The discovery and development of RNAi and CRISPR/Cas9 genetic screening technologies have provided researchers with invaluable tools for wide-scale and rapid genetic screening. A central goal of our research program has been to develop methodology for efficient application of these screening technologies in hematopoietic cell lineages, and to implement screens in both human and mouse hematopoietic cells to interrogate the mechanistic basis of immune cell responses to pathogenic stimuli. Our current efforts are focused on macrophages as they form the first line of defense against numerous bacterial and viral pathogens and characterization of these initial encounters are central to the LSB-wide efforts to generate quantitative models of host-pathogen interactions. We have previously generated reporter cell lines for mouse and human macrophages that provide a comprehensive range of biosensors for evaluation of the macrophage activation profile in response to pathogenic inputs. We have also developed robust small RNA delivery protocols that avoid non-specific activation of the macrophage response to dsRNA and we have described the utility of these macrophage cell systems for screening of pathogen responses (Li et al (2015) Sci. Rep. 5:9559). Using these assay platforms, we recently completed a comprehensive analysis of canonical TLR signaling components in human and mouse macrophages, which identified unexpected differences in signaling responses between species, particularly in use of the IRAK family of kinases (Sun et al (2016) Sci Signal. 9: ra3). In FY17, we have built on this work to complete and publish comprehensive reports of genome-wide siRNA screens of the LPS-induced TNF-alpha response in human macrophages and the LPS-induced NF-kB and TNF-alpha responses in mouse macrophages (Sun et al (2017) Sci. Data. 4:170007; Li et al (2017) Sci. Data. 4:170008). Activation of the TLR4 receptor by bacterial lipopolysaccharide (LPS) is the most widely studied TLR pathway due to its central role in host responses to Gram-negative bacterial infection and its contribution to endotoxemia and sepsis. The described screens targeted 18,110 human and 16,870 mouse genes. Secondary validation screens were conducted with six independent siRNAs per gene to facilitate removal of off-target screen hits, and microarray data from the same LPS-treated macrophage cells was used to facilitate downstream data analysis. The human screen identified 26 novel positive regulators and 13 negative regulators of LPS-induced TNF-a induction. Of these regulators, 24 and 8, respectively, were identified in a tertiary screen as having a regulatory role in the response to at least one additional TLR ligand. The mouse screen identified 82 robust novel regulatory candidates for the LPS response in mouse macrophages, 64 of these gene targets showing effects on both the NF-kB and TNF-a readouts. These data provide a valuable resource for analyzing gene function in the predominant pathway driving inflammatory cytokine expression in human and mouse macrophages. Beyond our continued study of the TLR4 pathway response to LPS, we are also extending our studies to interrogate the recently discovered cytosolic LPS sensing pathway, which activates the non-canonical inflammasome response and the release of IL-1 family inflammatory cytokines. Recent studies have shown this to be a critical component of the broader physiological response to LPS and a major contributor towards septic shock outcomes in Gram-negative bacterial infections. In FY17, using our robust siRNA delivery methods for mouse macrophages, we have completed the primary phase of a genome-scale screen of the IL-1 alpha response to cytosolic LPS. We find that the canonical hit rate for known components of this pathway is particularly high in this dataset, suggesting robust screen assay design and high potential for identifying novel regulators of this critical inflammatory pathway. RNAi screen data are susceptible to a myriad of experimental biases, some of which can be mitigated by computational analysis. During the analysis of the primary and secondary screen data from our genome-wide screens described above, we sought to improve the integration of the screen analysis process. In collaboration with Bhaskar Dutta in the LSB bioinformatics support team, we developed a user-friendly platform for integrated analysis and visualization of RNAi screen data, named CARD, for Comprehensive Analysis of RNAi Data (Dutta et al (2016) Nat. Commun. 7: 10578). In FY2017, we have further extended CARD to address the challenges in analyzing data from genome-scale perturbation studies, such as trade-offs in the selection of cutoff criteria and bias against novel gene identification when using database-dependent approaches. These challenges can limit the potential of studies attempting global identification of novel regulatory mechanisms in a given biological process. To address these challenges, we have leveraged the analysis tools within CARD to develop a novel bioinformatic method termed TRIAGE (Throughput Ranking from Iterative Analysis of Genomic Enrichment). This model applies multiple pathway and network enrichment steps on a screening dataset in an iterative manner, correcting for the biases of individual steps in a complementary fashion. To test and validate this approach, we used the primary and secondary screen data from our completed human and mouse screens of the LPS response. We compared TRIAGE to current analysis methods for; 1) ability to identify true positives at higher rates 2) overlap rate between orthogonal screens of similar biology 3) overlap rate between RNAi and CRISPR/Cas9 screens and 4) identification rate of canonical TLR pathway genes. In all of these cases, TRIAGE significantly outperformed established analysis methods. The signaling pathways and transcription factor responses induced in macrophages upon TLR stimulation are regulated by feedback loops that modulate the kinetics and magnitude of gene transcription. Among these, NF-kB has been a paradigm for a signal- responsive transcription factor that operates in a feedback regulatory network. We have previously described the use of our screening reporter cells, developed with dual assay readouts for NF-kB and TNF-alpha transcription, to identify a novel positive feedback loop in the macrophage NF-kB activation process which supports a robust inflammatory program at higher TLR ligand doses. Using genome-wide siRNA screen data, we discovered an important role for the transcription factor Ikaros in supporting this inflammatory response (Sung et al (2014) Sci Signal, 7: ra6). In FY17, we submitted a study for publication that provides further insight into the function and mechanisms of Ikaros action. We find unexpected dual repressor and activator functions for Ikaros in the macrophage LPS response using comprehensive genomic analysis of Ikaros-dependent transcription, DNA binding, and chromatin accessibility. Consistent with the known function of Ikaros as transcriptional repressor, Ikzf1-/- cells showed enhanced induction for select responses. In contrast, we observed a dramatic defect in expression of delayed response genes and ChIP-seq analyses support a key role for Ikaros in sustained NF-kB chromatin binding. Decreased Ikaros expression in Ikzf1+/- mice and human cells dampens these Ikaros-enhanced inflammatory responses, highlighting the importance of quantitative control of Ikaros protein level for its activator function. In the absence of Ikaros, a constitutively open chromatin state was coincident with dysregulation of LPS-induced chromatin remodeling, gene expression, and cytokine responses. Together, our data suggest a central role for Ikaros in coordinating the complex macrophage transcriptional program in response to pathogen challenge.
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