Quantifying the coordinated dynamics of single-gene transcription and translation in living cells

ABSTRACT Transcription and translation together form the two central processes around which the central dogma of molecular biology operates. Both processes are intimately coupled and dynamically regulated to allow cells to rapidly change or maintain their phenotypes in the face of environmental pressures. Failure to properly regulate either process can be fatal or lead to serious disease, such as cancer. Despite the fundamental roles of transcription and translation in gene regulation, the dynamics of the two processes have not yet been simultaneously imaged in living system at the single molecule level, leaving basic question about their coordinated regulation unanswered. Our goal is to address this shortcoming by developing and applying technology to light up in up to four colors both the transcription and the translation dynamics of individual sister alleles in single living cells. Over the past five years, my lab at Colorado State University has pioneered the imaging of single-allele transcription dynamics and single-mRNA translation dynamics. We will now combine these technologies to image the two processes together. Using advanced CRISPR genome engineering and multiplexed single molecule tracking, we will light up allele-specific transcription and translation dynamics in multiple colors on timescales ranging from sub-seconds to hours. Key questions we plan on addressing include: (1) How do histone modifications impact allele-specific transcription? (2) To what degree are allele-specific mRNA differentially regulated and translated in the cytoplasm? and (3) How coupled are allele-specific transcriptional and translational bursts? Using a homozygous CRISPR-engineered cell line harboring MS2 stem loops and repeat FLAG epitopes in the MYH9 locus (encoding myosin-2A, overexpression of which causes cancer), we will quantify bursts in MYH9 expression at the levels of transcription and translation to better understand their origins and coupling in both unperturbed and stimulated cells.