Nucleosome Avidities and Transcriptional Silencing in Yeast

Abstract

A classical example of "transcriptional silencing" is found in the yeast S. cerevisiae mating-type switch [1, 2]. The gene pairs a1/a2 and α1/α2, positioned at the loci HMR and HML, respectively, are silenced by Sir proteins recruited by proteins that bind sites flanking each locus. Transfer of either gene pair to the Sir-free MAT locus, or mutation of the Sirs, allows expression of those genes at levels sufficient to foster yeast mating. Here we confirm that, in the absence of Sirs, a1 and a2 at HMR are expressed at low levels [3]. This level is low because, we show, the relevant transcriptional activators, which work from regulatory sites located between the divergently transcribed genes, are weak. That property-weak activation-is a prerequisite for effective silencing upon recruitment of Sirs. We use our quantitative nucleosome occupancy assay to show that Sirs (which bind nucleosomes) increase the avidities with which those nucleosomes form at the promoters. That increase can account for at least part of the repressive effects of the Sirs and can explain why silencing is effective in countering weak activation only. We suggest that "silencing" in higher eukaryotes (e.g., by Polycomb or HP1) follows similar rules [4, 5] and note where such effects could be important.

The Nuts and Bolts of Transcriptionally Silent Chromatin in Saccharomyces cerevisiae

“…A protein complex containing Sir2, Sir3, and Sir4 is recruited to specific sites, known as silencers, by DNA-bound factors and Sir1. The Sir2/3/4 complex interacts with histones and DNA adjacent to silencers, thereby imposing greater nucleosome occupancy and more precise nucleosome positioning across silenced domains (Weiss and Simpson 1998;Ravindra et al 1999;Wang et al 2015). When Sir proteins are reconstituted with chromatin templates in vitro, nucleosomes aggregate; sometimes forming distinctive 20-nm fibers visible by electron microscopy (EM) (Onishi et al 2007;Johnson et al 2009).…”

Heterochromatin: Dark Matter or Variation on a Theme?

“…They observed that nucleosome occupancy of the HMR locus, which harbors the cell identity gene pair a1/a2, increased from 30-40% in the absence of Sirs (i.e., in a sir -mutant), to 50-60% in their presence [1]. Importantly, the authors observed this effect under circumstances in which the genes located at HMR were transcriptionally inactive irrespective of the presence of Sirs.…”

“…Over the past two decades, work from a number of laboratories has led to a revision of this 'dark matter' view of heterochromatin. The latest is a paper from Mark Ptashne and colleagues appearing in a recent issue of Current Biology [1]. Using as their model the Sir protein-regulated silent chromatin of the budding yeast Saccharomyces cerevisiae, these authors demonstrate that while Sir-heterochromatin can extinguish expression of weakly activated genes, it becomes increasingly less effective in repressing transcription as the strength of activation is increased.…”

“…Wang et al [1] through use of a powerful, quantitative micrococcal nuclease digestion assay developed in the Ptashne lab, show that Sir heterochromatin, and by implication other forms of heterochromatin, exert at least part of their repressive effects in the most fundamental way -by increasing the capacity of nucleosomes to form on the underlying DNA. They observed that nucleosome occupancy of the HMR locus, which harbors the cell identity gene pair a1/a2, increased from 30-40% in the absence of Sirs (i.e., in a sir -mutant), to 50-60% in their presence [1].…”

“…In a recent interview, Richard C. Lewontin, a central thinker in the field of evolutionary biology, succinctly noted that the ''admission of necessary ignorance of historically remote things is the first rule of intellectual honesty in evolution'' [1]. Possibly with reluctance, many biologists would admit to some level of ignorance in terms of our understanding of how different species responded to the extreme variation in climate over the past few million years.…”