NAD+-Dependent Sirtuin Activation Reduces Stochastic Heterochromatin Noise During iPSC Reprogramming

We hypothesize that elevating NAD+ levels during reprogramming lowers stochastic heterochromatin noise by activating SIRT1 and SIRT6, which promotes erasure of repressive marks and facilitates a more uniform transition to pluripotency. NAD+ serves as a cofactor for sirtuin deacetylases; SIRT1 deacetylates H3K9ac and H3K56ac, weakening HP1 binding and allowing chromatin relaxation, while SIRT6 promotes H3K9 demethylation and interacts with TET enzymes to drive DNA demethylation at late-replicating domains. By decreasing cell-to-cell variance in H3K9me3 and DNA methylation, NAD+ supplementation should sharpen the epigenetic landscape, reduce noisy intermediates, and increase the fraction of iPSCs that achieve complete reset.

To test this, we will reprogram human fibroblasts from aged donors using OSKM factors in parallel cultures treated with either nicotinamide riboside (NR) or vehicle control. At 0, 24, 48, 72, and 96 hours we will collect cells for single-cell ATAC-seq, single-cell bisulfite sequencing (scBS-seq), and CUT&Tag for H3K9me3. Noise will be quantified as the variance in accessibility at a set of pluripotency‑enhancer regions (e.g., distal Oct4 enhancers) and as the dispersion of methylation percentages at CpG islands within late‑replicating heterochromatin. We will also measure OCT4 ChIP‑seq signal and expression of pluripotency markers (NANOG, SSEA4) by flow cytometry.

Predictions: NR‑treated cultures will show a significant reduction (p<0.01, effect size >0.5) in accessibility and methylation variance compared with controls, accompanied by a >2‑fold increase in the proportion of cells exhibiting high OCT4 binding and SSEA4 positivity. Conversely, co‑treatment with the sirtuin inhibitors EX‑527 (SIRT1) or OSS_128167 (SIRT6) will abolish the NR‑induced noise reduction and fail to improve reprogramming efficiency, falsifying the hypothesis if noise remains unchanged despite NAD+ elevation.

This model extends the current view of epigenetic noise as a passive barrier by linking cellular metabolism directly to chromatin homogeneity. If validated, it would position NAD+ boosting as a scalable, metabolite‑based strategy to enhance reprogramming fidelity and potentially improve the quality of iPSCs derived from aged tissue.