Abstract

We have examined the chromatin structure of the HMRE/HSP82 and HMRa/HSP82 allels using three complementary approaches : DNase I chromating footprinting, micrococcal nuclease (MNase) nucleosome-protected ladder assay, and an in vivo E. coli dam methylase accessibility assay. The footprinting results indicate that the promoter and silencer sequences are assembled into nucleoprotein complexes which exhibit no detectable change in structure, despite a 70-fold range in expression levels. In addition, the promoter region of the HMRa/HSP82 allele is cleaved randomly by MNase in all cases, indicating the absence of anonical nucleosomes over this region irrespective of SIR4 or heat-shock. Finally, no discernible difference in the accessibility of the HMRE/HSP82 locus to dam methylase in SIR4 vs. sir4 cells was seen, which again suggests that the chromatin structure of HMRE/HSP82 allele is identical regardless of SIR4. Altogether, our results indicate that in contrast to other observations of the silent mating-type loci, no discernible structural alteration is detected at either HMR/HSP82 allele regardless of SIR genetic background or transcriptional state of the gene.

• Keywords: Transcriptional repression; chromatin structure; DNase I footprinting