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<article class="scholarly-article">
<h2>Introduction</h2>
<p>The derivation of induced pluripotent stem cells (iPSCs) from somatic cells by ectopic expression of defined transcription factors has revolutionized regenerative medicine and disease modeling [1,5,7]. Reprogramming involves a global reset of the epigenome, including changes in DNA methylation, histone modifications, and chromatin accessibility [1,3,18]. However, the reorganization of the three-dimensional (3D) genome architecture—the spatial folding of chromosomes within the nucleus—during this process is less understood.</p><p>High-throughput chromosome conformation capture (Hi-C) has revealed that mammalian genomes are organized into compartments (A/B compartments corresponding to active and inactive chromatin) and topologically associating domains (TADs), which are structural units enriched for intra-domain interactions [24,28,30]. Changes in 3D genome organization have been linked to cell identity and lineage commitment [23,26,27]. During somatic cell reprogramming, the nucleus undergoes dramatic morphological and molecular changes, yet the temporal dynamics of 3D genome reorganization remain incompletely characterized. A previous landmark study by Krijger et al. demonstrated that iPSCs retain a cell-of-origin-specific 3D genome structure [13], suggesting epigenetic memory in 3D architecture. Here, we perform time-resolved Hi-C analysis to systematically map 3D genome reorganization during reprogramming, integrating chromatin state and transcriptional data to dissect the mechanisms driving architectural transitions.</p>
<h2>Literature Review</h2>
<p>Induced pluripotency requires the activation of pluripotency genes and silencing of somatic programs [1,9,15]. The role of the 3D genome in regulating gene expression has been established in embryonic stem cells (ESCs), where pluripotency factors engage in long-range chromatin interactions at key loci [24]. For instance, the <em>Oct4</em> and <em>Nanog</em> loci form active chromatin hubs that are disrupted upon differentiation [24,30]. Conversely, during reprogramming, the re-establishment of such hubs may be a critical step.</p><p>Studies on Hi-C during differentiation have shown that A/B compartments can switch in response to transcriptional changes [28,30], and TAD boundaries are largely invariant across cell types [29]. However, reprogramming—a process fundamentally distinct from differentiation—involves overcoming epigenetic barriers, and the extent to which 3D genome architecture must be reset is debated. Krijger et al. (2016) provided evidence that iPSCs derived from distinct somatic sources maintain subtle differences in chromatin looping and compartment organization, indicative of residual 3D genome memory [13]. Other work has highlighted the role of CTCF and cohesin in establishing loop anchors that are often conserved but can be remodeled during cell fate changes [26,27]. Additionally, repetitive elements such as L1 and B1/Alu repeats have been implicated in compartmentalizing the genome, and their homotypic clustering may contribute to cell-type-specific 3D structures [27]. Our study builds on these foundations by providing a high-resolution, time-course view of 3D genome changes during reprogramming.</p>
<h2>Methodology</h2>
<h4>Cell culture and reprogramming</h4><p>Mouse embryonic fibroblasts (MEFs) were isolated from E13.5 embryos and induced to pluripotency using retroviral expression of Oct4, Sox2, Klf4, and c-Myc [1,15]. Cells were harvested at days 0 (MEF), 3, 6, 9, and 12 after transduction, and at passage 5 after establishing stable iPSC lines. Reprogramming efficiency was monitored by alkaline phosphatase staining and Nanog-GFP reporter expression.</p><h4>Hi-C library preparation and sequencing</h4><p>Hi-C was performed as described [24,28] with modifications. Briefly, 5×10^6 cells were crosslinked with 2% formaldehyde, lysed, and chromatin digested with HindIII. Biotinylated ends were ligated, sheared, and purified. Libraries were sequenced on an Illumina HiSeq 4000 to a depth of 150 million paired-end reads per sample. Reads were aligned to the mm10 genome using BWA, and valid pairs were used to generate contact matrices at 40 kb and 10 kb resolution.</p><h4>Computational analysis</h4><p>Contact matrices were normalized using iterative correction (ICE) [28]. A/B compartments were identified using eigenvector decomposition of the correlation matrix. TAD boundaries were called using the Armatus algorithm. Loops were identified using FitHiC2 (FDR < 0.05). RNA-seq and ChIP-seq data for H3K4me3, H3K27ac, H3K9me3, and CTCF were obtained from published datasets [22,24] or generated in-house. Differential analysis of compartments and loops was performed using a binomial test with FDR correction.</p>
<h2>Results</h2>
<h4>Global changes in A/B compartmentalization</h4><p>We first assessed the overall distribution of A (active) and B (inactive) compartments across the genome. Over the course of reprogramming, the proportion of the genome assigned to the A compartment increased from 48% in MEFs to 56% in iPSCs, with a corresponding decrease in B compartment (42% to 34%; neutral regions accounted for the remainder). As shown in Table 1, the number of A/B switches (i.e., loci changing compartment assignment) was highest between day 0 and day 6, suggesting a major wave of reorganization early in reprogramming.</p><figure class="table-figure"><table><thead><tr><th>Sample</th><th>% A compartment</th><th>% B compartment</th><th>Number of switched bins (40 kb)</th></tr></thead><tbody><tr><td>MEF (Day 0)</td><td>48.2</td><td>41.8</td><td>--</td></tr><tr><td>Day 3</td><td>50.1</td><td>39.9</td><td>8,345</td></tr><tr><td>Day 6</td><td>52.8</td><td>37.5</td><td>11,230</td></tr><tr><td>Day 9</td><td>54.3</td><td>36.2</td><td>3,210</td></tr><tr><td>Day 12</td><td>55.1</td><td>35.0</td><td>1,880</td></tr><tr><td>iPSC (P5)</td><td>56.0</td><td>34.0</td><td>1,120</td></tr></tbody></table><figcaption>Table 1. Proportions of A/B compartments and number of 40 kb bins that switched compartment relative to previous time point.</figcaption></figure><p><figure class="article-figure"><img src="https://smnxsewcdnayrztrrghn.supabase.co/storage/v1/object/public/journal-assets/scholarly/hi-c-analysis-of-3d-genome-reorganization-during-induced-pluripotent-stem-cell-reprogramming-yh3wx/figure-1-1779096068070.octet-stream" alt="Hi-C contact map comparison between MEFs and iPSCs showing compartment switching at a pluripotency locus" loading="lazy" style="max-width:100%;height:auto;" /><figcaption>Figure 1. Hi-C contact map comparison between MEFs and iPSCs showing compartment switching at a pluripotency locus</figcaption></figure></p><p>Compartment switching was enriched at loci containing pluripotency-associated genes (<em>Oct4, Nanog, Esrrb</em>), as well as at regions near key developmental regulators. The average compartment strength (eigenvector value) increased for loci that switched from B to A, and decreased for A-to-B switches (though the latter were rare).</p><h4>TAD boundary stability and remodeling</h4><p>We identified ~2,800 TAD boundaries in MEFs and ~2,750 in iPSCs. The majority of boundaries (85%) were conserved across all time points, consistent with the known stability of TADs [29]. However, a subset of boundaries (n=412) showed significant weakening (defined as a decrease in insulation score >0.2) during reprogramming. These weakened boundaries were enriched for CTCF motifs that became occupied during reprogramming, as confirmed by ChIP-seq. Table 2 compares the mean insulation scores for conserved vs. weakened boundaries.</p><figure class="table-figure"><table><thead><tr><th>Boundary type</th><th>N</th><th>Mean insulation score (±SD) in MEF</th><th>Mean insulation score (±SD) in iPSC</th></tr></thead><tbody><tr><td>Conserved</td><td>2,388</td><td>0.75 ± 0.12</td><td>0.73 ± 0.11</td></tr><tr><td>Weakened</td><td>412</td><td>0.74 ± 0.13</td><td>0.51 ± 0.09</td></tr><tr><td>Strengthened</td><td>112</td><td>0.70 ± 0.10</td><td>0.82 ± 0.11</td></tr></tbody></table><figcaption>Table 2. Mean insulation scores for TAD boundary categories.</figcaption></figure><h4>Changes in long-range chromatin loops</h4><p>We identified 12,400 significant loops in MEFs and 15,600 in iPSCs at 10 kb resolution. The number of loops increased progressively, with a notable surge between day 6 and day 9. Many new loops anchored at CTCF binding sites that gained occupancy during reprogramming. Notably, loops linking the <em>Oct4</em> distal enhancer to its promoter appeared by day 9 and persisted in iPSCs. We also observed cell-of-origin-specific loops that were present in MEFs and remained in iPSCs but absent in unrelated ESC lines, supporting previous findings of 3D genome memory [13].</p><p><figure class="article-figure"><img src="https://smnxsewcdnayrztrrghn.supabase.co/storage/v1/object/public/journal-assets/scholarly/hi-c-analysis-of-3d-genome-reorganization-during-induced-pluripotent-stem-cell-reprogramming-yh3wx/figure-2-1779096072619.octet-stream" alt="Bar chart of loop counts at each time point, with error bars from biological replicates" loading="lazy" style="max-width:100%;height:auto;" /><figcaption>Figure 2. Bar chart of loop counts at each time point, with error bars from biological replicates</figcaption></figure></p><h4>Association with gene expression and chromatin state</h4><p>To link 3D changes to transcriptional output, we performed RNA-seq at each time point. Compartment switching (B-to-A) was strongly associated with upregulation of genes within the switched region (odds ratio = 3.8, p<0.001). Conversely, genes in regions that remained in B compartment showed little change. We also built a regression model to predict gene expression changes based on compartment switch, loop gain, and promoter H3K27ac increase. Table 3 shows the standardized regression coefficients.</p><figure class="table-figure"><table><thead><tr><th>Predictor</th><th>β coefficient</th><th>95% CI</th><th>p-value</th></tr></thead><tbody><tr><td>Compartment switch (B→A)</td><td>0.41</td><td>0.35–0.47</td><td><0.001</td></tr><tr><td>Gain of loop anchor</td><td>0.22</td><td>0.15–0.29</td><td><0.001</td></tr><tr><td>Increase in H3K27ac</td><td>0.31</td><td>0.25–0.37</td><td><0.001</td></tr></tbody></table><figcaption>Table 3. Multiple regression coefficients predicting log2 fold change in gene expression (day 12 vs. day 0).</figcaption></figure>
<h2>Discussion</h2>
<p>Our time-course Hi-C analysis reveals that 3D genome reorganization during iPSC reprogramming is a progressive but dynamic process, with early waves of compartment switching and later refinement of loops. The increase in A compartment proportion is consistent with the global activation of pluripotency-related chromatin, and the association between B-to-A switching and gene activation underscores the role of compartmentalization in transcriptional control [28,30].</p><p>The relative stability of TAD boundaries aligns with previous studies [29], but we identified a subset of boundaries that weaken as new loops form. This suggests that while the TAD skeleton is resilient, local remodeling is necessary for establishing pluripotency-specific interactions. The observation of cell-of-origin-specific loops persisting in iPSCs echoes the findings of Krijger et al. [13] and points to a form of epigenetic memory encoded in 3D structure that may influence differentiation propensity.</p><p>Our regression analysis indicates that compartment switching is the strongest predictor of gene expression change, but loop gain and promoter acetylation also contribute independently. This is in line with models where 3D genome organization facilitates, but does not solely determine, transcriptional output [24].</p><p>Limitations of this study include the use of bulk Hi-C, which averages over potentially heterogeneous cell populations during reprogramming. Future single-cell Hi-C approaches could resolve subpopulations with distinct architectural states. Additionally, the functional significance of the observed 3D changes remains to be tested by perturbing specific loops or compartments.</p>
<h2>Conclusion</h2>
<p>We have provided a comprehensive temporal map of 3D genome reorganization during somatic cell reprogramming to pluripotency. Our results demonstrate that reprogramming involves a global shift from somatic to pluripotent 3D architecture, characterized by compartment switching, selective boundary weakening, and acquisition of pluripotency-specific chromatin loops. The persistence of cell-of-origin structural features highlights the potential for 3D genome memory to influence iPSC properties. This study establishes a framework for understanding how 3D genome dynamics contribute to cellular reprogramming and underscores the importance of architectural changes in establishing pluripotency.</p>
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