Journal: bioRxiv
Article Title: Differential Regulation of Large-scale Chromosome Conformations in Osteoblasts and Osteosarcoma
doi: 10.1101/2024.11.01.621571
Figure Lengend Snippet: (A) A schematic of CRISPR-Sirius. The CRISPR-Sirius single guide RNA (sgRNA) is engineered with an octet of PP7 hairpins (8XPP7). PP7 is labeled by binding to its coat protein (PCP) fused with the green fluorescent protein (GFP). The sgRNA targeted about 836 repeats spanning 17 Mb region on chromosome 19 q arm (C19q), which allows the visualization of chromosome 19 territory. (B) Images of C19q conformation in osteoblasts (OB, hFOB1.19) and osteosarcoma (OS, U2OS) cells. Osteoblasts have extended C19q conformations, whereas osteosarcoma cells demonstrated collapsed C19q conformations. (C) Schematic of C19q conformation quantitative analysis. The spline curves from C19q images in and were used to calculate gyration tensors that give rise to the asphericity (Δ) and aspect ratio (K) of C19q conformations in osteoblasts (upper row) and osteosarcoma cells (lower row). (D) Gaussian bimodal fitting of aspect ratio (K) distribution of C19q conformations for osteoblast (N C19q = 104) and osteosarcoma cells (N C19q =100). The blue lines represent fittings with two Gaussian peaks for whole distributions. Red and green lines represent the individual distributions for collapsed and extended conformations, respectively. (E) The percentages of collapsed and extended conformations from gaussian bimodal fittings. Histogram shows that osteosarcoma cells (N C19q =100) (***, p < 0.001) have significantly fewer C19q in the extended conformation as compared to those in osteoblasts (N C19q = 104). (F) Time lapse of C19q over 70 minutes in osteosarcoma under physiological conditions (37°C, 5% CO 2 and humidity)(G) Time lapse of C19q over 70 minutes in osteoblasts under physiological conditions (37°C, 5% CO 2 and humidity). The m denotes minutes. Bar in the enlarged inset, 2 µm. Each Z-stack was 3D deconvoluted and projected using maximal intensity. (H) Numbers of genes up– and down-regulated in chromosome 19 and in C19q, respectively. (I) Volcano plot of RNA-seq comparing differential expression of chromosome 19 genes in OB and OS. The significance test in (E) was calculated using Fisher’s exact test. All experiments were repeated at least three times.
Article Snippet: The sgRNA (pLH-sgRNA-Sirius-8XPP7, Cat#121940), dCas9 (pHAGE-TO-dCas9-P2A-HSA, Cat#121936), and GFP-fused PP7 coat protein (pHAGE-EFS-PCP-GFPnls, Cat#121938) expression constructs were obtained from Addgene.
Techniques: CRISPR, Labeling, Binding Assay, RNA Sequencing Assay, Expressing
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