RESUMO
Our research addresses the relationship between subnuclear localization and gene expression in fission yeast. We observed the relocalization of a heterochromatic region, the mating-type region, from its natural location at the spindle-pole body to the immediate vicinity of the nucleolus. Relocalization occurred in response to a DNA rearrangement replacing a boundary element (IR-R) with a ribosomal DNA repeat (rDNA-R). Gene expression was strongly silenced in the relocalized mating-type region through mechanisms that differ from those operating in wild type. Also different from the wild-type situation, programmed recombination events failed to take place in the rDNA-R mutant. Increased silencing and perinucleolar localization depended on Reb1, a DNA-binding protein with cognate sites in the rDNA. Reb1 was recently shown to mediate long-range interchromosomal interactions in the nucleus through dimerization, providing a mechanism for the observed relocalization. Replacing the full rDNA repeat with Reb1-binding sites, and using mutants lacking the histone H3K9 methyltransferase Clr4, indicated that the relocalized region was silenced redundantly by heterochromatin and another mechanism, plausibly antisense transcription, achieving a high degree of repression in the rDNA-R strain.
Assuntos
DNA Ribossômico/genética , Proteínas de Ligação a DNA/metabolismo , Inativação Gênica , Heterocromatina/fisiologia , Espaço Intranuclear/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/metabolismo , Fatores de Transcrição/metabolismo , Sítios de Ligação/genética , Proteínas de Ligação a DNA/genética , Reação em Cadeia da Polimerase Multiplex , Sequências Repetitivas de Ácido Nucleico/genética , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/genética , Fatores de Transcrição/genéticaRESUMO
A transcriptionally silent chromosomal domain is found in the mating-type region of fission yeast. Here we show that this domain is delimited by 2-kb inverted repeats, IR-L and IR-R. IR-L and IR-R prevent the expansion of transcription-permissive chromatin into the silenced region and that of silenced chromatin into the expressed region. Their insulator activity is partially orientation dependent. The silencing defects that follow deletion or inversion of IR-R are suppressed by high dosage of the chromodomain protein Swi6. Combining chromosomal deletions and Swi6 overexpression shows that IR-L and IR-R provide firm borders in a region where competition between silencing and transcriptional competence occurs. IR-R possesses autonomously replicating sequence (ARS) activity, leading to a model where replication factors, or replication itself, participate in boundary formation.