In a groundbreaking study published in Science Bulletin, researchers have unveiled the critical role of the helicase DHX36 in maintaining chromatin architecture and ensuring proper ribosomal RNA (rRNA) processing during mouse oocyte development. This discovery sheds new light on the molecular underpinnings of female fertility and early embryogenesis, highlighting DHX36’s essential function in resolving G-quadruplex (G4) structures that influence chromatin accessibility and gene expression. The implications of this research resonate deeply within the fields of molecular biology and reproductive science, opening avenues for better understanding infertility linked to chromatin dysregulation.
G-quadruplex structures, formed by guanine-rich DNA or RNA sequences through Hoogsteen hydrogen bonding, represent unique secondary configurations that can profoundly affect nucleic acid metabolism. DHX36, also known as RHAU, is a specialized helicase known for its capacity to unwind G4 structures, thus modulating transcriptional and post-transcriptional processes. While DHX36’s involvement in processes such as spermatogenesis and cardiac development is well documented, its specific role in oocyte maturation and early embryonic development has remained enigmatic—until now.
The research team embarked on a comprehensive investigation into the expression and localization patterns of DHX36 within mouse oocytes. They observed robust DHX36 expression in ovarian tissue, predominantly concentrated in the nucleus. Intriguingly, as oocytes progressed through developmental stages, the spatial distribution of DHX36 shifted from the nucleoplasm to the nucleolus, concomitant with nucleolar conformational changes. This dynamic localization hinted at a potential regulatory role linked intimately with nucleolar function and chromatin organization.
To explore DHX36’s functional impact, investigators engineered an oocyte-specific conditional knockout (CKO) mouse model employing the Cre-LoxP recombination system. The phenotypic consequences in DHX36-deficient females were striking: complete infertility, characterized by profound hormonal irregularities and impaired ovulatory responses. This phenotype underscored DHX36’s indispensable role in reproductive biology and prompted deeper mechanistic inquiries.
Detailed phenotypic analyses revealed multifaceted meiotic and post-fertilization defects in DHX36-CKO oocytes. Primarily, the majority of oocytes failed to progress past meiotic arrest, unable to execute germinal vesicle breakdown (GVBD) or extrude the first polar body. Among the minority that proceeded, chromosomal misalignments and disorganized spindle assemblies were prevalent, often culminating in erroneous ploidy maintenance. Moreover, oocytes from natural matings exhibited dramatically reduced fertilization efficiency, with fertilized embryos arresting predominantly at the zygote stage, rarely advancing beyond the two-cell stage.
To connect these cellular impediments with underlying molecular dysfunction, the team assessed chromatin organization and accessibility. They reported substantial enlargement of nucleolus-like bodies (NLBs) in DHX36-deficient oocytes, accompanied by diminished heterochromatin condensation and global reduction in key histone methylation marks—H3K4me3, H3K9me3, and H3K27me3. Concomitantly, the heterochromatin-associated protein HP1α adopted a diffuse nuclear distribution, indicative of disrupted heterochromatin maintenance. ATAC-seq data further demonstrated decreased chromatin accessibility at transcription start sites and ribosomal DNA (rDNA) regions, both abundant in G4 motifs, implicating DHX36-mediated G4 resolution in chromatin openness.
Exploring transcriptomic landscapes via Smart-seq2 RNA sequencing of oocytes across developmental timepoints, the researchers identified signatures of transcriptional insufficiency and defective maternal transcript clearance in DHX36-CKO samples. Gene ontology enrichment emphasized dysregulation in DNA transcription factor activity, ribonucleoprotein assembly, and rRNA biogenesis pathways. Notably, precursors of rRNA exhibited aberrant accumulation in fully grown oocytes (FGO) compared to growing oocytes (GO), paralleled by markedly reduced translational activity, suggesting an intricate link between DHX36 function, rRNA metabolism, and global protein synthesis.
At the heart of the mechanistic investigation was the demonstration of G4 structure accumulation within the nucleolus and nucleoplasm of DHX36-deficient oocytes using the G4-specific fluorescent probe CYTO and BG4 antibody. In vitro assays validated the formation of stable G4 structures within rDNA coding sequences corresponding to pre-rRNA transcripts, and biochemical interactions confirmed direct binding between DHX36 and pre-rRNA via its RSM domain. These findings led to the compelling hypothesis that DHX36 resolves G4 formations on rDNA, thereby facilitating proper transcription and processing of pre-rRNA.
To examine the consequences of stabilized G4 structures on rRNA processing, cells were treated with the G4 stabilizing ligand pyridostatin (PDS) in vitro. This treatment rendered pre-rRNA transcripts more resistant to RNase digestion, underscoring how G4 stabilization impedes normal RNA turnover. Spatial colocalization studies of DHX36, pre-rRNA, and G4 structures in early embryonic stages reinforced the functional interplay among these factors. Further, RNA immunoprecipitation coupled with qPCR substantiated direct DHX36-pre-rRNA interactions.
Critically, reintroduction of DHX36 into CKO oocytes reversed pre-rRNA accumulation, confirming its pivotal role in maintaining pre-rRNA homeostasis. The study culminated in the paradigm that in the absence of DHX36, excessive G4 structures accumulate, obstructing pre-rRNA cleavage, ribosome assembly, and translational competency—processes vital for oocyte maturation and embryonic progression.
This seminal work outlines a sophisticated mechanism whereby DHX36 governs chromatin remodeling and ribosome biogenesis through G4 resolution, intricately linking nucleic acid secondary structure dynamics to developmental competence in mammalian oocytes. The findings provide a molecular framework to understand infertility associated with disruptions in chromatin architecture and rRNA metabolism and suggest DHX36 as a potential therapeutic target for addressing reproductive dysfunctions linked to helicase deficiencies.
The study’s integration of advanced genomic, biochemical, and imaging methodologies provides a comprehensive perspective on the epigenetic and transcriptional consequences of DHX36 loss. By placing G4 structures at the nexus of chromatin accessibility and rRNA processing, it prompts future research into G4-targeted interventions for correcting developmental defects. Moreover, the revelations about nucleolar biology and ribosomal assembly underscore the broader significance of helicase-mediated nucleic acid remodeling in cellular differentiation and early life formation.
In conclusion, the meticulous dissection of DHX36’s role in mouse oocytes underscores the helicase’s indispensable function in facilitating proper chromatin conformation, empowering transcriptional resilience, and safeguarding the fidelity of ribosome biogenesis. This study builds a compelling narrative where DHX36 emerges as a guardian of genomic and epigenomic integrity, orchestrating the molecular choreography essential for oocyte viability and embryonic success.
Subject of Research: The role of the helicase DHX36 in chromatin remodeling and pre-rRNA processing during mouse oocyte development and early embryogenesis
Article Title: DHX36 deficiency disrupts chromatin structure and impairs pre-rRNA processing in mouse oocytes
News Publication Date: Not specified
Web References: http://dx.doi.org/10.1016/j.scib.2025.02.017
Image Credits: ©Science China Press
Keywords: DHX36, G-quadruplex, chromatin accessibility, nucleolus, pre-rRNA processing, oocyte development, meiosis, ribosome biogenesis, infertility, mouse model, ATAC-seq, RNA-seq
