(598e) A Novel Four-Dimensional Omics Approach for Mapping Large Targetomes of Global Regulatory Networks

Manipulation of global regulators is a new frontier for metabolic engineering. Before global regulators can be explored fully for this application, however, a deep and more cohesive understanding of both the extent to which they exert their cellular effects and their mechanisms of action is required. High-throughput omics experiments have been used to begin to gain this understanding, revealing vast yet diverse and variable targetomes for global regulators. Sorting through the high volumes of target information associated with omics experiments is challenging and false positives are common. For these reasons, we hypothesize that applying only a single omics experiment is insufficient to capture a high-confidence quantification of a global regulatorâ??s targetome.

In this work, we have proposed and demonstrated an integrative four-dimensional omics (InFO) approach that evaluates changes in gene expression after an (i) imposed stress as a function of (ii) time in (iii) multiple genetic backgrounds by (iv) multiple omics methods (transcriptomics, proteomics, and high throughput sequencing crosslinking immunoprecipitation). We apply this approach to the Carbon storage regulator (Csr) system in Escherichia coli as proof of concept. Collecting and integrating 126 sets of omics data revealed a well defined set of genes that likely have direct regulatory interactions with CsrA, the main Csr protein regulator. These systems-scale predictions were confirmed via the use of two in vivo follow-up experiments that tested both regulation effect of the Csr regulator on its presumed identified targets as well as the ability for physical interactions. A key element of this analysis is the classification of omics-detected genes by potential regulatory mechanisms (up- or down-regulation at the transcript or protein level). Additionally, this novel approach has served to demonstrate the variety of ways that a global protein regulator can regulate its mRNA targets at the systems-level as well as the selectivity that it can exhibit in regulation to yield directional control of biological pathways. Overall, this highly integrative approach has led to a core set of genes that can be confidently called true Csr targets, illustrating the advantages of combining multi-dimensional omics experiments in elucidating targetomes of global regulators.