​ Following mammalian fertilization, chromatin undergoes extensive reorganization into active and repressed compartments, yet the role of subnuclear organelles in this process remains unclear. This research generated high-resolution maps of nuclear speckle-genome interactions in mouse preimplantation embryos, revealing step-wise establishment of Nuclear Speckle-Associated Domains (SPADs). SPADs emerge at the PN5 zygote stage with allelic specificity, persisting until the 4-cell stage. These domains are categorized into primary (pre-zygotic genome activation, ZGA) and secondary (post-ZGA) SPADs, which sequentially preconfigure gene expression programs. Intriguingly, heterochromatin marker H3K9me3 within SPADs fine-tunes transcriptional precision. SPAD formation correlates with sperm-derived A compartments, suggesting inheritance of 3D chromatin features. Cohesin loader Nipbl and RNA transcription critically regulate secondary SPADs, while stage-specific speckle-associated factors like Gata6 drive lineage-restricted SPAD organization. These findings highlight a dynamic, hierarchical SPAD framework that integrates chromatin architecture, transcription, and developmental cues to meet embryonic demands.
Key findings from the study include:

1. Dynamic Reorganization of SPADs During Embryogenesis​​: This study reveals that nuclear SPADs undergo step-wise reorganization during mouse embryonic development. SPADs are reestablished shortly after fertilization, with distinct temporal patterns: paternal genomes exhibit stronger SPADs signals than maternal genomes prior to the 4-cell stage, correlating with paternal genome activation. Post-8-cell stage, paternal X chromosomes show rapid SPADs signal attenuation, coinciding with X chromosome inactivation.
2. Stage-Specific SPADs Subtypes and Functional Roles​​: SPADs are categorized into two subtypes: primary SPADs (pSPADs), formed before ZGA, and secondary SPADs (sSPADs), formed post-ZGA. While pSPADs exhibit stable chromatin activity, sSPADs are transcriptionally dynamic and developmentally regulated. Chemical inhibition of ZGA (e.g., α-amanitin) abolishes ~80% of sSPADs but minimally affects pSPADs, highlighting their differential dependencies on transcriptional activity.
3. Spatial and Structural Coordination with 3D Genome Architecture​​: SPADs are predominantly localized within transcriptionally active A compartments. Early TAD boundaries align closely with SPAD boundaries, and overlapping TAD-SPAD junctions exhibit reduced insulation scores. This suggests SPADs contribute to TAD formation and chromatin compartmentalization during embryogenesis, linking nuclear speckle interactions to higher-order genome organization.
4. Regulatory Mechanisms Underlying SPAD Formation​​: The study identifies transcription factors (e.g., Gata6) as critical regulators of SPAD dynamics. Motif enrichment analyses and knockdown experiments demonstrate that lineage-specific transcription factors drive SPAD formation in a stage-dependent manner. Additionally, cohesin loading factor Nipbl modulates sSPAD intensity, implicating chromatin loop extrusion in SPAD organization.

This work provides a comprehensive framework for understanding how SPADs orchestrate spatiotemporal gene expression and nuclear architecture to meet the demands of early mammalian embryogenesis. The work entitled “Step-wise organization of genomic nuclear speckle-associated domains during mammalian embryonic development” was published on Protein & Cell (published on Feb. 18, 2025).

DOI: 10.1093/procel/pwae015