Heterochromatin is classically perceived to be refractory to transcription because of its compact structure. Our work on RNAi and other RNA-turnover pathways challenged this paradigm by demonstrating the synthesis of heterochromatic transcripts even when heterochromatin is normally packaged. However, mechanisms also exist that ensure highly efficient and specific elimination of heterochromatic RNAs.
We have shown that specificity for this RNA degradation pathway relies on one of the best-known heterochromatin proteins, HP1. Our work in S. pombe revealed that HP1 efficiently captures heterochromatic RNAs, which thereby become translationally incompetent and are eventually degraded. Importantly, we discovered that RNA binding via the hinge region of HP1 competes with methylated histone H3K9 binding mediated by the chromodomain of HP1. Hence, HP1 binding to RNA is incompatible with stable heterochromatin association in fission yeast. Based on our current results, we propose a model for the action of the HP1 ensemble, which dynamically exchanges with the bulk in a maintenance cycle. Free RNA is captured in the eviction cycle and passed on to the degradation machinery. Constant flux of RNA-unbound HP1 from the bulk ensemble prevents saturation of heterochromatin with RNA. Competition between RNA and methylated H3K9 for HP1 binding at the ensemble level guarantees that RNA-free HP1 is preferably recruited to heterochromatin, which sustains a functional checkpoint on the H3K9-methylated nucleosome and leads to constant turnover of heterochromatic RNAs.
Our work on HP1 provides answers to many open questions in the field, such as why the association of HP1 proteins with heterochromatin is so highly dynamic, why classical PEV screens have not revealed the involvement of the RNA degradation machinery in heterochromatin silencing, and what distinguishes heterochromatic mRNA from euchromatic mRNA. We hypothesize that our mechanistic insights into yeast heterochromatin silencing might also be relevant to transposon silencing through the piRNA pathway in animals, a possibility that is subject of current investigations.
Following up our observation that RNA binding to HP1 is incompatible with H3K9methyl binding, we discovered a class of ncRNAs that counteract encroachment of heterochromatin into neighboring euchromatin. We have identified a long ncRNA, termed BORDERLINE, that prevents the spreading of HP1 and H3K9 methylation beyond the pericentromeric repeat region of fission yeast chromosome 1. Notably, BORDERLINE RNAs function in a sequence-independent but locus-dependent manner.
That ncRNAs demarcate distinct epigenetic domains by counteracting the deposition of a repressive histone modification is unexpected and adds another layer of complexity to the crosstalk between RNA and chromatin. Considering the high conservation of HP1 proteins and the production of ncRNAs from silent chromatin in higher eukaryotes, we anticipate that similar mechanisms shaping the epigenome also operate in other organisms. Therefore, we have intensified our efforts to characterize RNA-mediated HP1 regulation in different eukaryotes.
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