Single-Cell RNA-seq Uncovers Dynamic Processes Orchestrated by RNA-Binding Protein DDX43 in Chromatin Remodeling during Spermiogenesis

Abstract

Advances in single-cell RNA sequencing (scRNA-seq) have allowed for elucidating biological mechanisms at cell state level. Mammalian spermatogenic process showcases dynamic switches of gene expression pattern with delicate morphological and functional alterations of germ cells, but it is unclear how such dynamics is genetically controlled. Here we demonstrate that mouse testis-enriched RNA helicase DDX43, as well as its ATP hydrolysis site, is required for spermiogenesis. Genetic mutation of Ddx43 renders spermatids heterogeneously defective in multiple steps of chromatin remodeling, resulting in incomplete substitution of transition protein by protamine and less condensed sperm nucleus. Through scRNA-seq analyses of testicular cells derived from adult wild-type and Ddx43 mutant testes in mice, we reveal that the DDX43 deficiency-elicited perturbation in the dynamic RNA regulatory processes underlies the differentiation deficiency of spermatids. Further, focused analyses on early-stage spermatids combined with enhanced CLIP sequencing (eCLIP-seq) identify Elfn2 as DDX43-targeted hub gene, whose in vivo knockdownshows similar phenotypic defects as Ddx43 mutant. Our study illustrates an essential role for DDX43 in post-meiotic chromatin remodeling and highlights the single cell-based strategy for a refined dissection of stage-specific regulation of germline differentiation.

Integrated single cell transcriptome sequencing analysis reveals species‐specific genes and molecular pathways for pig spermiogenesis

“…Molecular events, such as chromatin remodelling and transcriptional reprogramming, are essential to spermatid maturation and function. The RNA helicase DDX43 regulates spermatid chromatin remodelling and differentiation (Tan et al., 2023), and the phosphorylation of DDX25 is crucial to the differentiation of round spermatid to elongated spermatid and acrosome biogenesis (Kavarthapu et al., 2023). CTCF also regulates the transcriptional programme during spermiogenesis, and its depletion produces abnormal sperm and infertility (Rivero‐Hinojosa et al., 2021; Torres‐Flores et al., 2023).…”

“…In primates, transcriptional similarity and difference were discovered, and the DNA damage response pathway was found to be important to genome integrity and meiotic sex chromosome inactivation (MSCI) (Lau et al., 2020; Ramaswamy et al., 2017). In mice, the testicular cell atlas to study germ cell development and in vivo gametogenesis was also built (Green et al., 2018; Grive et al., 2019), and functional analyses on key genes in spermatogenesis were also performed (Hong et al., 2022; Song et al., 2022; Tan et al., 2023). Across‐species evolutionary comparisons also revealed conserved and divergent molecular cues fundamental for spermatogenesis (Hermann et al., 2018; Murat et al., 2023; Shami et al., 2020).…”

Effect of DNMT3A R882H Hot Spot Mutations on DDX43 Promoter Methylation in Acute Myeloid Leukemia

“…DDX43 was first identified together with sarcoma antigen (SAGE) as a tumor-specific CTA gene in a human sarcoma cell line [21]. DDX43 has been identified as being overexpressed in a range of solid tumors including those affecting the salivary gland, colon, brain, lung, and prostate, as well as in hematologic malignancies like chronic myeloid leukemia and multiple myeloma [22][23][24][25][26][27][28][29]. Recently, abnormal hypomethylation of DDX43 promoter has frequently been reported in hematologic malignancies, including CML, MDS, and AML [30].…”

Single‐cell transcriptome characteristics of testicular terminal epithelium lineages during aging in the Drosophila

“…Single‐cell transcriptome sequencing (scRNA‐seq) technology is used for the high‐throughput detection of transcription signatures at single‐cell resolution, and is widely used in studies of the reproductive system (Shi et al., 2021; Tan et al., 2023; Zhao et al., 2020). Recently, several scRNA‐seq studies were performed to generate a single‐cell atlas or cellular landscapes in different study models in Drosophila testis (Witt et al., 2019; Yu, Fu, et al., 2023; Yu, Li, et al., 2023).…”