Sokol Lab

The Sokol lab exploits the genetically tractable fly model system to study the heterochronic pathway, which includes the LIN-28 RNA binding protein and the let-7 microRNA and controls stem cell fate and differentiation in response to environmental cues. Broadly conserved across the animal kingdom, LIN-28 is found in large quantities in early embryos and is also present in adult stem cells. In contrast, let-7 and related microRNAs are absent in early stages of animal development but abundant in differentiated cells at later stages. We study the activities of both LIN-28 and let-7 microRNAs within the context of fundamental cell properties (metabolism, reproduction, and identity), with particular attention to their roles in the timing mechanisms that prompt the emergence of specific cell types during development and in response to environmental cues. We take advantage of key strengths of the fly system, including well characterized populations of stems cells that underlie tissue construction, remodeling, and regeneration, a biology that facilitates the integration of detailed biochemical and genetic analyses (including CRISPR-mediated genome editing), and large-scale resources and techniques that enable rapid and comprehensive exploratory screens to identify novel components of conserved genetic pathways. Given that LIN-28 and let-7 microRNAs are also present in humans, we believe that our approach in flies will build a foundation for better understanding the cellular basis of human development, tissue homeostasis and regeneration, and cancer.

Stem cells switch from asymmetric to symmetric division to expand in number during tissue growth or after injury. The cellular factors that influence this switch are largely unknown, but our recent findings indicates that LIN-28 is a central player in this decision (Chen, 2015). The conserved RNA-binding protein Lin-28 provides a unique entry point into these regulatory RNA pathways, since it is a general stemness factor – abundant in embryonic stem cells and adult stem cells but absent in most differentiated cells – and its elevated but uncharacterized activity enhances tissue regeneration and pluripotent cell programming in mice and human cells, respectively . While an attractive candidate for therapeutic intervention, essential details about the Lin-28 pathway – its mRNA targets, its mechanism of action, its regulation – are currently unknown and, furthermore, not easily determined in available vertebrate system. Because of this, there is a critical need for a genetically tractable in vivo model system that can be used to decipher the Lin-28-related regulatory RNA mechanism that controls adult stem cell behavior. Drosophila offers such a system, providing a proven platform to identify Lin-28 targets and cofactors whose regulation and function can be rigorously and rapidly characterized at the cellular level. Because the behavior of fly and human stem cells are remarkably similar, such analysis should illuminate fundamental principles of stem cell behavior underlying tissue homeostasis and regeneration in animals including humans.

The let-7 and miRNA-125 microRNAs regulate stem cell identity and differentiation and are deregulated in human diseases, including cancer. In flies and vertebrate systems, let-7 and miRNA-125 are co-transcribed with each other and a third miRNA, miRNA-100, from a single locus, termed the let-7- Complex (let-7-C) (Sokol et al., 2008). Despite intensive investigation of the molecular mechanisms controlling microRNA biogenesis, the regulation and processing of polycistronic microRNA transcripts like that produced by let-7-C has received comparatively little attention. Combining the strengths of the fly as a premier genetic and biochemical model system, the Sokol lab has established methods to identify novel regulators of let-7-C microRNA production and activity (Chawla and Sokol, 2012, 2014; Luhur et al., 2014) as well as analyze the role of these factors in controlling stem cell identity and differentiation in vivo (Wu et al., 2012). Our future studies will define the molecular mechanisms that modulate miRNAs in order to control stem cell behavior during development and adulthood and in response to environmental cues.

Using a large-scale genetic approach to screen for mutations that affect expression of a joint let-7 and miR- 125 reporter transgene, we recovered mutations in ~100 different genes (see Luhur et al., 2014 for a description of methods). Analysis of a subset of these has identified novel properties of known microRNA regulators as well as new microRNA regulators. We will now use biochemical and molecular methods to distinguish whether identified factors affect let-7 and/or miRNA-125 production or activity. In addition, in vivo genetic analysis will determine the role of these factors in neural stem cell differentiation. Finally, we will map, clone and characterize the remaining alleles identified from the screen, many of which are homozygous viable, using a novel mapping scheme we recently developed in collaboration with Dr. Kevin Cook (Bloomington Drosophila Stock Center).