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Sperm chromatin structure and reproductive fitness are altered by substitution of a single amino acid in mouse Protamine 1 [MNase-Seq]

ABSTRACT: Conventional dogma presumes that protamine-mediated DNA compaction in sperm is achieved by electrostatic interactions between DNA and the arginine-rich core of protamines. However, phylogenetic analysis reveals several non-arginine residues conserved within, but not across species. The significance of these residues or post-translational modifications are poorly understood. Here, we investigated the functional role of K49, a rodent-specific lysine residue in mouse protamine 1 (P1) that is acetylated early in spermiogenesis and retained in sperm. In vivo, alanine substitution (P1 K49A) results in loss of programmatic histone retention, decreased sperm motility, decreased male fertility, and in zygotes, premature P1 removal from paternal chromatin, altered DNA replication, and embryonic arrest. In vitro, P1 K49A decreases protamine-DNA binding and alters DNA compaction/decompaction kinetics. Hence, a single amino acid substitution outside the P1 arginine core is sufficient to profoundly alter protein function and developmental outcomes, suggesting that protamine non-arginine residues are essential for reproductive fitness.

ORGANISM(S): Mus musculus

PROVIDER: GSE225268 | GEO | 2023/05/09

REPOSITORIES: GEO

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Sperm chromatin structure and reproductive fitness are altered by substitution of a single amino acid in mouse Protamine 1 [scRNA-Seq]

Project description:Conventional dogma presumes that protamine-mediated DNA compaction in sperm is achieved by electrostatic interactions between DNA and the arginine-rich core of protamines. However, phylogenetic analysis reveals several non-arginine residues conserved within, but not across species. The significance of these residues or post-translational modifications are poorly understood. Here, we investigated the functional role of K49, a rodent-specific lysine residue in mouse protamine 1 (P1) that is acetylated early in spermiogenesis and retained in sperm. In vivo, alanine substitution (P1 K49A) results in loss of programmatic histone retention, decreased sperm motility, decreased male fertility, and in zygotes, premature P1 removal from paternal chromatin, altered DNA replication, and embryonic arrest. In vitro, P1 K49A decreases protamine-DNA binding and alters DNA compaction/decompaction kinetics. Hence, a single amino acid substitution outside the P1 arginine core is sufficient to profoundly alter protein function and developmental outcomes, suggesting that protamine non-arginine residues are essential for reproductive fitness.

2023-05-09 | GSE225270 | GEO

Sperm chromatin structure and reproductive fitness are altered by substitution of a single amino acid in mouse Protamine 1

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Project description:Histone ubiquitination has been suggested to serve as a “tag” for nucleosome removal during histone-to-protamine exchange that is essential for chromatin packaging in round spermatids. Here, we screen for putative E3 ligase and identify that the PHF7, containing both RING finger and PHD domains, is critical for H2A ubiquitination and histone removal. Mechanistically, its PHD domain as a histone code reader can specifically bind H3K4me3/me2 and its RING domain as a histone writer can ubiquinate H2A. PHF7 deficiency results in male infertility in mice by generating Phf7 knockout mice using CRISPR-Cas9 technology. Futhermore, we find impaired histone-to-protamine exchange leads to reduced chromatin compaction in Phf7 knockout sperm. Immunostaining and western blot further confirme abnormal retention of core histones and reduced levels of protamine PRM1 and PRM2 in Phf7 knockout sperm. In order to exclude the possibility that Phf7 regulate the expession of protamine, we sorte the round spermatids from WT and Phf7 knockout females for RNA-seq. The transcriptomes in round spermatids are very similar between WT and Phf7 knockout mice. There are few differentially expressed genes in the two groups and the expression of protamine is also unchanged even if PHF7 deficiency. Therefore we find PHF7 has no effects on gene regulation in histone-to-protamine exchange.

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DNA methylation analysis for target regions in swim-up sperm and HRCS.

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