Histone Mutations Project

Name of Research Sponsor: Dr. David Engelke

Dates of Involvement: Summer 2010

Histone mutations that alleviate tRNA gene mediated silencing

Sara Rivera, Dr. David Engelke, James Ignatz-Hoover, Paul Good

Purpose:

The 274 tRNA genes in Saccharomyces cerevisiae are scattered throughout the linear maps of the 16 chromosomes, but the genes are clustered together in the nucleolus in 3-demensional space. tRNA gene mediated (tgm) silencing, in which transcription of RNA polymerase III from tRNA gene promoters suppresses nearby RNA polymerase II (pol II) transcription when the RNA pol II is downstream, is an effect of this localization. Because tRNA genes are found upstream of many ORFs, tgm silencing could impact a large group of genes. Little is known about the mechanism of tgm silencing; however, some mutations have previously been found to alleviate the silencing while leaving the spatial organization of tRNA genes essentially the same. These mutations suggest that additional events take place to achieve tgm silencing outside of tRNA gene localization. Moreover, the mechanisms of numerous forms of silencing in yeast are strongly dependent on chromatin structure. Thus, it is reasonable to hypothesize that tgm silencing also would be dependent on chromatin structure.

Methods:

To test this hypothesis, we tested a large collection of mutants (SHIMA library) in which each amino acid in the four core histones of chromain nucleosomes, H2A, H2B, H3, and H4, was mutated to alanine, one at a time. Assays tested whether the mutated histones released tgm silencing of an artificial test gene, NAT1, that confers antibiotic resistance in yeast.

Results:

Of the 442 mutations, 18 of which are lethal, 63 were found to alleviate tgm silencing.

Conclusions:

Currently, these mutations are being verified. A few of the amino acid positions are known to be modified in chromatin, but the majority of residues that alleviate tgm silencing are not known to be modified. Mechanisms will be discussed relating nucleosome structure to the mechanism of tgm silencing.

To view with better resolution click here: SHIMA Project Poster