UASrpg can function as a heterochromatin boundary element in yeast

Area of Science:

• Molecular Biology
• Yeast Genetics
• Chromatin Biology

Background:

• The HM loci in Saccharomyces cerevisiae are known to establish gene-nonspecific repression domains, silencing various heterologous genes.
• Certain promoters, including those of Ashbya gossypii TEF and S. cerevisiae TEF1/TEF2, resist transcriptional silencing by HM silencers.

Purpose of the Study:

• To investigate the mechanism by which specific promoter segments resist transcriptional silencing.
• To identify and characterize elements that can block the spread of silencing and function as chromatin boundary elements in yeast.

Main Methods:

• Assessed the silencing resistance of Ashbya gossypii TEF and S. cerevisiae TEF promoters when placed within HM loci.
• Interposed promoter segments between HML alpha genes and an E silencer to evaluate their effect on repression.
• Analyzed the DNA sequences of active fragments for conserved elements, such as Rap1-binding sites within UASrpg.
• Tested the insulating and orientation-dependent properties of a specific 149-bp TEF2 promoter segment.

Main Results:

• Segments from TEF promoters, particularly those containing UASrpg with Rap1-binding sites, were found to block HM silencer-mediated repression.
• A 149-bp fragment from the yeast TEF2 UASrpg, containing three tandem Rap1-binding sites, demonstrated significant silencer-blocking activity.
• This element exhibited insulating activity and orientation dependence, characteristic of chromatin boundary elements, and blocked heterochromatin spread.

Conclusions:

• The identified 149-bp segment from the yeast TEF2 UASrpg functions as a novel chromatin boundary or insulator element in Saccharomyces cerevisiae.
• This element prevents the spread of heterochromatin, offering protection against silencing mediated by HM loci.
• This discovery provides the first example of a chromatin domain boundary element in yeast, characterized by Rap1-binding sites.