Chromatin Remodeling During Reprogramming of Somatic Cells to Induced Pluripotent Stem Cells
DOI:
https://doi.org/10.5281/zenodo.22756698Keywords:
induced pluripotent stem cells, reprogramming, SWI/SNF, BAF complex, H3K9me3 barrier, chromatin accessibility, Yamanaka factorsAbstract
Reprogramming of somatic cells to induced pluripotent stem cells (iPSCs) by the transcription factors OCT4, SOX2,
KLF4 and MYC (OSKM) requires large-scale chromatin remodeling to convert a differentiated, lineage-restricted
accessibility landscape into the open, pluripotency-associated configuration of embryonic stem cells, yet the relative
contribution of specific remodeling complexes to this transition remains incompletely defined. Here we combined a
doxycycline-inducible OSKM reprogramming system in mouse embryonic fibroblasts with time-resolved ATAC-seq,
ChIP-seq for H3K9me3 and H3K27me3, single-cell RNA-sequencing, and short hairpin RNA depletion of the BAF
complex ATPase SMARCA4 (BRG1) to dissect the chromatin remodeling trajectory of reprogramming and its
rate-limiting barriers. ATAC-seq across a 12-day reprogramming time course resolved three accessibility phases:
an early, transient wave of accessibility gain at somatic enhancers within 48 hours of OSKM induction, a mid-phase
accessibility loss at fibroblast identity genes coinciding with H3K9me3 accumulation, and a late-phase acquisition of
accessibility at pluripotency-associated loci restricted to cells that successfully completed reprogramming.
SMARCA4 depletion did not block the early somatic enhancer response but severely impaired late-phase opening
of pluripotency loci and reduced iPSC colony formation efficiency by 83%, without preventing H3K9me3
accumulation at silenced somatic genes, indicating that BAF-dependent remodeling acts specifically at the late,
pluripotency-establishing step. Single-cell RNA-sequencing identified a discrete subpopulation of cells at day 8 with
elevated Smarca4 and reduced H3K9me3-associated repressor expression that preferentially progressed to a
pluripotent state by day 12, while cells retaining high H3K9me3-pathway expression stalled in a partially
reprogrammed state. These results define a three-phase chromatin remodeling trajectory in which H3K9me3
deposition acts as a barrier independent of BAF complex activity, while BAF-dependent remodeling is specifically
required to open pluripotency loci once that barrier is cleared, reconciling BAF and heterochromatin-barrier models
within a single ordered framework.
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