Abstract
Effective boundary mechanisms halt the spread of repressive histone methylation. In the fission yeast Schizosacchromyces pombe, two factors/elements required for boundary function have been described, the jmjC protein Epe1 and binding sites for the RNA polymerase III transcription factor TFIIIC. Perplexingly, individual mutation of Epe1 or TFIIIC sites produces only mild boundary defects, and no other boundary factors have been identified. To approach these issues, we developed a synthetic reporter gene tool that uses a tethered Clr4 histone H3K9 methyltransferase and monitors the ability of a DNA element to block heterochromatin spread. The inverted repeat (IR) that flanks the mat2/3 silent mating-type cassette region demonstrates strong boundary activity compared to sequences that flank pericentromeric heterochromatic repeats. Rather than acting in the same inhibitory pathway, Epe1 and TFIIIC sites mediate boundary function of the IR via the two parallel and largely redundant pathways. We also use the system to demonstrate that HP1/Swi6 promotes boundary activity in addition to promoting silencing and acts in the same pathway as Epe1. Inhibition of heterochromatin spread at the endogenous IR element also requires either Epe1 or TFIIIC sites. Strikingly, mutation of both mechanisms results in growth inhibition that is associated with the spread of heterochromatin over many kilobases to the nearest essential gene and the near-complete silencing of several intervening euchromatic genes. The growth defect is reversed by deletion of clr4+, indicating that the redundant boundary mechanisms protect cells from intrinsic toxicity caused by the spread of heterochromatin.
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