Investigating the role of SALL4 in embryonic stem cells and early development.

Abstract

Early mammalian development requires precise coordination between transcription factor networks and chromatin state to ensure orderly progression from pluripotency to lineage commitment. SALL4 is a conserved transcription factor essential for this process, yet its mechanistic role in pluripotent cells has remained unresolved and debated. Although traditionally linked to pluripotency maintenance and timing of differentiation, emerging evidence suggests that SALL4 preferentially binds AT-rich regulatory regions that are intrinsically prone to premature activation. Whether SALL4 exerts its regulatory influence by recruiting chromatin remodelling complexes such as NuRD, or through direct modulation of chromatin accessibility, has remained unclear. To address this, I used acute FKBP–dTAG degradation of SALL4 in mouse embryonic stem cells, integrating ATAC-seq, CUT&RUN and RNA-seq profiling with a complementary gastruloid model that captures early embryonic patterning. Acute SALL4 depletion induced rapid and widespread chromatin accessibility changes within one hour, predominantly representing increased accessibility at AT-rich, intergenic SALL4-bound regions. These early chromatin changes were largely uncoupled from detectable changes in steady-state RNA at 1 h, supporting a primary chromatin-level consequence of acute SALL4 loss. Transcriptional responses emerged only at later timepoints (4h and 24h), consistent with the general principle that chromatin remodelling can precede measurable shifts in steady-state RNA. Notably, despite the large number of SALL4-dependent accessibility changes, the transcriptional response remained comparatively modest in 2iL conditions, likely reflecting the stabilising influence of this pluripotency-supportive environment. CUT&RUN profiling for NANOG, SOX2, CHD4 and MBD3 further showed that SALL4 loss produces more pronounced effects at intergenic regulatory regions than at established 2iL-active enhancers, underscoring the selective sensitivity of these sites to SALL4. This contrast highlights how CUT&RUN can define the specific chromatin contexts in which SALL4 exerts its strongest regulatory influence. Given SALL4’s essential role in mouse embryonic development, I next sought to determine how these molecular changes influence early embryonic organisation. To this end, I adapted the FKBP–dTAG system to free-floating gastruloids. Transient depletion of SALL4 during a critical Day-3 to Day-4 window disrupted axial elongation and germ-layer patterning, alongside transcriptional changes indicative of altered lineage allocation. Because SALL1 is the only other SALL‑family member expressed during the earliest stages of mouse development, comparisons between gastruloids with or without SALL1 revealed that SALL1 and SALL4 contribute in overlapping but distinct ways to early tissue patterning. Together, these findings establish SALL4 as a chromatin‑level regulator that interprets DNA sequence composition to constrain accessibility at susceptible intergenic regions and stabilise transcription factor binding landscapes. By integrating acute protein depletion with complementary chromatin and transcriptional readouts, alongside a gastruloid model that recapitulates early patterning events, this work refines the mechanistic understanding of SALL4 function in pluripotent cells and demonstrates how these molecular effects translate into developmental outcomes. More broadly, these results place SALL4 within the wider context of SALL‑family regulation and help clarify how related transcription factors coordinate chromatin and transcriptional dynamics to support robust lineage progression during early development.