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. 2025 Jan 15;20(1):e0317718.
doi: 10.1371/journal.pone.0317718. eCollection 2025.

Nucleosome repositioning in cardiac reprogramming

Affiliations

Nucleosome repositioning in cardiac reprogramming

Sonalí Harris et al. PLoS One. .

Abstract

Early events in the reprogramming of fibroblasts to cardiac muscle cells are unclear. While various histone undergo modification and re-positioning, and these correlate with the activity of certain genes, it is unknown if these events are causal or happen in response to reprogramming. Histone modification and re-positioning would be expected to open up chromatin on lineage-specific genes and this can be ascertained by studying nucleosome architecture. We have recently developed a set of tools to identify significant changes in nucleosome architecture which we used to study skeletal muscle differentiation. In this report, we have applied these tools to understand nucleosome architectural changes during fibroblast to cardiac muscle reprogramming. We found that nucleosomes surrounding the transcription start sites of cardiac muscle genes induced during reprogramming were insensitive to reprogramming factors as well as to agents which enhance reprogramming efficacy. In contrast, significant changes in nucleosome architecture were observed distal to the transcription start site. These regions were associated with nucleosome build-up. In summary, investigations into nucleosome structure do not support the notion that fibroblasts to cardiac muscle cell reprogramming involves chromatin opening and suggests instead long-range effects such as breaking closed-loop inhibition.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Expression of muscle and non-muscle genes during the direct reprogramming of fibroblasts into cardiac muscle cells.
(A) To determine the minimum number of genes for analysis of nucleosome architecture, an experiment was conducted whereby genes were additively compared to their full-group. The comparisons were made on nucleosome density at a 1bp resolution across the full promoter and the R2 value noted after the addition of each gene. Five random sequences of genes were investigated. (B) RNA-seq data derived from fibroblasts four days after transfection with the reprogramming cocktail miR combo were investigated for the expression of the members of each listed gene-group. The data is expressed as a fold change in expression when compared to cells transfected with a control miRNA. N = 3. Significances were determined by Wilcoxon Signed Rank Test (Pratt method, median = 1); ns–not significant, **P<0.01.
Fig 2
Fig 2. Direct reprogramming of fibroblasts into cardiac muscle cells is associated with significant changes in nucleosome architecture.
(A) Our PRJNA837987 MNase-seq dataset was analyzed to determine the effects of direct reprogramming on nucleosome positioning. To compare nucleosome patterns within each group and across the three groups, the read counts were normalized by dividing the read count at each base-pair of the promoter by the sum of read counts across the promoter (a full description is provided in the methods). The schematic shows those regions in cardiac and common muscle gene promoters where nucleosomes were significantly enriched or depleted in response to reprogramming as well as in response to the reprogramming enhancers PolyIC and 3p-hpRNA. (B) Regions of significant enrichment or depletion (A, B, C and E) were analyzed further alongside a region encompassing the TSS (D) by determining the nucleosome correlations.
Fig 3
Fig 3. Cardiac and common muscle genes show significant nucleosome re-positioning away from the TSS.
For each analyzed promoter, the change in read number (Δ normalized read count) following direct reprogramming (miR combo versus control miR) was calculated at a 1bp resolution and summed over the genomic region analyzed. Significances were determined by Wilcoxon Signed Rank Test (Pratt method, median = 0); ns–not significant, **P<0.01.
Fig 4
Fig 4. TLR3 and Rig1 agonists affect nucleosome architecture differently.
For the whole promoter as well as regions A to E, the change in read number (Δ normalized read count) following direct reprogramming (miR combo plus PolyIC/3p-hpRNA versus miR combo) was calculated at a 1bp resolution and compared between the various gene groups. The matrices report the correlations between the various groups.
Fig 5
Fig 5. TLR3 and Rig1 agonists have opposing effects on nucleosome architecture in muscle and non-muscle genes.
For the whole promoter as well as regions A to E, the change in read number (Δ normalized read count) following direct reprogramming (miR combo plus PolyIC/3p-hpRNA versus miR combo) was calculated at a 1bp resolution and summed. Significances were determined by Wilcoxon Signed Rank Test (Pratt method, median = 0); ns–not significant, *P<0.05, **P<0.01, ***P<0.001.

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