which bind to the L1 RNA in cis, forming a ribonucleoprotein (RNP) complex that is critical for retrotransposition. Interactions with both permissive and repressive host factors pervade every step of the L1 life cycle. Until recently, limitations in detection and production precluded in-depth characterization of L1 RNPs. Inducible expression and recombinant engineering of epitope tags have made detection of both L1 ORFs routine. Here, we describe large-scale production of L1-expressing HEK-293T cells in suspension cell culture, cryomilling and affinity capture of L1 RNP complexes, sample preparation for analysis by mass spectrometry, and assay using the L1 element amplification protocol (LEAP) and qRT-PCR.