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 various pathogenic inputs. We have also developed robust siRNA 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 siRNA screening of pathogen responses (Li et al (2015) Sci. Rep. 5:9559). Using the platforms described above, this year we completed a comprehensive screening analysis of the canonical TLR signaling components in human and mouse macrophages (Sun et al (2016) Sci Signal. 9: ra3). While confirming the expected specificity for the TLRs and some of the core pathway components, this study identified unexpected selectivity in signaling responses through different TLR pathways, and also some particularly striking differences between human and mouse macrophages. Mouse cells are more sensitive than human cells to depletion of CD14, IRAK4, IRAK2, PI3K signaling, IKKb and MEK1 and 2, while human cells are more sensitive to depletion of IRAK1, TRAF6, the TAK1/TAB complex and Tpl2 among others. Among the gene-specific phenotypes, we describe further investigation of differences in IRAK protein usage in the 2016 report for project AI001107. 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, we sought to improve the integration of the screen analysis process. This year, in collaboration with Bhaskar Dutta in the LSB bioinformatics support team, we described 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). We have applied CARD to several published screen datasets and demonstrated both increased hit validation rates and improved hit gene overlap between related screens. The flexible design of the software will also allow application of many of the integrated features to CRISPR/Cas9 genome-editing screens. We are further implementing CARD to gain more detailed insight to the similarities and differences in the LPS response network of mouse and human macrophages from our genome-wide siRNA screens. This has led to the development of a sophisticated Iterative Analysis Model (IAM) for genome scale screen data that incorporates metadata from Network and Pathway databases to identify enriched cellular modules and processes involved in the biological process being studied in the screen. A manuscript describing this analysis method and its application to genome scale RNAi and CRISPR/Cas9 screens of the LPS pathway is currently in preparation. This year, we also submitted comprehensive reports of genome-wide siRNA screens of the LPS-induced TNF-a response in human macrophages and the LPS-induced NF-kB and TNF-a responses in mouse macrophages (Nature Scientific Data, under review). 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. 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. Despite the considerable literature on NF-kB activation and function, there remains a lack of data on NF-kB single cell dynamics in hematopoietic cells (especially macrophages) responding to pathogenic stimuli. We have previously described the use of our screening reporter cells, developed with dual assay readouts for NF-kB and TNF-a 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). This year, to gain further insight into the function and mechanisms of Ikaros action, we performed ChIP-seq, RNA-seq and DNase-seq analysis of LPS stimulated bone marrow derived macrophages from WT and Ikaros KO mice. We find that the induction of important subsets of LPS-induced genes are altered in Ikaros KO macrophages, and that LPS-induced dynamic changes in chromatin accessibility are strongly perturbed by Ikaros deficiency. Our data suggests that Ikaros has a dual role as a transcriptional repressor and activator in the LPS-induced gene program, acting predominantly as a transcriptional activator as evidenced by the larger proportion of genes whose expression is attenuated in Ikaros KO cells. We observe robust recruitment of Ikaros to numerous sites on macrophage chromatin in response to LPS, and a high percentage of RelA-bound NF-kB sites overlap with Ikaros sites, particularly at sites which show sustained binding several hours after LPS activation. This late RelA binding is almost completely ablated in Ikaros KO macrophages, suggesting a key role for Ikaros in modulating NF-kB chromatin occupancy to sustain the macrophage TLR-activated gene program. We show that Ikaros and the repressive histone modifier HDAC2 form a complex and co-precipitate in untreated cells, however this interaction diminishes after extended LPS treatment, consistent with a more positive regulatory role for Ikaros during the late-phase sustained transcriptional response to LPS. We have also determined that Ikaros KO cells are deficient in their response to the gram-negative bacterium B. cenocepacia, showing substantially increased intracellular bacterial replication after infection. A manuscript describing this data is in preparation.
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