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A NOVEL PROCESS SAFEGUARDS GENOME INTEGRITY IN THE MAMMALIAN GERM LINE

Award Number: R01GM143308
ORGANIZATION: NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES
OPDIV: NIH
AWARD CLASS: DISCRETIONARY
AWARD ACTIVITY TYPE: SCIENTIFIC/HEALTH RESEARCH (INCLUDES SURVEYS)
PERIOD OF PERFORMANCE START DATE: 06/20/2022
PERIOD OF PERFORMANCE END DATE: 05/31/2027

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A NOVEL PROCESS SAFEGUARDS GENOME INTEGRITY IN THE MAMMALIAN GERM LINE - 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 protein protects genome integrity and targets de novo DNA methylation by controlling Z-DNA structure in the mammalian germ line.


Issue Date FY	Funding FY	Legal Entity Name	Legal Entity Address	Legal Entity City	Legal Entity State	Legal Entity Zip Code	Legal Entity COUNTY	Legal Entity COUNTRY	Assistance Listing	Award Code	Budget Year	Action Date	Action Type	Action Amount

Issue Date FY: 2026 ( Subtotal = $0 )
2026	2025	VAN ANDEL RESEARCH INSTITUTE	333 BOSTWICK AVE NE	GRAND RAPIDS	MI	49503	KENT	USA	Biomedical Research and Research Training	000	4	3/25/2026	NON-COMPETING CONTINUATION	$0
														Subtotal = $0

Issue Date FY: 2025 ( Subtotal = $519,036 )
2025	2025	VAN ANDEL RESEARCH INSTITUTE	333 BOSTWICK AVE NE	GRAND RAPIDS	MI	49503	KENT	USA	Biomedical Research and Research Training	000	4	5/29/2025	NON-COMPETING CONTINUATION	$519,036
														Subtotal = $519,036

Issue Date FY: 2024 ( Subtotal = $519,036 )
2024	2024	VAN ANDEL RESEARCH INSTITUTE	333 BOSTWICK AVE NE	GRAND RAPIDS	MI	49503	KENT	USA	Biomedical Research and Research Training	000	3	5/8/2024	NON-COMPETING CONTINUATION	$519,036
														Subtotal = $519,036

Issue Date FY: 2023 ( Subtotal = $531,386 )
2023	2023	VAN ANDEL RESEARCH INSTITUTE	333 BOSTWICK AVE NE	GRAND RAPIDS	MI	49503	KENT	USA	Biomedical Research and Research Training	001	1	5/15/2023	SUPPLEMENT FOR EXPANSION	$12,350
2023	2023	VAN ANDEL RESEARCH INSTITUTE	333 BOSTWICK AVE NE	GRAND RAPIDS	MI	49503	KENT	USA	Biomedical Research and Research Training	002	2	6/1/2023	NON-COMPETING CONTINUATION	$519,036
2023	2022	VAN ANDEL RESEARCH INSTITUTE	333 BOSTWICK AVE NE	GRAND RAPIDS	MI	49503	KENT	USA	Biomedical Research and Research Training	000	1	10/24/2022	NEW	$0
														Subtotal = $531,386

Issue Date FY: 2022 ( Subtotal = $519,036 )
2022	2022	VAN ANDEL RESEARCH INSTITUTE	333 BOSTWICK AVE NE	GRAND RAPIDS	MI	49503	KENT	USA	Biomedical Research and Research Training	000	1	6/20/2022	NEW	$519,036
														Subtotal = $519,036

Grand Total All Awards = $2,088,494

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A NOVEL PROCESS SAFEGUARDS GENOME INTEGRITY IN THE MAMMALIAN GERM LINE

Award Number: R01GM143308

ORGANIZATION: NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES

OPDIV: NIH

AWARD CLASS: DISCRETIONARY

AWARD ACTIVITY TYPE: SCIENTIFIC/HEALTH RESEARCH (INCLUDES SURVEYS)

PERIOD OF PERFORMANCE START DATE: 06/20/2022

PERIOD OF PERFORMANCE END DATE: 05/31/2027

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