Friday, April 4, 2025
4/4/2025
RNA Interference and Heterochromatic Silencing in Replication and Quiescence - Gene regulation by RNA interference is usually attributed to microRNA, but RNAi has a more ancient and fundamental role in heterochromatic silencing and genome stability. Heterochromatin comprises condensed repetitive regions of eukaryotic chromosomes, and mediates transcriptional silencing, chromosome segregation and genome integrity. We have found that heterochromatin is unexpectedly transcribed, and that subsequent RNAi guides histone modification. In the fission yeast S. pombe, “co- transcriptional” silencing occurs during the S phase of the cell cycle, followed by transcriptional silencing thereafter. Release of RNA polymerase II (Pol II) during replication prevents DNA damage, but without RNAi, replication forks stall and are repaired by homologous recombination (HR), causing genome instability. We have found that RNAi regulates genome stability through R-loops, RNA-DNA hybrid structures at transcription-replication collisions that promote HR. Silencing depends on histone modification, but recent studies show this may be an oversimplification. First, histone methylation recruits Heterochromatin Protein 1, which mediates liquid-liquid phase separation (LLPS) and may limit access to Pol II. Second, RNAi also recruits ubiquitin ligase, and we recently found that ubiquitin promotes these phase transitions. RNAi guides histone modification in C. elegans and Drosophila, but conservation in mammalian systems has been controversial. For example, piRNAs mediate histone modification in the germline but do not depend on Dicer, while genome instability in Dicer mutant mouse embryonic stem cells (mESC) depends on transcription of satellite repeats. Strikingly, we have found genome instability in Dicer-/- mESC depends on the transcriptional co- activator BRD4, and identical in-frame bromodomain deletions rescue Dicer mutants in fission yeast. S. pombe is an outstanding model system for cell cycle research, and we were the first to show that RNAi is essential for quiescence (G0). Genetic screens have revealed that nucleolar RNA silencing and histone modifications mediate this novel function. Our goals in the next five years are to determine the elusive mechanism by which RNAi guides each aspect of heterochromatin from repeat instability, to silencing, chromosome segregation and DNA repair as well as survival in quiescence. Our recent work suggests a central role for long non-coding RNA, R-loops and the activity of RNAse H in the upstream steps in this pathway. Downstream events include histone modification and LLPS that may underlie the classically “condensed” properties of heterochromatin. We will use our stem cell model to assess the conservation and relevance of these mechanisms in health and disease, especially in cancer.
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