# Understanding DNA methylation reprogramming dynamics during preimplantation development using single-cell sequencing

> **NIH NIH R01** · UNIVERSITY OF CALIFORNIA SANTA BARBARA · 2021 · $278,597

## Abstract

PROJECT SUMMARY
During mammalian preimplantation development, one of the most dramatic epigenetic events is the global
erasure of DNA methylation (5-methylcytosine or 5mC) from the parental genomes. This reprogramming is
critical to reset the methylation status of the gametes and restore developmental potency to pluripotent cells in
the blastocyst. Elucidating the molecular mechanisms that regulate 5mC reprogramming is central to gaining
deeper insights into normal embryonic development as well as to better understand aberrant 5mC patterns and
imprinting disorders associated with assisted reproductive technologies (ART). While early studies showed that
the paternal genome undergoes ‘active’ demethylation through conversion of 5mC to 5-hydroxymethylcytosine
(5hmC) and the maternal genome undergoes ‘passive’ demethylation through a lack of maintenance DNA
methyltransferase (Dnmt1) activity during replication, more recent studies have suggested that 5mC erasure
from the parental genomes is a combination of these two demethylation pathways. Unraveling the mechanisms
that regulate 5mC erasure has been challenging due to our inability to directly distinguish between these
pathways. This limitation can be overcome by making genome-wide strand-specific measurements of 5mC in
single cells. In addition, this epigenetic reprogramming coincides temporally with the first cell fate specification
in the embryo towards the trophectoderm (TE) and inner cell mass (ICM) lineages. However, it remains unclear
how 5mC reprogramming influences this cell fate decision. To address these questions, in Specific Aims 1 we
propose to develop a novel single-cell sequencing technology to simultaneously quantify 5mC strand-specifically
together with mRNA from the same cell. Preliminary experiments in mouse embryonic stem cells and mouse
embryos show that we can detect both 5mC and mRNA from the same cell. To test our central hypothesis that
the mechanisms regulating the erasure of 5mC are parent- and stage-specific, in Specific Aims 2 we plan to
quantify the genome-wide strand-specific distribution of 5mC in hybrid mouse embryos from the 2- to 64-cell
stage of embryogenesis. Further, through stochastic mathematical modeling and knockdown experiments, we
will elucidate how different molecular factors regulate active and passive demethylation pathways during
preimplantation development. Finally, while cell-to-cell variability in a variety of factors have been shown to bias
the cell fate potential of early blastomeres towards the TE or ICM lineages, it remains unclear how 5mC
reprogramming tunes or reinforces these decisions. To address this question, in Specific Aims 3 we plan to
quantify both 5mC and mRNA from the same cell to directly correlate how heterogeneity in genome-wide strand-
specific patterns of 5mC influence the decision between symmetric vs. asymmetric cell divisions and the resulting
cell fate choices. Thus, through the development of novel single-cell methods we w...

## Key facts

- **NIH application ID:** 10208919
- **Project number:** 5R01HD099517-03
- **Recipient organization:** UNIVERSITY OF CALIFORNIA SANTA BARBARA
- **Principal Investigator:** Siddharth Subhas Dey
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $278,597
- **Award type:** 5
- **Project period:** 2019-09-15 → 2024-06-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10208919

## Citation

> US National Institutes of Health, RePORTER application 10208919, Understanding DNA methylation reprogramming dynamics during preimplantation development using single-cell sequencing (5R01HD099517-03). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/10208919. Licensed CC0.

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