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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

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 | GSE225271 | GEO

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

2023-05-09 | GSE225270 | GEO

Mouse sperm protamine mass spectrometry

Project description:Conventional dogma presumes that protamine-mediated DNA compaction in sperm is achieved by passive electrostatics between DNA and the arginine-rich core of protamines. However, phylogenetic analysis reveals several non-arginine residues that are conserved within, but not across, species. The functional significance of these residues or post-translational modifications are poorly understood. In this experiment, we investigated the presence of post-translational modifications on mouse protamine 1 to begin to dissect their function in vivo.

2023-05-17 | PXD028917 | Pride

Characterization of human sperm protamine proteoforms through a combination of top-down and bottom-up mass spectrometry approaches

Project description:Protamine 1 (P1) and protamine 2 family (P2) are the most abundant nuclear basic spermspecific proteins, packing 85-95% of the paternal DNA. P1 is synthesized as a mature form, whereas P2 components (HP2, HP3 and HP4) arise from the proteolysis of the precursor (pre-P2). Due to the particular protamine physical-chemical properties, their identification by standardized bottom-up mass spectrometry (MS) strategies are not straightforward. Therefore, the aim of this study was to identify the sperm protamine proteoforms profile including P1 and P2 members and their post-translational modifications in normozoospermic individuals using two complementary strategies, a top-down MS approach and a proteinase K-digestion based bottom-up MS approach. By top-down MS approach, both described and new truncated P1 and pre-P2 proteoforms were identified. Likewise, intact P1, pre-P2, and P2 mature forms and their phosphorylation pattern were detected, as well as a +61 Da modification in different proteoforms. Additionally, the bottom-up MS approach revealed phosphorylated residues for pre-P2 and the new P2 isoform 2, which is not annotated at UniProtKB database. Implementing these strategies in comparative studies of different infertile phenotypes would permit to determine alterations in the protamine proteoforms pattern and elucidate the role of phosphorylation/dephosphorylation dynamics in male fertility.

2019-11-11 | PXD014618 | Pride

Loss of Prm1 leads to defective chromatin protamination, impaired PRM2 processing, reduced sperm motility and subfertility in male mice [PRM1]

Project description:One of the key events during spermiogenesis is the hypercondensation of chromatin by substitution of the majority of histones by protamines. In humans and mice, protamine 1 (PRM1/Prm1) and protamine 2 (PRM2/Prm2), are expressed in a species-specific ratio. Using CRISPR-Cas9-mediated gene editing we generated Prm1-deficient mice and demonstrate, that Prm1+/- mice are subfertile while Prm1-/- are infertile. Prm1-/- and Prm2-/- sperm show high levels of reactive oxygen species (ROS)-mediated DNA damages and increased histone retention. In contrast, Prm1+/- sperm display only moderate DNA damages. The majority of Prm1+/- sperm were CMA3 positive indicating protamine-free DNA. This is not due to increased histone retention in Prm1+/- sperm. Additionally, we found that sperm from Prm1+/- and Prm1-/- mice contain high levels of incompletely processed PRM2. Further, the PRM1:PRM2 ratio is skewed from 1:2 in WT to 1:5 in Prm1+/- animals. Our results reveal that PRM1 is required for proper PRM2 processing to produce the mature PRM2 which, together with PRM1 is able to hypercondense DNA. Hence, the species specific PRM1:PRM2 ratio has to be precisely controlled in order to retain full fertility.

2022-04-06 | GSE200050 | GEO

CTCF contributes in a critical way to spermatogenesis and male fertility

Project description:The CCCTC-binding factor (CTCF) is an architectural protein that governs chromatin organization and gene expression in somatic cells. Here, we show that CTCF regulates chromatin compaction necessary for packaging of the paternal genome into mature sperm. Inactivation of Ctcf in male germ cells in mice (Ctcf-cKO mice) resulted in impaired spermiogenesis and infertility. Residual spermatozoa in Ctcf-cKO mice displayed abnormal head morphology, aberrant chromatin compaction, impaired protamine 1 incorporation into chromatin and accelerated histone depletion. Thus, CTCF regulates chromatin organization during spermiogenesis, contributing to the functional organization of mature sperm.

2016-06-03 | GSE76439 | GEO

Protamine characterization by top-down proteomics: Boosting proteoform identification with DBSCAN

Project description:Protamines replace histones as the main nuclear protein in sperm cells having a crucial role in compacting the paternal genome. Human spermatozoa contain the protamine 1 (P1) and the family of protamine 2 (P2) proteins. Alterations in protamine PTMs or on the P1/P2 ratio could be associated with male infertility. Top-down proteomics enables large-scale analysis of intact proteforms derived from alternative splicing, missense or nonsense genetic variants or PTMs. In contrast to current gold standard techniques, top-down allows a more in-depth analysis of protamine PTMs and proteoforms, opening up new perspectives to unravel their impact on male fertility. We analyzed two normozoospermic semen samples by top-down and discussed in detail the difficulties we found in the data analysis and the suggested solutions, as this step is one of the current bottlenecks in top-down proteomics with currently available bioinformatic tools. Our strategy for the data analysis combines two different software, ProSight PD (PS) and TopPIC suite (TP), with a clustering algorithm to decipher protamine proteoforms. We identified up to 32 protamine proteoforms at different levels of characterization. This in-depth analysis of the protamine proteoform landscape of an individual boosts personalized diagnosis of male infertility.

2021-05-13 | PXD024405 | Pride

DOT1L regulates chromatin reorganization and gene expression during sperm differentiation

Project description:Spermatozoa have a unique genome organization: their chromatin is almost completely devoid of histones and is formed instead of protamines which confer a high level of compaction and preserve paternal genome integrity until fertilization. Histone-to-protamine transition takes place in spermatids and is indispensable for the production of functional sperm. Here we show that the H3K79-methyltransferase DOT1L controls spermatid chromatin remodelling and subsequent reorganization and compaction of spermatozoon genome. Using a mouse model in which Dot1l is knocked-out (KO) in postnatal male germ cells, we found that Dot1l-KO sperm chromatin is less compact and has an abnormal content, characterized by the presence of transition proteins, immature protamine 2 forms and a higher level of histones. Proteomics and transcriptomics analyses performed on spermatids reveal that Dot1l-KO modifies the chromatin prior to histone removal, and leads to the deregulation of genes involved in flagellum formation and apoptosis during spermatid differentiation. As a consequence of these chromatin and gene expression defects, Dot1l-KO spermatozoa have less compact heads and are less motile, which results in impaired fertility.

2023-03-30 | PXD030734 | Pride

PHF7 Links H3K4 Methylation to H2A Ubiquitination During Spermiongenesis [RNA-seq]

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.

2019-07-05 | GSE119701 | GEO

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

Project description:Majority of histones are replaced by protamine during spermatogenesis, but small amount of histones are retained in mammalian spermatozoa. Since nucleosomes in spermatozoa influence epigenetic inheritance, it is important how histones are distributed on sperm genome. We have found that the swim-up sperm used in many studies contains about 10% of immature sperm which do not yet complete the histone-to-protamine replacement. We have developed the novel method to purify the histone replacement-completed sperm (HRCS), and to completely solubilize nucleosomes from cross-linked HRCS without MNase digestion. Here, we analyzed H3 binding profiles in total sperm and HRCS by Chromatin IP – sequencing (ChIP-seq) analysis, and DNA methylation profiles in swim-up sperm and HRCS by target bisulfite sequencing (TGSB).

2018-07-26 | GSE113137 | GEO

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