Co-Evolution of Transcriptional Silencing Proteins and the DNA Elements Specifying Their Assembly

Abstract

As shown by genetic assays in Saccharomyces interspecies hybrids, the co-evolution of heterochromatin assembly proteins with silencer elements allows transcriptional silencing functions to be maintained in rapidly evolving regions of the genome.

Abstract

Co-evolution of transcriptional regulatory proteins and their sites of action has been often hypothesized but rarely demonstrated. Here we provide experimental evidence of such co-evolution in yeast silent chromatin, a finding that emerged from studies of hybrids formed between two closely related Saccharomyces species. A unidirectional silencing incompatibility between S. cerevisiae and S. bayanus led to a key discovery: asymmetrical complementation of divergent orthologs of the silent chromatin component Sir4. In S. cerevisiae/ S. bayanus interspecies hybrids, ChIP-Seq analysis revealed a restriction against S. cerevisiae Sir4 associating with most S. bayanus silenced regions; in contrast, S. bayanus Sir4 associated with S. cerevisiae silenced loci to an even greater degree than did S. cerevisiae's own Sir4. Functional changes in silencer sequences paralleled changes in Sir4 sequence and a reduction in Sir1 family members in S. cerevisiae. Critically, species-specific silencing of the S. bayanus HMR locus could be reconstituted in S. cerevisiae by co-transfer of the S. bayanus Sir4 and Kos3 (the ancestral relative of Sir1) proteins. As Sir1/Kos3 and Sir4 bind conserved silencer-binding proteins, but not specific DNA sequences, these rapidly evolving proteins served to interpret differences in the two species' silencers presumably involving emergent features created by the regulatory proteins that bind sequences within silencers. The results presented here, and in particular the high resolution ChIP-Seq localization of the Sir4 protein, provided unanticipated insights into the mechanism of silent chromatin assembly in yeast.

Author Summary

As eukaryotic species evolve, transcriptionally silent portions of their genomes—termed “heterochromatin”—mutate rapidly. To maintain the “off” state of certain genes in silenced regions, regulatory DNA sequences called silencers, which reside within a rapidly mutating region, must co-evolve with the regulatory proteins that bind these sequences to turn off transcription. Although hypothesized to occur widely in nature, such “molecular co-evolution” of genetic regulators has been demonstrated in only a few cases. Unlike previous examples of gene regulatory co-evolution, we found that the transcription factors that bind silencers in two budding yeast species are, in fact, functionally interchangeable, even though the silencers are not. Surprisingly, the Sir1 and Sir4 silencing proteins, which are heterochromatin components that bind the transcription factors rather than the silencer DNA sequences per se, are the proteins engaged in rapid co-evolution with the silencers. Silencer sequences therefore contain additional, evolutionarily labile information directing the assembly of heterochromatin. As mutations in Sir1 and Sir4 over evolutionary time can compensate for changes in the silencers, this “extra information” likely involves cooperative assembly of the transcription factors with the Sir1 and Sir4 “adaptor” proteins. The localization patterns of two species' Sir4 proteins across both species' genomes in interspecies yeast hybrids illuminate unexpected features of heterochromatin structure and assembly.