# RNA Interference and Heterochromatic Silencing in Replication and Quiescence

> **NIH NIH R35** · COLD SPRING HARBOR LABORATORY · 2022 · $435,072

## Abstract

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.

## Key facts

- **NIH application ID:** 10330828
- **Project number:** 1R35GM144206-01
- **Recipient organization:** COLD SPRING HARBOR LABORATORY
- **Principal Investigator:** ROBERT A MARTIENSSEN
- **Activity code:** R35 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $435,072
- **Award type:** 1
- **Project period:** 2022-08-05 → 2027-07-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10330828

## Citation

> US National Institutes of Health, RePORTER application 10330828, RNA Interference and Heterochromatic Silencing in Replication and Quiescence (1R35GM144206-01). Retrieved via AI Analytics 2026-05-22 from https://api.ai-analytics.org/grant/nih/10330828. Licensed CC0.

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