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bioRxiv · 10.1101/2025.06.19.660366

The eutherian-specific histone H3.4 promotes germ cell development and reproductive fitness

Komarov, P. A.·Bammer, P.·Liang, C.-Y.·Hotz, H.-R.·Fanourgakis, G.·Chun, S.·Kohler, H.·Buchholz, T.-O.·Spetz, J.-F.·Peters, A. H. F. M.
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Abstract

Many genes encoding chromatin proteins are subject to evolutionary selection driving reproductive fitness. In mice and men, the histone H3.4 variant is essential to spermatogenesis. Here we define the evolutionary origin and molecular-physiological roles of sequence variation in H3f4 for male germ cell development in mice. Our phylogenetic analyses indicate that eutherian H3f4 orthologs originate from an ancestral H3.2 gene existing prior to the divergence of eutherian and marsupial mammals over 100 million years ago. Positioned in truncated histone gene clusters, eutherian H3f4 orthologs show increased non-synonymous and synonymous substitution rates compared to orthologous marsupial H3.2 loci located in typically large histone clusters. To determine the impact of sequence divergence on reproductive fitness, we reverted non-synonymously substituted residues in H3f4 to those present in canonical H3.1 (H3f4V24A, H3f4H42R, H3f4S98A). Hemizygous expression of such triply reverted H3f4H3.1 allele on a H3f4-deficiency background caused an >40% reduction in testis weight associated with impaired meiotic DNA double strand break repair, death of pachytene spermatocytes, impaired differentiation of spermatids and aberrant expression of thousands of genes during spermatid elongation. Hemizygous expression of individual residue substitution alleles revealed residues V24 and H42 of H3.4 to promote spermatogenesis, while residue S98 is neutral. Together, our study shows that H3f4 has been subject to positive evolutionary selection, promoting male reproductive fitness.

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Komarov, P. A., Bammer, P., Liang, C.-Y., Hotz, H.-R., Fanourgakis, G., Chun, S., Kohler, H., Buchholz, T.-O., Spetz, J.-F., Peters, A. H. F. M.. 2025-06-24. The eutherian-specific histone H3.4 promotes germ cell development and reproductive fitness. https://doi.org/10.1101/2025.06.19.660366

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