Transcription and chromatin dynamics controlling stochastic cell fate specification

Johns Hopkins University, Baltimore MD

Investigators

Abstract

Project Summary: Cell fate specification is generally considered to be a tightly controlled process, guided by lineage and signaling mechanisms that lead to reproducible outcomes. However, some cell fate decisions rely on stochastic mechanisms in which cells randomly choose between two or more fates with a set probability. Stochastic cell fate specification arises from inherent molecular noise in gene expression. Very little is known about how variability in gene expression drives fate decisions during metazoan development. This project will investigate stochastic cell fate decisions in the developing visual system of Drosophila melanogaster, which contains a stochastic, binary fate choice. The fly eye consists of a mosaic of two R7 photoreceptor subtypes that are defined by their expression of light-detecting Rhodopsin 3 (Rh3) or Rhodopsin 4 (Rh4) proteins. The choice to express Rh3 or Rh4 is controlled by a transcription factor called Spineless (Ss), which is randomly expressed in 67% of R7 photoreceptors (SsON R7s). ss is expressed in two distinct phases during eye development. The early and late expression of ss is regulated by two enhancers (the “early” enhancer and “late” enhancer). Early expression is noisy, with R7 precursor cells expressing ss at different levels. Interestingly, about 67% of precursors express ss at high levels while 33% express at low levels, which corresponds to the proportion of terminally differentiated SsON/ SsOFF R7s. Manipulating DNA elements (silencers and enhancers) and the transcriptional repressor Klu changes the level of early ss expression and the proportion of SsON/SsOFF R7s. From these data, I hypothesized that variable expression of ss early sets the ratio of SsON/SsOFF R7s late. Based on this model, I will investigate how regulation of transcription and chromatin dynamics produce variable ss expression to determine the stochastic R7 fate choice. Gene expression involves inherently noisy molecular processes, resulting in fluctuations in the amount of mRNA produced within a cell at a given time. Transcription is a major source of gene expression noise, occurring in bursts that differ in size, duration, and frequency. These bursts are strongly influenced by chromatin architecture and transcription factors. This work will use the MS2/MCP and DNA labeling lacO/LacI and parS/ParB systems to monitor and characterize ss transcription and chromatin in live developing cells (Aim 1 & 2). These experiments will establish the link between early ss transcription, chromatin dynamics, and terminal fate by monitoring endogenous expression and compaction from the precursor stage to terminal differentiation (Aim 2). Ultimately, this work will uncover generalizable mechanisms that regulate stochastic cell fate specification.