Towards the Identification of Genomic Targets of MED 31 in Tetrahymena thermophila.

Authors:
C. ThuppuMudalige 1 ; J. Garg 2 ; J. Lambert 3,4 ; A. Gingras 3 ; R. Pearlman 2 ; J. Fillingham 1


Institutes
1) Ryerson University, Toronto, Ontario, Canada; 2) York University, Toronto, Ontario, Canada; 3) Samuel Lunenfeld Research Institute, Toronto, Ontario, Canada; 4) University of Toronto, Ontario, Canada.


Abstract:

Tetrahymena thermophila is a unicellular protist that has a nuclear dimorphism with a polyploid somatic macronucleus (MAC) and a diploid germline micronucleus (MIC) within its single cell. During the vegetative growth of Tetrahymena, the two nuclei replicate and divide independently of each other; MIC divides by mitosis and the MAC divides by amitosis. The MIC undergoes meiosis during the sexual life cycle of Tetrahymena known as conjugation. The MIC is transcriptionally inactive during the vegetative state and active during meiosis while the MAC is active during vegetative growth and conjugation. The MIC genome is extensively remodeled to form the transcriptionally active MAC genome.  The Med31 protein belongs to the transcriptional coactivator complex, Mediator.  Med31 protein is small and highly conserved in T. thermophila and most eukaryotes at the amino acid level.  While Mediator complexes are well characterized in plants, fungi and animals, it is yet to be characterized in protists. In humans and yeast, Mediator has been shown to interact with the C-terminal domain (CTD) of RPB1 subunit of RNA Polymerase II. Since T. thermophila lacks a canonical CTD in its RPB1 subunit, it raises the question whether the organism has a canonical Mediator.  Affinity purification using epitope tag added to the Med 31 and subsequent mass spectrometry analysis represented a potential Mediator complex in T. thermophila. Indirect immunofluorescence has indicated that during vegetative growth, Med 31 localizes to the MAC and to the MIC during meiosis, consistent with function in transcription. To test this hypothesis, we are working towards mapping the genomic targets of Med31 in growth and development using Chromatin Immunoprecipitation combined with next generation sequencing (ChIP-Seq). ChIP is a powerful molecular method where crosslinked chromatin extracts are immune depleted of DNA-protein complexes with specific antibodies against the DNA-binding proteins.  The DNA is subsequently analyzed using a variety of methods including direct sequencing.  The ChIP method was initially developed for Tetrahymena model organism by Dedon et al. (1991) using purified MACs as starting material. We have modified the protocol to use whole cells as starting material and intend to use highthroughput sequencing to identify the genomic targets of Med31.