EAPSI: Developing Super-resolution Microscopy to Image 'Jumping Genes' in Living Cells

Warneford-Thomson Robert R, Philadelphia PA

Investigators

Abstract

Surprisingly, repetitive 'junk' DNA makes up almost half of the human genome. One of the most common types of these repetitive elements are retrotransposons that can copy-and-paste themselves to other locations. While most are now inactive, there is a subset of these elements that are still mobile. These transposition events can lead to a reorganization of DNA in the nucleus, and have been linked to both aging and cancer, yet how they function remains poorly understood. In order to unravel the function and impact of retrotransposons on the genome new approaches are necessary. Under the direction of Dr. Yujie Sun of Peking University, China, an expert in single-molecule super-resolution optical microscopy, this project aims to develop imaging methods to capture these 'jumping genes' in action in living cells. Recent advances in targeted genome editing have provided tools to recruit proteins to precise sequences of DNA. A specific locus can be imaged by expression of a nuclease-deficient Cas9 enzyme fused to a fluorescent protein and an RNA adaptor that is complementary to the sequence of interest, referred to as a guide RNA. Transposition events can be captured in cultured human cells by designing guide RNAs to an ectopically expressed long interspersed element-1 transposable element. These events then be captured using single molecule super-resolution techniques such as stochastic optical reconstruction microscopy (STORM), providing information on the kinetics of retrotransposition. This approach can also assess the effects of retrotransposon activity on higher order chromosome structure and nuclear architecture, both of which are important regulators of the transcriptional landscape of the nucleus. These methods can aid in the development of a more dynamic view of the genome, supplementing the wealth of information obtained from DNA sequencing. This NSF EAPSI award supports the research of a U.S. graduate student and is funded in collaboration with the Chinese Ministry of Science and Technology.