Abstract
Chromatin boundary elements serve as cis-acting regulatory DNA signals required to protect genes from the effects of the neighboring heterochromatin. In the yeast genome, boundary elements act by establishing barriers for heterochromatin spreading and are sufficient to protect a reporter gene from transcriptional silencing when inserted between the silencer and the reporter gene. Here we dissected functional topography of silencers and boundary elements within circular minichromosomes in Saccharomyces cerevisiae. We found that both HML-E and HML-I silencers can efficiently repress the URA3 reporter on a multi-copy yeast minichromosome and we further showed that two distinct heterochromatin boundary elements STAR and TEF2-UASrpg are able to limit the heterochromatin spreading in circular minichromosomes. In surprising contrast to what had been observed in the yeast genome, we found that in minichromosomes the heterochromatin boundary elements inhibit silencing of the reporter gene even when just one boundary element is positioned at the distal end of the URA3 reporter or upstream of the silencer elements. Thus the STAR and TEF2-UASrpg boundary elements inhibit chromatin silencing through an antisilencing activity independently of their position or orientation in S. cerevisiae minichromosomes rather than by creating a position-specific barrier as seen in the genome. We propose that the circular DNA topology facilitates interactions between the boundary and silencing elements in the minichromosomes.
Highlights
The DNA in the nuclei of eukaryotic cells is packed into nucleosomes, establishing the primary level of chromatin packing
We report here that the URA3 reporter gene was efficiently silenced by the E and/or I silencers in the absence of heterochromatin boundary elements and the URA3 reporter was de-repressed in the presence of STAR and TEF2-UASrpg elements in circular minichromosomes similar to previous studies in the yeast genome [19,20]
We examined if the URA3 reporter gene would be protected from silencing by two heterochromatin boundary elements, STAR and TEF2-UASrpg that are able to counteract the silencing in an orientation-dependent manner in the yeast genome [19,20]
Summary
The DNA in the nuclei of eukaryotic cells is packed into nucleosomes, establishing the primary level of chromatin packing. Previous research has focused on identifying cis-acting genetic elements termed ‘‘boundary elements’’ that demarcate the heterochromatin from the euchromatin [5,6]. Such DNA elements were identified in evolutionary diverse organisms ranging from yeast to humans [7,8,9]. The heterochromatin boundary elements establish boundaries of chromatin domains by limiting the spread of silencing signals to the adjoining regions [10] These elements are especially important when transcriptionally active genes are surrounded by condensed heterochromatin as they stop the incursion of silencing signals from the surrounding regions thereby protecting the genes from position-dependent variegation
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