Adam Norris, Ph.D. - US grants

ABSTRACT Biological processes are controlled by multiple genes working in concert to achieve a given function. This phenomenon is apparent in genetic interactions, defined as a phenotype observed in a double mutant not easily explained by the phenotypes in the respective single mutants. While genetic interactions have long been recognized as important drivers of animal phenotypes, it has not been possible to perform genetic interaction analysis in animals in a systematic, null allele, reverse-genetics fashion. This is a critical gap, because understanding healthy and disease states in animals requires an appreciation of how multiple genes coordinately affect a given phenotype. To overcome this gap, we have developed a CRISPR/Cas9 toolkit that enables targeted genome modification and subsequent genetic interaction analysis in the nematode worm Caenorhabditis elegans, thus enabling for the first time systematic targeted genetic interaction profiling in animals. We will focus on genetic interactions among factors regulating gene expression. Proper gene expression is controlled by multiple layers of regulation (e.g. transcription, RNA processing, translation) but little is known about how these layers are coordinated at the level of single cells. The first direction of the lab therefore is to profile genetic interactions between different layers of gene expression, specifically focusing on transcription factors (TFs) and RNA binding proteins (RBPs). Double mutant combinations with unexpected phenotypes will be the entry point to mechanistic understanding of how combinations of TFs and RBPs coordinately control gene expression. The second direction of the lab will be to understand the regulation of alternative splicing at the single cell level by combinations of TFs and RBPs. Individual cell types can be defined by the presence of TFs and the resulting gene expression patterns, but can also be further refined by the presence of splicing factors and the resulting isoforms expressed. We have created a large number of in vivo splicing reporters in C. elegans and found extensive alternative splicing at the single cell level. Using a combination of forward and reverse genetics we have identified a number of splicing factors, as well as a surprising number of TFs, important for specific alternative splicing regimes at the single cell level. We now plan to investigate the mechanisms by which these factors combine to control splicing at the single cell level, as well as the functional consequences of such splicing. Together these directions will represent a key advance in our understanding of combinatorial action of gene regulatory factors and how they coordinately ensure proper gene expression.

The development and function of individual neurons are defined by their unique transcriptomic properties, but despite recent efforts cataloguing single neuron transcriptomes, there remains a gap in our understanding of the causal mechanisms by which gene regulatory factors specify individual neuronal transcriptomes. In particular, little is known about how factors regulating various layers of gene expression, e.g. transcription factors (TFs) and RNA binding proteins (RBPs), coordinately control the transcriptomes of single neurons. This proposal aims to fill the gap by leveraging unique properties of the nematode Caenorhabditis elegans to mechanistically investigate coordinated transcriptomic regulation of specific model neurons in vivo. The well-described and invariant lineage of the C. elegans nervous system, combined with powerful genetic techniques, will enable detailed dissection of TF-RBP control over neuronal development. Additional tools recently developed and adapted in the lab, including combinatorial CRISPR/Cas9, single-neuron in vivo alternative splicing reporters, and neuron-specific FACS sorting followed by RNA Seq, will reveal mechanisms and consequences of coordinated regulation of single neurons in vivo. The objective of this proposal is to define TF-RBP pairs that genetically interact and combinatorially shape neuron-specific transcriptomes. The hypothesis is that cell-specific combinations of TFs and RBPs converge on specific target networks to define neuronal transcriptomes. This hypothesis is supported by preliminary in vivo data in C. elegans showing that (a) certain TFs and RBPs combinatorially define splicing choices including splicing of the conserved neuronal kinase sad-1 in individual neurons such as the touch-sensing neurons, and (b) neuronal TFs and RBPs genetically interact to affect neuronal function and behavior. The hypothesis will be further tested by the experiments proposed in the following aims: 1) Determine molecular mechanisms by which the neuronal TFs and RBPs we have identified coordinately control sad-1 alternative splicing in touch neurons, 2) Define functional consequences of dysregulated touch neuron transcriptomes when these regulatory factors or their target transcripts are lost, and 3) Systematically identify neuronal TFs and RBPs coordinately controlling neuron fate and function in specific tractable neuronal cell types. The expected outcomes of the proposed work are to determine mechanisms and functional consequences of coordinate TF-RBP control over single neuron transcriptomes. The proposed approach is innovative as it departs from the status quo by examining causal mechanisms and consequences of single-neuron transcriptomic regulation across multiple layers of gene regulation in vivo. It is significant because it is expected to advance the field of single-neuron transcriptomics into causal mechanisms, functional consequences, and coordinated regulation in single neurons in vivo. Ultimately, these findings will inform our understanding of how nervous systems develop and are specified.