Fig. 1: E-P distances and contact frequencies versus gene expression.
a, Schematic representation of the reporter gene consisting of the HBG1 promoter (P) driving GFP expression and the enhancer (E) comprising of four of the DNase I hypersensitivity sites (HS1–4) of the human β-globin locus control region (µLCR)41. b, Genomic context of integrated transgene in K562 cells: Knight-Ruiz (KR) normalized HiC contact map at 5-Kb resolution taken from ref. 16, with the different enhancer integration sites (E) indicated. For reference, yellow triangles demarcate the ‘left boundary’ (see text) and right domain boundary. Tracks below show: ChIP–seq tracks61 for SMC3; CTCF binding sites (CBS), forwardly and reversely orientated CBS in red and blue triangles, respectively; H3K27ac; H3K27me3; RNA-seq; and Ref-seq genes. Genomic positions on chromosome 18 are in Mb. c, GFP fluorescence intensity in arbitrary units (FI) of multi-clonal (bulk) cell populations carrying no enhancer (no E) or the µLCR enhancer at 0 kb, 11 kb, 47 kb or 100 kb (E0, E11, E47, E100, respectively) upstream of the GFP-reporter, without hemin (green) or with hemin (purple) in the culture medium. d, Percentage of remaining GFP-positive cells in multi-clonal (bulk) cell populations carrying the enhancer at the indicated distances, after long-term cell culturing. At each time point, cells were first treated with hemin for 2 days prior to FACS analysis. n = 1 replicate per time point per clone. e, Same as in c, but for selected clonal cell lines carrying the µLCR enhancer at indicated distances. f, GFP fluorescence intensity of an E407 cell line, after deleting intervening sequences to have the µLCR enhancer placed at 0 kb from the reporter gene. g. Same as in d, but for selected E-lines carrying the µLCR enhancer at indicated distances. n = 1 replicate per time point per clone. h–j, Median reporter gene expression (GFP fluorescence) plotted against linear E-P distance (h), and mean 4C-seq-measured E-P contacts (mean normalized (per 1 million cis-reads) 4C-seq signal at enhancer) plotted against E-P linear distance (i) or against median GFP fluorescence (j). Cells were untreated (circles), treated with hemin (squares) or long-term silenced (triangles). n = at least 2 technical replicates/clone.
