Son haploinsufficiency in hematopoietic cells disrupts the crosstalk between splicing fidelity and chromatin accessibility, impairing lymphoid lineage commitment Free

Hematopoietic lineage specification and differentiation rely on precise gene regulation, integrating transcription factors with RNA splicing and chromatin remodeling. The SON gene encodes a multi-domain protein essential for transcriptional regulation, RNA splicing, and nuclear speckle organization. We previously showed that SON enhances accurate splicing of transcripts with weak splice sites and modulates chromatin by repressing H3K4me3 through Menin sequestration, thereby inhibiting the MLL1/2 complex. Heterozygous SON loss-of-function mutations cause Zhu-Tokita-Takenouchi-Kim (ZTTK) syndrome, a developmental disorder with multiple symptoms, including hematological issues such as immunodeficiency and poor vaccine responses. Aberrant SON levels are also observed in hematologic malignancies. Despite growing recognition of SON's role in gene regulation and disease, its cell-intrinsic function in hematopoietic lineage commitment remains largely unexplored.

To investigate SON's role in hematopoiesis, we created a mouse model with hematopoietic-specific Son deletion using Son-floxed mice crossed to Vav-iCre transgenics. We analyzed hematopoietic stem and progenitor cells (HSPCs) by flow cytometry, single-cell RNA sequencing (scRNA-seq), bulk RNA deep-seq, and ATAC-seq of bone marrow Lin⁻Sca-1⁺cKit⁺ (LSK) cells to determine phenotypic changes, differential gene expression, alternative RNA splicing, and chromatin accessibility caused by Son haploinsufficiency.

Flow cytometry of fetal liver LSK cells from homozygous Son deletion mice (Vav-iCre; Son⁻/⁻) showed a marked reduction in lymphoid-primed multipotent progenitors (LMPP/MPP4) and embryonic lethality at E17.5–E18. Heterozygous Son deletion mice (Vav-iCre; Son⁺/⁻) were viable, and flow cytometry and scRNA-seq of Son⁺/⁻ bone marrow LSK cells revealed a consistent, cell-intrinsic reduction in LMPP/MPP4, suggesting impaired lymphoid commitment.

scRNA-seq showed downregulation of lymphoid-related genes, including transcription factors (Foxp1, Irf9, Notch1,) and genes involved in antigen presentation (B2m, H2-D1, H2-Q4,6,7, Tap1, H2-K1, H2-T). Notably, several genes encoding epigenetic factors and chromatin remodelers (Atrx, Tet2) were also downregulated.

Splicing analysis using rMATS of bulk RNA Seq identified extensive alternative splicing events in Son +/- LSK cells, and exon skipping/inclusion events are most prevalent. Atrx and Ehmt2 (G9a) were identified as prominent targets, linking SON-dependent splicing to epigenetic regulation. Loss of ATRX is known to cause de-repression of repetitive DNA elements and heterochromatic regions, leading to their transcription and contributing to genomic instability. Similarly, EHMT2, a histone methyltransferase responsible for H3K9me2 deposition, plays a critical role in establishing repressive chromatin. These findings suggest that SON deficiency may impair heterochromatin maintenance via both reduced expression and mis-splicing of chromatin-modifying enzymes.

Interestingly, ATAC-seq analysis revealed widespread increases in chromatin accessibility in Son⁺/⁻ LSK cells. Motif analysis further showed that binding sites for the AP-1 family transcription factors (TFs) JUN, FOS, and ATF3, which promote myeloid differentiation, became more accessible in Son⁺/⁻ LSK cells. In contrast, binding sites for ETS family TFs (PU.1, BACH2), which are critical for lymphoid lineage development, exhibited reduced accessibility. This shift is likely due to the pioneering activity of AP-1 TFs, which occupy and stabilize open chromatin regions, thereby limiting the access of chromatin remodelers to ETS-regulated loci and resulting in functional closure.

Our findings demonstrate that hematopoietic-specific SON haploinsufficiency disrupts both transcription and RNA splicing of key chromatin remodeler and epigenetic regulator genes in HSPCs. The increased open chromatin status in Son +/- HSPCs shifts chromatin control from ETS to AP-1 TFs, which might link to silencing lymphoid lineage genes. These results establish SON as a pivotal regulator in transcriptional and epigenetic programs required for lymphoid specification in hematopoietic progenitors. Our work provides a mechanistic link between SON insufficiency and immune dysfunction in ZTTK syndrome and highlights the critical importance of proper SON dosage in coordinating splicing and chromatin regulation in HSPCs.