A Genetic Screen For Histone H3 Mutations That Interfere With Transcription Elongation By RNA Polymerase II

Eukaryotes package their genomes into chromatin.  The basic repeating subunit of chromatin, the nucleosome, permits the orderly storage of DNA in the nucleus, but also poses a physical barrier to elongating RNA polymerases. One factor proposed to help RNA polymerase II overcome this barrier is Spt5, a universally conserved and essential transcription elongation factor.  Previously, our lab described a mutation in the S. cerevisiae SPT5 gene [spt5CS-] that reduces the rate of transcription elongation by RNA polymerase II.  We also found that the spt5CS- mutation can be genetically suppressed by mutations in histones, several histone modifying enzymes and chromatin remodeling enzymes implicated in transcription elongation.  We hypothesize that these mutations suppress spt5CS- by alleviating the nucleosomal barrier to efficient transcription elongation.  We further hypothesize that histone mutations that increase the stability of nucleosomes on DNA should have the opposite phenotype of our histone suppressors, i.e. we expect this class of histone mutation to enhance the phenotypes of spt5CS-.  To test this idea, we are performing a genetic screen for histone H3 mutations that are lethal when combined with spt5CS-.  As is the case for the histone H3 mutations that suppress spt5Cs-, we predict that such mutations will define a novel class of histone H3 defects, and thus a novel function of histone H3 and nucleosomes in transcription.