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. 2020 Nov 16;48(21):12135–12150. doi: 10.1093/nar/gkaa1051

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© The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research.

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com

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Figure 3. — A novel intrachromosomal loop in the NTS locus. (A) A chromosomal conformation capture (3C) assay was performed to detect intrachromosomal interactions between the NTS promoter and enhancer regions in OCM1, OCM1a and OM431 cells. Right panel: the intrachromosomal interactions between 1–2, 1–3, 1–4 and 1–5 by PCR. M, DNA marker. Numbers under 1–5: distance from the translation start site (TSS). The interaction frequency was determined by normalizing the 3C PCR signal to that of the positive control (input DNA). *P < 0.05 compared to negative control human Schwann cells (HSCs), arising retinal pigment epithelia 19 (ARPE19) cells and pigmented (PIG1) cells. (B) Schematic diagram of variant primer sets in 3C assay. L1–L4: primers in LRRIQ1 intronic region, N1-N5: primers in NTS promoter region. (C) The interaction frequency was determined in OCM1, OCM1a and OM431 cells by normalizing the 3C PCR signal to that of the positive control (input DNA). *P < 0.05 compared to negative control retinal pigment epithelial (RPE) cells and pigmented (PIG1) cells. L2 was set as 3C bait. (D). Identification of the NTS upstream interacting region as an NTS enhancer. Enhancer activity was measured as the relative luciferase units in 293T cells. The NTS promoter and its enhancer inserted upstream of pGL2-promoter-Luc; Mock, empty pGL2-promoter-Luc vector; 293T, wild-type 293T cells. For comparison, the luciferase expression of the mock insert at 48 h was arbitrarily set as 1 in the calculation. *P < 0.05 compared to mock luciferase expression. (E) ChIP assay of the CTCF status in the enhancer (LRRIQ1 intron) in UM (OCM1, OCM1a and OM431) and normal control (ARPE19 and PIG1) cells. * P < 0.05. (F) A 3C assay was performed to detect the existence of chromosomal looping in clinical samples.