Interrogating Putative Roles for R-Loops in dsRNA Formation and Transcription Regulation

R-loops are three-stranded nucleic acid structures consisting of an RNA-DNA hybrid and a displaced ssDNA. Their formation is a conserved feature of the genomes of all living organisms, induced naturally by RNA transcription and specific nucleotide-sequence features. Intriguingly, R-loops have been implicated in transcription termination and RNAi-mediated heterochromatin formation through dsRNA formation at specific gene terminators. Yet this role has never been confirmed or tested on a genome-wide scale, and the underlying mechanism is still unclear. Here, I proposed two models for R-loop-mediated dsRNA formation and examined them using S. pombe cells. The first model is based on bidirectional double R-loop formation while the second is based on R-loops coupled with overlapping free asRNA. For a comprehensive and complementary R-loop profiling, I used three different S9.6-based and DRIP-like methods for consistent, high resolution and directional R-loop mapping. Beside the ultra-high-resolution double-stranded DNA-based ChIP-exonuclease (dsChIP-exo) technique, I developed directional and single stranded DNA-based ChIP-exonuclease (ssChIP-exo) to map the DNA strand of the hybrid. Moreover, I adopted the DRIPc method combined with the SMART-seq technology (SMART-DRIPc) to sequence the RNA strand of R-loops. To examine a possible role for R-loops in heterochromatin formation, I mapped the heterochromatic H3K9me2 mark using a high-sensitivity version of ChIP-exo and studied the impact of either RNase H depletion or overexpression on levels of this histone mark. dsChIP-exo revealed bidirectional signals mapping to both DNA strands. Differently, both ssChIP-exo and SMART-DRIPc revealed single R-loops forming in transcription direction. Although my data pointed toward double R-loops formation over tRNAs and rRNAs, it revealed a global anti-correlation between template and non-template strand R-loops even over the double R-loop forming genes. I found that R-loops over 60 to 70% of forming regions were extremely sensitive to physiological RNase H levels, which weakens both models of R-loop-dependent dsRNA formation. However, a category of RNase H-insensitive R-loops were associated with dsRNA-forming sites of sense-antisense transcription. Unexpectedly, RNase H depletion decreased R-loop signals over template strand of protein synthesis and ribosome biogenesis genes. R-loops of these genes are RNase H-insensitive and form on reverse strand. Excitingly, RNase H deletion, globally, increased forward-strand but decreased reverse-strand R-loop signals. Independently, no R-loops were detected over gene terminators or enriched over any of the heterochromatic repeats except tRNA genes and chromosome III telomeres rich in rRNA genes. Strikingly, either RNase H depletion or overexpression, respectively, disrupted H3K9me2 over heterochromatin in fission yeast and depleted global H3K9me2 in mammalian cells. R-loops may have been overrated as contributors for transcription termination and heterochromatin formation as they stand as marks of transcriptionally-active rather than heterochromatic domains. Despite challenges for the currently proposed models, R-loops coupled with overlapping asRNAs seems to be a more plausible model for dsRNA formation compared to double R-loops model. Nevertheless, practical involvement of R-loops in dsRNA formation still needs to be biochemically confirmed. My results suggest that perturbing RNase H levels, independent from R-loops, may alter the transcriptome and proteome, and impact cellular activities. Interestingly, R-loop orientation seems to modulate its response to RNase H. Excitingly, my observations suggest a role for RNase H in regulation of gene expression through suppressing formation of forward-strand R-loops and maintaining expression of sense genes. Transcriptome profiling and quantitative single-cell R-loop mapping using mimic and spike-in R-loop standards are required to confirm these results

Phage Display in the Quest for New Selective Recognition Elements for Biosensors

Phages are bacterial viruses that have gained a significant role in biotechnology owing to their widely studied biology and many advantageous characteristics. Perhaps the best-known application of phages is phage display that refers to the expression of foreign peptides or proteins outside the phage virion as a fusion with one of the phage coat proteins. In 2018, one half of the Nobel prize in chemistry was awarded jointly to George P. Smith and Sir Gregory P. Winter "for the phage display of peptides and antibodies." The outstanding technology has evolved and developed considerably since its first description in 1985, and today phage display is commonly used in a wide variety of disciplines, including drug discovery, enzyme optimization, biomolecular interaction studies, as well as biosensor development. A cornerstone of all biosensors, regardless of the sensor platform or transduction scheme used, is a sensitive and selective bioreceptor, or a recognition element, that can provide specific binding to the target analyte. Many environmentally or pharmacologically interesting target analytes might not have naturally appropriate binding partners for biosensor development, but phage display can facilitate the production of novel receptors beyond known biomolecular interactions, or against toxic or nonimmunogenic targets, making the technology a valuable tool in the quest of new recognition elements for biosensor development.This study was supported by the Ministry of Economy and Competitiveness (Ministerio de Ciencia, Innovación y Universidades RTI2018-096410-B-C21). R.P. acknowledges UCM for a predoctoral grant and R.B. the PI17CIII/00045 grant from the AES-ISCIII program.S

Implementation and Optimization of an in vivo Photo-crosslinking Methodology to Define Direct Targets of Transcriptional Activators.

Protein-protein interactions are primarily used to accomplish many biological processes. Understanding protein-protein interactions, particularly, the direct interacting proteins and mechanism for interaction, is instrumental to offering therapeutic interventions for diseases they facilitate. For example in the transcription, many human diseases have strong correlations with alterations in gene expression. Thus, there is intense interest in the development of chemical agents to restore aberrant gene expression to normal levels. To properly define transcriptional activation and ultimately find therapies for diseases associated with altered transcription profiles, there needs to be an in-depth understanding of how transcriptional activators interact with their transcriptional machinery partners (coactivators). However, very few direct binding partners of transcriptional activators are known and structurally characterized, making the generation of tailored screens for inhibitors of activator−coactivator interactions challenging. To better understand activator−coactivator interactions, we probed for direct binding partners of activators in vivo, using an enhanced tRNA/tRNA synthetase pair, developed to site specifically incorporate the nonnatural amino acid pbenzoyl- L-phenylalanine (pBpa) into the amphipathic activators. Initially we started with the model prototypical yeast transcriptional activator, Gal4, and later expanded our studies to two other prototypical activators, Gcn4 and VP16. First, we used a powerful method, nonsense suppression, to incorporate pBpa, which has a crosslinking moiety, into Gal4. Using pBpa-containing constructs of Gal4 we carried out in vivo photo-crosslinking experiments in the yeast strain LS41. Crosslinked activator−coactivator complexes were immunoprecipitated and analyzed by Western blotting. Before identifying the binding partners of Gal4, we determined whether pBpa was readily incorporated into the Gal4 TAD and if these photo-crosslinkable constructs were transcriptionally active. Results showed that all Gal4Bpa constructs were permissive for the incorporation of pBpa, produced the full length protein and were transcriptionally functional. Our initial in vivo crosslinking experiments revealed a well-characterized binding partner of Gal4, the masking protein Gal80. Further, using in vivo photo-crosslinking again, we were able to capture other targets that engage in modest-affinity and/or transient interactions with transcriptional activators (Gal4, Gcn4 and VP16) including Med15, Taf12, Tra1 and Snf2. In the future, in vivo photo-crosslinking methodology can be used to define both tight and modest-affinity protein-protein interactions.Ph.D.Medicinal ChemistryUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/91515/1/adaora_1.pd

Identification of Novel RPA-Protein Interactions Using the Yeast Two Hybrid Assay and Identification of Regions Important for Interaction Between RPA and Rad24

Replication Protein A (RPA) [Replication Factor A (RFA) in yeast] is an ssDNA binding protein composed of Rpa1, Rpa2, and Rpa3 and involved in numerous DNA processing pathways such as Replication, Recombination, and Repair. It participates in such diverse pathways by its ability to interact with numerous proteins. The goal of my project was to find novel RPA-protein interactions using the yeast two hybrid assay. Using this method, we identified several known and unknown proteins that interact with Rfa1 and showed that these interactions were dependent on the phosphorylation state of Rfa2. Next, we determine the region important for interaction between Rfa1 and Rad24. Rad24 is a checkpoint protein important for initiation of the DNA damage checkpoint signaling. By using the β- galactosidase assay, we determined the N-terminal region of Rfa1 (DBD-F) and the C-terminal region of Rad24 (460-660 aa) to be necessary for their interaction

Regulatory Signaling Networks Governing Budding Yeast Filamentous Growth.

In response to specific stresses, the budding yeast Saccharomyces cerevisiae undergoes a morphogenetic program wherein cells elongate and interconnect, forming pseudohyphal filaments. This filamentous growth transition has been studied extensively as a model signaling system with relevance to fungal pathogenicity. Classic studies have identified core pseudohyphal growth signaling modules in yeast; however, the scope of regulatory networks that control yeast filamentation is broad and incompletely defined. In this work, we address the genetic basis of yeast pseudohyphal growth by implementing a systematic analysis of 4909 genes for overexpression phenotypes in a filamentous strain of S. cerevisiae. Our results identify 551 genes conferring exaggerated invasive growth upon overexpression under normal vegetative growth conditions. In particular, overexpression screening suggests that nuclear export of the osmoresponsive MAPK Hog1p may enhance pseudohyphal growth. The function of nuclear Hog1p is unclear from previous studies, but our analysis using a nuclear-depleted form of Hog1p is consistent with a role for nuclear Hog1p in repressing pseudohyphal growth. In a second study, we interrogate the kinase signaling network regulating filamentous growth using a quantitative phosphoproteomic approach. The filamentous growth transition is controlled by at least three kinase signaling pathways; however, the global scope of filamentous growth kinase signaling networks is not presently understood. We engineered kinase-dead mutations in a core set of eight regulatory protein kinases and identified differentially phosphorylated proteins relative to wild type by SILAC-based mass spectrometry. Our analysis reveals 752 significantly differentially phosphorylated phosphopeptides, including many that are previously unsurveyed in any yeast strain. From this set of significantly differentially abundant phosphopeptides, we identify novel functional regulatory phosphorylation events crucial for proper filamentation. This collective phosphoproteomic data also reveals novel contributions of two cellular processes during filamentous growth: First, genetic analysis suggests that the components of a translationally repressive mRNA decay complex regulate MAPK signaling downstream of the MAPKKK Stellp. Second, null mutants of the inositol kinase pathway genes indicate an unexpected regulatory role for soluble inositol polyphosphates in the regulation of filamentous growth. In sum, this collective work more completely defines the genomic complement and kinase signaling networks contributing to this model stress response.PHDCellular & Molecular BiologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/108918/1/chashive_1.pd