# A NOVEL PROCESS SAFEGUARDS GENOME INTEGRITY IN THE MAMMALIAN GERM LINE

> **NIH NIH R01** · VAN ANDEL RESEARCH INSTITUTE · 2022 · $519,036

## Abstract

PROJECT SUMMARY
Simple purine–pyrimidine (Pu/Py) repeats (PPRs) are known to form the left-handed, fragile Z-DNA structure.
Such repeats are also known to be highly mutagenic, inducing large chromosomal deletions and rearrangements
in the cells of higher organisms. The mutagenic effects of Z-DNA would be the most detrimental to a species if
breaks occurred uncontrollably in the germ line and mutations occurring at unrepaired breaks were passed on
to the next generation. We discovered a novel biological process in the mammalian germ line that controls Z-
DNA structure at PPRs. Using the Zbtb43 mutant mouse genetic model and our transgenic mouse line that
allows us to isolate germ cells, we found that a previously uncharacterized DNA binding protein, ZBTB43
remodels Z-DNA structure and protects from double-strand breaks in fetal male germ cells in vivo. By
biochemical assays we found that ZBTB43 binds to PPR-rich DNA sites in the genome in vitro. ZBTB43 binding
sites form Z-DNA and cause large genomic rearrangements in mammalian cells. By in vivo epigenome mapping
we detected Z-DNA in mutant germ cells at the locations where ZBTB43 binding occurs in wild-type
prospermatogonia. We hypothesize, therefore, that ZBTB43 safeguards genome integrity in the germ line by
binding and eliminating Z-DNA at PPRs. In addition, we found that by eliminating Z-DNA, ZBTB43 promotes de
novo methylation at PPRs during the time of global epigenetic remodeling. We propose to pursue the following
Aims, using a combination of genetic, cell biology, biochemistry, and epigenomic approaches. In Aim 1, we will
test the working hypothesis that ZBTB43 eliminates Z-DNA structure in vivo by directly binding to PPRs in fetal
male germ cells. We will determine 1) the spatial and temporal changes of the Z-DNA structure in fetal male
germ cells in the presence or absence of ZBTB43 protein in vivo; 2) the dependence of the Z-DNA remodeling
process on the direct binding of ZBTB43 to the Z-DNA structure in vivo; and 3) the molecular requirements of
ZBTB43 action on Z-DNA. In Aim 2, we will test the working hypothesis that ZBTB43 facilitates de novo DNA
methylation in prospermatogonia indirectly by eliminating Z-DNA, thus revealing the sequences as substrates
for de novo methyltransferases. We will map DNA methylation in the presence and absence of ZBTB43 during
the epigenome remodeling process in fetal male germ cells. We will test whether de novo DNMTs methylate Z-
DNA substrates in vitro. We will test whether ZBTB43 affects nucleosome occupancy at PPRs. In Aim 3, we will
test the hypothesis that Z-DNA is mutagenic in the germ line and that ZBTB43 has evolved to manage that
burden. We will map double-strand breaks in mutant fetal germ cells, test the anti-mutagenic effect of ZBTB43
in cell culture, and search for genomic rearrangements in sperm of Zbtb43 mutant males. By the end of the grant
period, we will have identified and characterized the first example of how a DNA binding protei...

## Key facts

- **NIH application ID:** 10445720
- **Project number:** 1R01GM143308-01A1
- **Recipient organization:** VAN ANDEL RESEARCH INSTITUTE
- **Principal Investigator:** Piroska Edit Szabó
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $519,036
- **Award type:** 1
- **Project period:** 2022-06-20 → 2026-05-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10445720

## Citation

> US National Institutes of Health, RePORTER application 10445720, A NOVEL PROCESS SAFEGUARDS GENOME INTEGRITY IN THE MAMMALIAN GERM LINE (1R01GM143308-01A1). Retrieved via AI Analytics 2026-05-22 from https://api.ai-analytics.org/grant/nih/10445720. Licensed CC0.

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