Fig. 4: An immediately flanking CTCF site helps a distal gene to compete for a shared enhancer.

a, Schematic representation of the two dual reporter lines. The µLCR enhancer was integrated 50 kb downstream of the GFP reporter gene, while a dsRed reporter gene driven by the same HBG1 promoter was integrated 100 Kb upstream of the GFP reporter gene. To place the genes and enhancer in a smaller domain, the 400-kb region between the dsRed reporter gene and the right domain boundary was deleted. Plots show hemin-induced expression (fluorescent intensity, FI) of both genes measured by FACS in the large domain cell line (purple) and the small domain cell line (red), 3 days after sorting each line for double-positive (GFP⁺dsRed⁺) cells. Lower left panel highlights that, in the context of a small domain, more cells manage to keep both genes active, and expressed at higher levels. Middle panel shows GFP expression (FI) for both clones, and right panel shows dsRed expression (FI) for both clones, highlighting that particularly the expression of the distal dsRed reporter gene benefits from being part of a smaller domain. b, The small domain protects against gene silencing, particularly of the distal dsRed reporter gene. Percentage of remaining GFP-positive (green lines) and dsRed-positive cells (red lines) after sorting the ‘small domain’ cells (triangles) and the ‘large domain’ cells (squares) each for double positive (GFP⁺dsRed⁺) cells and culturing them for the indicated period. 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. c, Transgene silencing is not the consequence of enhancer inactivation. Percentages of double-positive (orange), GFP-positive (green), dsRed-positive (red) and double-negative (gray) cells at 0, 3, 10 or 17 days after double-positive sorting of the indicated clones, as measured by flow cytometry.