Hypothesis

Specific genomic loci resist epigenetic erasure during iPSC reprogramming because they are replicated late in S‑phase, allowing parental histones carrying repressive marks (H3K9me3, H3K27me3) to be preferentially retained via a replication‑coupled inheritance mechanism.

Mechanistic Rationale

Recent work shows that chromatin accessibility changes during reprogramming are tightly linked to DNA replication, with replicated regions opening at OCT4/SOX2/KLF4 binding sites while unreplicated regions shift only gradually {[https://pmc.ncbi.nlm.nih.gov/articles/PMC12985388/]}. Moreover, histone modifications such as H3K9me3 and H3K27me3 are partially retained after reprogramming {[https://www.frontiersin.org/journals/cell-and-developmental-biology/articles/10.3389/fcell.2025.1559183/full]}. If a locus is replicated late, the parental nucleosomes carrying these marks have a higher chance of being recycled onto the nascent strand before reprogramming factors can access the DNA, creating a deterministic barrier to erasure. This model also explains the observed genotype‑dependent methylation at sites like cg27577782 near USP36 {[https://doi.org/10.1101/2024.12.13.627515]}, as genetic variants that alter local replication timing (e.g., by affecting origin firing) would change the probability of parental histone retention.

Predictions

1. Loci identified as resistant to erasure (e.g., those retaining donor‑specific DNA methylation or H3K9me3/H3K27me3 in iPSCs) will show significantly later replication timing in the donor cell type compared with loci that are fully erased.
2. Pharmacologically advancing replication timing (e.g., low‑dose CDK2 inhibition to promote early‑origin firing) will increase the efficiency of erasure at resistant loci without compromising pluripotency.
3. Conversely, delaying replication timing (e.g., CDK1 overexpression) will exacerbate the retention of age‑associated marks and widen the rejuvenation gap.

Experimental Design

• Cell system: Human dermal fibroblasts from donors with known genotypes at cg27577782 and matched iPSC lines generated via OSKM transduction.
• Replication timing profiling: Perform Repli‑Seq on donor fibroblasts and on day‑0, day‑3, day‑7, and day‑14 reprogramming intermediates to assign early/medium/late S‑phase fractions to each CpG and nucleosome region.
• Epigenetic read‑out: Whole‑genome bisulfite sequencing (WGBS) and ChIP‑seq for H3K9me3/H3K27me3 at the same time points to quantify retention of donor‑specific marks.
• Intervention: Treat parallel reprogramming cultures with either a CDK2 inhibitor (e.g., CVT‑313) at a concentration that advances early‑origin firing, or a CDK1 overexpression lentiviral vector, alongside appropriate controls.
• Analysis: Compare the fraction of resistant loci that lose donor methylation or histone marks between conditions. Use linear modeling to test whether replication timing predicts erasure efficiency independent of genotype.

Potential Outcomes

• Support: A significant negative correlation between replication timing (later S‑phase) and persistence of donor epigenetic marks; CDK2 inhibition leads to a measurable reduction (e.g., >20%) in resistant loci and a corresponding decrease in epigenetic age metrics (e.g., Horvath clock) toward the level seen in fully erased iPSCs.
• Refutation: No relationship between replication timing and mark retention, or manipulation of replication timing fails to alter the resistant locus fraction, indicating that other mechanisms (e.g., transcription factor shielding or nuclear compartmentalization) dominate.

Significance

Linking replication timing to the rejuvenation gap provides a testable, mechanistic framework that integrates genetic variability, chromatin dynamics, and epigenetic inheritance. If validated, it suggests that transient modulation of S‑phase progression could be a safe, non‑integrative strategy to enhance epigenetic reset during reprogramming, thereby narrowing the gap between chronological and biological age without increasing tumorigenic risk.