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ABSTRACT Eukaryotic gene expression occurs in the context of structurally distinct chromosomal domains such as the relatively open, gene-rich, and transcriptionally active euchromatin and the condensed and gene-poor heterochromatin where its specific chromatin environment inhibits transcription. To study gene silencing by heterochromatin, we created a minichromosome reporter system where the gene silencer elements were used to repress the URA3 reporter gene. The minichromosome reporters were propagated in yeast Saccharomyces cerevisiae at a stable copy number. Conduction of gene silencing through nucleosome arrays was studied by placing various repeats of clone-601 DNA with high affinity for histones between the silencer and reporter in the yeast minichromosomes. High-resolution chromatin mapping with micrococcal nuclease showed that the clone-601 nucleosome positioning downstream of the HML-E gene silencing element was not significantly altered by chromatin silencing. Using URA3 reporter assays, we observed that gene silencing was conducted through arrays of up to eight nucleosomes. We showed that the shorter nucleosome repeat lengths, typical of yeast (167 and 172 bp), were more efficient in conducting silencing in vivo compared to the longer repeats (207 bp) typical of higher eukaryotes. Both the longer and the shorter repeat lengths were able to conduct silencing in minichromosomes independently of clone-601 nucleosome positioning orientations vs. the silencer element. We suggest that the shorter nucleosome linkers are more suitable for conducting gene silencing than the long repeats in yeast due to their higher propensity to support native-like chromatin higher-order folding.
EUKARYOTIC DNA is repeatedly coiled by histone De- tainers into nucleosome cores, the primary structural units of chromatin (Richmond and Davey 2003). The nucle- osome cores are connected by linker DNA-forming nucleo- some arrays that fold into compact higher-order structures (huger et al. 2012). One of the critical biological questions has been deciphering the chromatin structure-function re- lationship in epigenetic regulation of gene expression. Eu- karyotic gene expression occurs mainly in the context of the structurally open and transcriptionally active state (euchro- matin) while, in the repressive state (heterochromatin), its specific chromatin organization inhibits transcription (Grewal and Moazed 2003). A combination of transcription factors, DNA modifications, histone modifications, noncod- ing RNA, and chromatin compaction distinguishes hetero- chromatin from the transcriptionally active euchromatin (Moazed 2011). Recently, nucleosome positioning in the genome and intrinsic affinity of DNA...