Journal: bioRxiv

Article Title: A developmentally regulated long-range enhancer-promoter contact mediates human neural development

doi: 10.64898/2025.12.01.691702

Figure Lengend Snippet: (A) Genomic organization of a 1 Mb domain of the SOX2 TAD in human NSCs. 4C-sequencing with a SOX2 bait, ATAC-Sequencing, and H3K27ac and H3K27me3 ChIP-sequencing are shown. (B) Comparison of hESC and NSC HiC sequencing, ChIP-sequencing for CTCF, H3K27ac, and H3K27me3, RNA-sequencing transcript reads, and virtual 4C-Sequencing visualizations (derived from HiC sequencing) with a viewpoint of either the SOX2 gene promoter or the putative enhancer region. See also Figure S1A,B. (C) Genome browser view of HS1332 and adjacent HS1332b sequences corresponding to the putative enhancer in the human genome with H3K27ac ChIP-sequencing and ATAC-sequencing from NSCs, PhyloP score for 100 vertebrate genomes, and view of conserved sequences (green bars) for 6 select species. See also Figure S1C. (D) Comparison of mouse ESC and NSC HiC sequencing, ChIP-sequencing for CTCF, H3K27ac, and H3K27me3, and virtual 4C-Sequencing visualizations (derived from HiC sequencing) with a viewpoint of either the SOX2 gene promoter or the putative enhancer region. (E) Schematic demonstrating the hypothesis tested.

Article Snippet: Arima-HiC sequencing libraries were prepared by first shearing purified proximally ligated DNA and then size-selecting 200-600 bp DNA fragments using SPRI (Solid Phase Reversible Immobilization) beads.

Techniques: Sequencing, ChIP-sequencing, Comparison, RNA Sequencing, Derivative Assay

Journal: bioRxiv

Article Title: A developmentally regulated long-range enhancer-promoter contact mediates human neural development

doi: 10.64898/2025.12.01.691702

Figure Lengend Snippet: (A) Graphical representation of teratoma formation experiments. (B) Immunofluorescence microscopic analysis of ROSA26 , 1 HS1332 , and 1 nMotif teratomas harvested after 6 weeks. Representative images are shown for GFP, SOX2, PAX6, HNF3B, and smooth muscle actin (SMA). (C) Quantification of teratoma immunofluorescence markers: GFP (n=4 biological replicates, one-way ANOVA F 2,8 =28.84, p=0.0002; Tukey’s post hoc multiple comparison test: ROSA26 vs 1 HS1332 **p < 0.01, ROSA26 vs 1 nMotif ***p < 0.001); PAX6 (n=5 biological replicates, ANOVA F 2,13 = 6.030, p=0.0140, Tukey’s post hoc multiple comparison test: ROSA26 vs 1 HS1332 *p<0.05, ROSA26 vs 1 nMotif *p<0.05); SOX2 (n=4 biological replicates, ANOVA F 2,11 =4.934, p=0.0295; Tukey’s post hoc multiple comparison test: ROSA26 vs 1 HS1332 *p < 0.05, ROSA26 vs 1 nMotif *p<0.05); HNF3B (n=5 biological replicates, ANOVA F 3,16 =5.297, p=0.0100; Tukey’s post hoc multiple comparison test: ROSA26 vs 1 HS1332 **p<0.01, ROSA26 vs 1 nMotif p<0.05); SMA (n=3 biological replicates, F 2,6 =0.09587, p=0.9099; Tukey’s post hoc multiple comparison test: ROSA26 vs 1 HS1332 p>0.05, ROSA26 vs 1 nMotif p>0.05). ns, not significant. (D) Single-cell RNA sequencing data from teratomas in UMAP representations. Clusters are labeled by cell identity (left). A comparison of ROSA26 vs 1 HS1332 (center) and ROSA26 vs 1 nMotif (right) clusters with color coding is shown. Epi, epithelium; CycProg, cycling progenitors; Prog, progenitors; MSC, mesenchymal stem cells; Fib, fibroblasts; Myofib, myofibroblasts. (E) Bar graph representing the proportion of cells sequenced in each condition falling into an ectodermal, endodermal, or mesodermal identity. (F) scRNA-seq UMAP representation in which only ectodermal clusters from the prior UMAP (D) were extracted and re-clustered with identities assigned (left). Comparisons of ectodermal sub-clustering in ROSA26 vs 1 HS1332 (center) and ROSA26 vs 1 nMotif (right) with color coding are shown.

Article Snippet: Arima-HiC sequencing libraries were prepared by first shearing purified proximally ligated DNA and then size-selecting 200-600 bp DNA fragments using SPRI (Solid Phase Reversible Immobilization) beads.

Techniques: Immunofluorescence, Comparison, RNA Sequencing, Labeling

Journal: bioRxiv

Article Title: A developmentally regulated long-range enhancer-promoter contact mediates human neural development

doi: 10.64898/2025.12.01.691702

Figure Lengend Snippet: (A) Genome browser view of H3K27ac ChIP-sequencing in the SOX2 TAD in hESCS using RPGC-normalized signal track and rosettes using input-normalized ppois signal track. (B) Genome browser view of RPGC-normalized H3K27me3 ChIP-sequencing in the SOX2 TAD in hESCS and rosettes. Additional data for peaks 1-3 are shown in Figure S6C. (C) 4C-sequencing with a SOX2 promoter bait in hESCS and rosettes. (D) Significant interacting peaks, as determined by Benjamini-Hochberg multiple testing procedure (red), in 4C-sequencing data of rosettes from the ROSA26 and 1 nMotif conditions (*p<0.05).

Article Snippet: Arima-HiC sequencing libraries were prepared by first shearing purified proximally ligated DNA and then size-selecting 200-600 bp DNA fragments using SPRI (Solid Phase Reversible Immobilization) beads.

Techniques: ChIP-sequencing, Sequencing

Journal: bioRxiv

Article Title: A developmentally regulated long-range enhancer-promoter contact mediates human neural development

doi: 10.64898/2025.12.01.691702

Figure Lengend Snippet: (A) Agarose gel electrophoresis of PCR product containing the n Motif domain in control (NT-gRNA) vs 1 nMotif NSCs (left). The Sanger sequencing results of the two PCR products is also shown (right). (B) qRT-PCR for SOX2 transcript in control and 1 nMotif NSCs (n=3 biological replicates, t-test **p<0.01) (C) Immunofluorescence microscopy for SOX2 and NESTIN in control vs 1 nMotif NSCs. (D) Growth/viability measurement by WST8 absorbance assay in control and 1 nMotif NSCs (n=3 biological replicates, two-way ANOVA n=3, F 3,6 =32.1 p<0.01; Sidak’s multiple comparisons test **p<0.01 at indicated timepoints). (E) Immunofluorescence microscopy for TUJ1 and GFAP in neuronal and astrocytic differentiation of control and 1nMotif NSCs. (F) qRT-PCR of neuronal transcript RBFOX3 and astrocytic transcript SLC1A2 after neuronal and astrocytic differentiation of control and 1 nMotif NSCs (n=3 biological replicates, t-test p=0.0091 and p=0.0171, respectively). (G) Genome browser view of SOX2 TAD comparing the following assays in NeuN+ or NeuN- cells isolated from postmortem DLPFC in PsychEncode: ATAC-Sequencing, H3K27ac ChIP-sequencing, SOX2 promoter virtual 4C-sequencing, and HS1332 enhancer virtual 4C-sequencing. (H) HiC plots of the SOX2 TAD in NeuN+ or NeuN- cells of postmortem DLPFC show preservation of 3D architecture in adult neurons and glia.

Article Snippet: Arima-HiC sequencing libraries were prepared by first shearing purified proximally ligated DNA and then size-selecting 200-600 bp DNA fragments using SPRI (Solid Phase Reversible Immobilization) beads.

Techniques: Agarose Gel Electrophoresis, Control, Sequencing, Quantitative RT-PCR, Immunofluorescence, Microscopy, Isolation, ChIP-sequencing, Preserving