Building a unified framework for understanding bacterial gene regulation and chromosomal architecture

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ABSTRACT Transcriptional regulation via protein-DNA interactions plays an important role in the regulatory networks of all known organisms. Bacterial regulatory networks are now an especially fruitful target for detailed investigation: as antibiotic- resistant bacteria continue to emerge as a global health threat, new and innovative approaches to either preventing virulence or impairing bacterial growth are required. As our ability to predict and exploit bacterial behavior for therapeutic purposes hinges on our understanding of the logic behind their regulatory networks, it is of great utility to fully map those networks and the molecular mechanisms underlying them. Several challenges, both old and newly recognized, stand in the way of a comprehensive understanding of regulatory logic even in well-studied models such as Escherichia coli. In additional to classical cis-regulatory logic by transcription factors and sigma factors, recent work by our laboratory and others has revealed contributions due to chromosomal context, large heterochromatin-like regions of repressive occupancy of nucleoid-associated proteins, overall three-dimensional chromosomal structure, and epigenetic modifications of both DNA-binding proteins and the DNA itself that further modulate transcriptional regulation. In addition, for non-model bacteria even the fundamental logic of classical transcriptional regulation is often poorly characterized. Thus, the fundamental regulatory logic behind cellular decisions such as metabolic switches, motility, and induction of virulence is often under-characterized. We have developed several innovative technologies to assist in rapid characterization of bacterial transcriptional regulatory logic, including: IPOD-HR, which allows overall profiling of protein occupancy on bacterial; transposon based methods for rapidly profiling genome-wide effects of genetic context on transcription; and a method based on the transposable phage Mu for crosslinking-free measurement of the 3D structure of the genome. Using these methods alongside classical approaches such as bacterial genetics and ChIP-seq, we are pursuing several avenues of research to investigate bacterial transcriptional regulatory networks. Key areas of interest include: Rapid elucidation of new transcriptional regulatory networks: Leveraging the IPOD-HR technology, which we have shown can be readily applied to new bacterial species, we are mapping the set of cis regulatory interactions driving important environmental responses in several clinically relevant bacterial species. Dynamics and composition of extended protein occupancy domains (EPODs): We have shown that highly protein occupied, heterochromatin like EPODs are present in a broad range of bacterial species, and play key roles in regulating prophages, virulence genes, and metabolic genes. We will continue to investigate the regulatory roles of EPODs, the proteins that comprise them, and the factors dictating their formation/dissociation across a range of bacterial taxa. Interplay of bacterial epigenetic marks with TF binding and EPODs: By interfacing our IPOD-HR and ChIP-seq experiments with tracking of covalent DNA and DNA-binding protein modifications, we will elucidate how these modifications modulate both local and large-scale protein occupancy and transcription in bacteria.