Heterochromatic gene silencing by activator interference and a transcription elongation barrier

Heterochromatin silences transcription, contributing to development, differentiation, and genome stability in eukaryotic organisms. Budding yeast heterochromatic silencing is strictly dependent on the silent information regulator (SIR) complex composed of the Sir2 histone deacetylase and the chromatin-interacting proteins Sir3 and Sir4. We use reconstituted SIR heterochromatin to characterize the steps in transcription that are disrupted to achieve silencing. Transcriptional activator binding is permitted before and after heterochromatin assembly. A comprehensive proteomic approach identified heterochromatin-mediated disruption of activator interactions with coactivator complexes. We also find that if RNA polymerase II (Pol II) is allowed to initiate transcription, the SIR complex blocks elongation on chromatin while maintaining Pol II in a halted conformation. This Pol II elongation barrier functions for even one nucleosome, is more effective when assembled with multiple nucleosomes, and is sensitive to a histone mutation that is known to disrupt silencing. This dual mechanism of silencing suggests a conserved principle of heterochromatin in assembling a specific structure that targets multiple steps to achieve repression.