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. 2021 Jan 22;12:537. doi: 10.1038/s41467-020-20757-1

Fig. 2. Nanobody-mediated recruitment of endogenous chromatin regulators can silence gene expression and confer memory.

Fig. 2

a Construct for constitutive coexpression of H2B-mIFP and nanobody against CR (antiHP1 or antiDNMT1) fused with the rTetR DNA-binding protein (blue box, top) were expressed in HEK293T cells containing a TagRFP reporter (bottom). b Left: fluorescence distributions of the TagRFP reporter after 5 days of recruitment (dox treatment) in cells stably expressing the nanobody constructs or rTetR-KRAB were analyzed by flow cytometry to determine the percentage of cells with the reporter silenced (left of the red dotted line). Right: means of percentages cells silent were calculated from 3 replicates. Statistical analysis by two-tailed Tukey’s test (antiHP1 vs. antiDNMT1: ****p = 5.6e − 6; antiHP1 vs. antiDNMT1-antiHP1: ****p = 2.5e − 13; antiDNMT1 vs. antiDNMT1-antiHP1: ****p = 1.8e − 11; antiDNMT1-antiHP1 vs. KRAB: ****p = 1.7e − 7). c Targeted bisulfite sequencing of the reporter after 5 days of recruitment with antiDNMT1, DNMT1, and DNMT3B (+dox), compared to untreated cells expressing the same effectors (−dox). Dox-treated cells were sorted based on the silencing of the TagRFP reporter labeled as +dox ON and +dox OFF (see Supplementary Fig. 2a for representative gating). CpG positions are calculated relative to the start of the most upstream TetO site. Positive and negative controls for DNA methylation are shown in Supplementary Fig. 2b. d Experimental design for investigating epigenetic memory: rTetR-effectors were recruited to the reporter for 5 days (+dox) and then released (−dox). Memory was monitored after dox removal via flow cytometry throughout 30 days. e Silencing and memory dynamics data (right) for the experiment described in d with representative flow cytometry histogram for antiDNMT1 at day 0, 15, 30 after dox removal (left). The percentage of cells silenced was normalized to the no dox control to adjust for any background silencing (“Methods”). Means are from three replicates. SDs are plotted but are too small to show for most data points. Statistical analysis by two-tailed Tukey’s test at day 30 after dox removal (antiHP1 vs. antiDNMT1: **p = 0.0076; antiDNMT1 vs. KRAB: ***p = 0.00026; KRAB vs. antiDNMT1-antiHP1: *p = 0.043). Source data are available in the Source Data file.