# Mechanisms of Gene Silencing

> **NIH NIH R01** · UNIVERSITY OF CALIFORNIA, SAN FRANCISCO · 2021 · $386,854

## Abstract

The majority of cytosine in the human genome is methylated on the 5-position (5mC) where it plays key roles
in development and in disease, including cancer, through its potent gene repression activity. In vertebrates,
5mC is established by de novo enzymes (Dnmt3a/b-Dnmt3L) and maintained epigenetically in the context of
symmetric sequence contexts (e.g. CpG) by the maintenance enzyme Dnmt1 and its cofactor Uhrf1. Because
S. cerevisiae and S. pombe lack 5mC, facile yeast experimental systems have not been developed to
investigate this important DNA mark. We have now developed such a system, namely the human fungal
pathogen C. neoformans. This tractable, haploid budding yeast harbors symmetric CpG methylation at
centromeres and subtelomeric regions. 5mC production is catalyzed by an enzyme called Dnmt5. In addition to
a methyltransferase domain, Dnmt5 harbors a chromodomain that recognizes histone H3 modified on lysine 9
(H3K9me) and an ATPase domain. H3K9me promotes DNA methylation in vivo as does a homolog of the
human hemimethyl-DNA-binding protein Uhrf1. Purified Dnmt5 is ATP-dependent and displays extraordinary
specificity for hemimethylated DNA in vitro, with no detectable enzymatic activity on unmethyated substrates.
In vivo, once 5mC is lost, it is not globally re-established, either in vegetatively-growing cells or during sexual
reproduction. These data indicate that Dnmt5 is a maintenance-type enzyme. Surprisingly, Dnmt5 is the only
cytosine DNA methyltransferase encoded by the C. neoformans genome. This finding raises the intriguing
question of how 5mC can be established in an organism lacking a de novo enzyme. Through phylogenetic
analysis of whole genome sequences, we determined that predicted Dnmt enzyme, DnmtX, was present in an
ancestral species, but that its gene was lost at least 50 million years ago. To test whether DnmtX was a de
novo enzyme, we introduced versions of DnmtX from existing species into a C. neoformans strain lacking 5mC
but harboring Dnmt5. We find that 5mC is deposited by these enzymes de novo and is then maintained. Thus,
DnmtX has de novo activity. Our results thus suggest a radical possibility, namely that 5mC has been
maintained epigenetically by Dnmt5 since the DnmtX de novo methylase was lost. Such a model requires a
combination of a high fidelity of epigenetic inheritance of 5mC together with some level of natural selection. We
will test predictions of this model and use the power of a yeast system to investigate epigenetic inheritance
fidelity and function. Specifically, we will measure and investigate 5mC inheritance fidelity, elucidate the
evolution of 5mC patterns, investigate roles of 5mC in centromere function and genome defense, and identify
determinants of 5mC maintenance and action. This work will probe the limits of epigenetic memory and
illuminate how cytosine methylation of DNA is accurately maintained and how it functions.

## Key facts

- **NIH application ID:** 10074146
- **Project number:** 5R01GM071801-15
- **Recipient organization:** UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
- **Principal Investigator:** Hiten D Madhani
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $386,854
- **Award type:** 5
- **Project period:** 2005-04-01 → 2022-12-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10074146

## Citation

> US National Institutes of Health, RePORTER application 10074146, Mechanisms of Gene Silencing (5R01GM071801-15). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10074146. Licensed CC0.

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