Fig. 3: Activated CTCF sites that reduce domain size support enhancer-dependent expression levels and stability. | Nature Structural & Molecular Biology

Fig. 3: Activated CTCF sites that reduce domain size support enhancer-dependent expression levels and stability.

From: Building regulatory landscapes reveals that an enhancer can recruit cohesin to create contact domains, engage CTCF sites and activate distant genes

Fig. 3

a, GFP fluorescent intensity (FI) of multi-clonal (bulk) cell lines carrying the enhancer without (E, in green) and with (EC, in orange) the co-integrated 3×-hCTCF cassette, cultured without (left) or with hemin (right). Yellow triangles symbolize 3×-hCTCF. b, Percentage of remaining GFP-positive cells in multi-clonal (bulk) cell populations carrying the enhancer without (solid line) or with flanking 3×-hCTCF (dashed line) at the indicated distances. At each time point, cells were first treated with hemin for 2 days prior to FACS analysis. c, 4C-seq contact profile overlays, comparing contacts of the integrated gene promoter (P) and µLCR enhancer (E) in the E0 versus EC0 cell line (left), and in the E100 versus EC100 cell line (right), highlighting in orange their gained contacts and in green their lost contacts due to the presence of the 3×-hCTCF binding sites (CBS). y axis: 4C coverage normalized per 1 million cis-reads. n = 2 technical replicates/clone. VP: 4C-seq viewpoint. Also shown at the top right is the overlay of their FACS profiles, demonstrating that 3×-hCTCF at position 0 mildly supports enhancer-mediated GFP expression. n = 1 replicate per time point per clone. d, 4C-seq contact profile overlays comparing contacts of the integrated gene promoter (P, top) and 3×-hCTCF (CBS, bottom) in the EC0 versus C0 cell line (left) and the EC100 versus C100 cell line (right), with contacts gained and lost due to the enhancer shown in orange and yellow, respectively. n = 2 technical replicates/clone. Top right plots show the overlay of their FACS profiles, demonstrating that both in C0 and in C100, the 3×-hCTCF cassette itself does not activate GFP expression. e, 4C-seq contact profile overlays comparing contacts of the integrated gene promoter (P, top) and enhancer (E, bottom) in the C-0E100 versus E100 cell line, with contacts gained and lost due to the presence of 3×-hCTCF sites shown in orange and green, respectively. n = 2 technical replicates/clone. Top right plots show the overlay of their FACS profiles, demonstrating that the downstream 3×-hCTCF cassette only mildly supports enhancer-mediated GFP expression.

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