DNA elimination mechanisms in Tetrahymena

Washington University, Saint Louis MO

Investigators

Abstract

The genomes of many organisms contain DNA with no known function. In some organisms, this so-called junk DNA is eliminated during development by a complex rearrangement of the genome. However, it is not clear how the cell can tell what is junk and what is not. This research will study how the ciliate, Tetrahymena thermophila, establishes what parts of the genome should be thrown away as junk and what should be retained as functionally important. Because junk DNA is found in the genomes of many organisms, this work could have broad scientific significance on understanding how cells deal with extraneous, non-essential DNA. To accomplish the research, a tool for visualizing genome-related datasets will be created and made accessible for use by the broader scientific community, thereby improving the current scientific infrastructure. The project will enhance the future scientific workforce by engaging trainees from the undergraduate to post-doctoral level in hypothesis-driven research. The research efforts will be coordinated with curricular development activities of the principle investigator, and thus will facilitate best teaching practice by bringing authentic research into the university biology classroom. Students engaged in the classroom research, in turn, will gain first-hand experience in the practice of scientific discovery and add to a growing body of scientific knowledge. This project will elucidate mechanisms that eukaryotic cells use to establish boundaries between functionally distinct regions of the genome by exploiting the model experimental organism, Tetrahymena thermophila, which eliminates much of its non-coding DNA from the somatic copy of its genome. Using a combination of genetic and biochemical approaches, the work will uncover mechanisms that Tetrahymena cells use to define the boundaries of the DNA segments that are eliminated during its development. The fact that Tetrahymena eliminates these sequences allows one to unambiguously identify those that get packaged as junk by comparing the content of the reorganized somatic genome and the intact germline genome. The project will use available Tetrahymena genome sequences to generate a genome browser that will map the retained and eliminated regions, using the intact genome as a reference. Previous research revealed that a regulatory protein called Lia3 is critical for the accurate removal of a subset of the eliminated sequences. The genome browser will be used to compare the DNA of normal cells to DNA from cells lacking LIA3 to identify all the sequences for which elimination boundaries are inaccurately defined when this protein is absent. Preliminary experiments have revealed that the sequences controlled by Lia3 contain guanine (G)-rich sequences positioned about 50 base pairs from each boundary. Lia3 binds these sequences when they form G-quadruplex DNA, a four-stranded DNA structure. This represents a novel DNA binding activity, and the project will thoroughly characterize both the ability of Lia3 to bind this non-standard DNA structure and elucidate how this structure can serve to organize the genome. In addition, the Tetrahymena genome encodes three other proteins that are similar to Lia3 in sequence and gene expression, and the project will test the hypothesis that each of these proteins identifies the boundaries of a distinct subset of eliminated DNA. Together, these approaches will characterize a previously unknown mechanism that eukaryotic cells use to define boundaries between genes and non-coding DNA and provide clear evidence that G-quadruplex structures have important regulatory roles.