A Single Heterochromatin Boundary Element Imposes Position-Independent Antisilencing Activity in Saccharomyces cerevisiae Minichromosomes

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.

Nucleosome-Positioning Sequence Repeats Impact Chromatin Silencing in Yeast Minichromosomes

“…We found that, in both orientations tested, the STAR and TEF2-UASrpg acted as antisilencer elements even in the presence of a monomer nucleosome-positioning sequence and were able to derepress URA3 (Figure 2, A-D, spotting assays). This experiment demonstrates that clone-601 DNA does not perturb the ability of an antisilencer placed upstream of the E-silencer to activate transcription of the reporter gene (Chakraborty et al 2011). In further work, we used this property of the antisilencer elements to activate transcription without changing the sequence of chromatin block including the silencer, the array of clone-601 nucleosomes, and the reporter.…”

“…In the prototype constructs, either the HML-E (positioned upstream of the URA3 reporter) or HML-I (positioned downstream of the URA3 reporter) alone were capable of silencing the expression of the URA3 reporter gene (Chakraborty et al 2011). Here we examined if placing a clone-601 DNA sequence in combination with linker DNA of variable lengths between the silencer and the reporter would impede or facilitate silencing of the URA3 reporter gene.…”

“…Furthermore, it has been reported that DNA sequences that do not favor nucleosome formation and have the ability to disrupt chromatin structure can also function as barriers to the propagation of transcriptionally silent chromatin (Bi et al 2004). Here we used our recently established URA3-based yeast minichromosome reporter system containing silencer and antisilencer elements (Chakraborty et al 2011) to investigate if arrays of nucleosomes with high DNA affinity to histones and varying nucleosome number and repeat lengths will conduct silencing from the HML-E and HML-I elements to a reporter gene. In this study, we employed the clone-601 DNA sequences that have the highest affinity for the histone octamer and positions the nucleosome core with a single-base precision (Lowary and Widom 1998).…”

“…NC_001136.10; SGD: chromosome IV, 461842-462516); and a pBR322 vector-derived sequence with an AmpR gene for propagation in Escherichia coli. The S. cerevisiae minichromosome constructs ( Figure 1A) containing the URA3 reporter gene, the HML-E, and the HML-I silencer elements were generated as previously described (Chakraborty et al 2011). …”

“…Yeast colonies grown on 2Trp media were selected for minichromosomes containing a TRP1 marker gene in the construct backbone. The expression of the URA3 reporter gene and function of the regulatory elements in the minichromosome constructs were determined in the presence or absence of the silencer elements using different selective media (Chakraborty et al 2011). Yeast colonies were grown on complete synthetic media (CSM), 2Trp (lacking tryptophan), 2Trp/2Ura (lacking both tryptophan and uracil), or 2Trp/5-FOA+ (lacking tryptophan, but containing 5-fluroorotic acid).…”