R-loop landscape in mature human sperm: Regulatory and evolutionary implications

Scheuren, Maurice; Möhner, Jonas; Zischler, Hans

doi:10.3389/fgene.2023.1069871

Cited by 3 publications

(1 citation statement)

References 78 publications

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“…The work employed data from the Gene Expression Omnibus, which was converted to GRCh38 using UCSC Liftover if necessary. The dataset PRJNA480448 was obtained from the Sequence Read Archive and reanalyzed according to ENCODE standards for the corresponding sequencing strategy ( Supplementary Table S4 ) ( Hammoud et al, 2014 ; Lesch et al, 2016 ; Jung et al, 2019 ; Bae and Lesch, 2020 ; Oikawa et al, 2020 ; Scheuren et al, 2023 ).…”

Section: Methodsmentioning

confidence: 99%

DSB profiles in human spermatozoa highlight the role of TMEJ in the male germline

Scheuren,

Möhner,

Müller

et al. 2024

Front. Genet.

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The male mammalian germline is characterized by substantial chromatin remodeling associated with the transition from histones to protamines during spermatogenesis, followed by the reversal to nucleohistones in the male pronucleus preceding the zygotic genome activation. Both transitions are associated with the extensive formation of DNA double-strand breaks (DSBs), requiring an estimated 5 to 10 million transient DSBs per spermatozoa. Additionally, the high transcription rate in early stages of spermatogenesis leads to transcription-coupled damage preceding meiotic homologous recombination, potentially further contributing to the DSB landscape in mature spermatozoa. Once meiosis is completed, spermatozoa remain haploid and therefore cannot rely on error-free homologous recombination, but instead depend on error-prone classical non-homologous end joining (cNHEJ). This DNA damage/repair-scenario is proposed to be one of the main causes of the observed paternal mutation propensity in human evolution. Recent studies have shown that DSBs in the male pronucleus are repaired by maternally provided Polθ in Caenorhabditis elegans through Polθ-mediated end joining (TMEJ). Additionally, population genetic datasets have revealed a preponderance of TMEJ signatures associated with human variation. Since these signatures are the result of the combined effect of TMEJ and DSB formation in spermatozoa and male pronuclei, we used a BLISS-based protocol to analyze recurrent DSBs in mature human sperm heads as a proxy of the male pronucleus before zygotic chromatin remodeling. The DSBs were found to be enriched in (YR)n short tandem repeats and in evolutionarily young SINEs, reminiscent to patterns observed in murine spermatids, indicating evolutionary hotspots of recurrent DSB formation in mammalian spermatozoa. Additionally, we detected a similar DSB pattern in diploid human IMR90 cells when cNHEJ was selectively inhibited, indicating the significant impact of absent cNHEJ on the sperm DSB landscape. Strikingly, regions associated with most retained histones, and therefore less condensed chromatin, were not strongly enriched with recurrent DSBs. In contrast, the fraction of retained H3K27me3 in the mature spermatozoa displayed a strong association with recurrent DSBs. DSBs in H3K27me3 are associated with a preference for TMEJ over cNHEJ during repair. We hypothesize that the retained H3K27me3 may trigger transgenerational DNA repair by priming maternal Polθ to these regions.

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Section: Methodsmentioning

confidence: 99%

DSB profiles in human spermatozoa highlight the role of TMEJ in the male germline

Scheuren,

Möhner,

Müller

et al. 2024

Front. Genet.

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Helicases in R-loop Formation and Resolution

Yang,

Winstone,

Mondal

et al. 2023

Journal of Biological Chemistry

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Autism-Related Cc2d1a Heterozygous Mice: Increased Levels of miRNAs Retained in DNA/RNA Hybrid Profiles (R-Loop)

Sener,

Dana,

Tahtasakal

et al. 2024

Biomolecules

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Autism spectrum disorder (ASD) is a complex neurodevelopmental disorder with a highly variable expression of phenotypes (restricted interest or activity and repetitive behavior in communication and social interactions), genes (mutation), markers (alteration of transcription) and pathways. Loss of function of the CC2D1A gene appears to primarily affect the brain, leading to a range of behavioral problems in humans. In our study published in 2020, we found that the expressions of miR-19a-3p, miR-361-5p, miR-150-5p, miR-3613-3p, miR-126-3p and miR-499a-5p were downregulated in the serum samples of autistic patients, their families and mouse models (Cc2d1a +/− and valproic acid treated males). Here, acquired non-Mendelian hereditary character in a genetically defined mouse model of autism (Cc2d1a +/−) correlates with the transcriptional alteration of five miRNAs. We seek to test the hypothesis that miRNA levels vary by changes in RNA/DNA structure during development, thereby creating transcription alteration and cell memory. Behavioral tests were conducted on the offspring of Cc2d1a (+/−) mutant and control mice, such as novel object, social interaction, marble burying and tail suspension behavior. Two RNA fractions were isolated from mouse hippocampal tissues and sperm cells via standard TRIzol extraction: free RNA and the fraction of RNA bound to DNA in the form of a DNA/RNA hybrid (R-loop). The expression levels of miR-19a-3p, miR-361-5p, miR-150-5p, miR-126-3p and miR-499a-5p were investigated by quantitative real-time RT-PCR. We report differences in the distribution of five miRNAs in the hippocampus between male and female mice, particularly in colonies of Cc2d1a (+/−) mice. Furthermore, the number of miRNAs engaged in the DNA/RNA hybrid fraction is generally higher in the mutant pedigree than in the control group. On the other hand, in sperm, both fractions are at lower levels than in controls. R-loops contribute to the physiology and pathology of organisms including human disease. Here, we report a variation in five miRNA levels between gender and tissue. Our results suggest that the transcription levels of these five miRNAs are directly regulated by their RNA.

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R-loop landscape in mature human sperm: Regulatory and evolutionary implications

Cited by 3 publications

References 78 publications

DSB profiles in human spermatozoa highlight the role of TMEJ in the male germline

DSB profiles in human spermatozoa highlight the role of TMEJ in the male germline

Helicases in R-loop Formation and Resolution

Autism-Related Cc2d1a Heterozygous Mice: Increased Levels of miRNAs Retained in DNA/RNA Hybrid Profiles (R-Loop)

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