Figure 4: RNA helicases DDX5 and DDX17 stabilize long-term chromatin loops. | Nature Communications

Figure 4: RNA helicases DDX5 and DDX17 stabilize long-term chromatin loops.

From: Manipulation of nuclear architecture through CRISPR-mediated chromosomal looping

Figure 4

(a) MS/ChIP of dimerized CLOuD9 cells after 72 h and 10 day treatments demonstrate differential enrichment of novel proteins at the induced looping loci after 10 days. (b) Immunoprecipitation of CLOuD9 complexes demonstrates that CTCF and cohesin were not found to be localized to the induced loops. (c) shRNA knockdown of DDX5 and DDX17 in K562s containing CLOuD9 constructs. (d,e) 3C demonstrates that while DDX5 and DDX17 knockdown do not affect induction of β-globin-LCR contacts, the induced chromatin loops are no longer stabilized following 10 days of dimerization and subsequent washout. 3C values were normalized to tubulin, and interaction frequencies between the anchor fragment and the fragment encompassing the β/HS fragment were set to zero. Error bars indicate s.d. n=3. (f) Endogenous β-globin expression is restored following short- and long-term dimerization and subsequent ligand washout in DDX5 and DDX17 knockdown cells. Significance given relative to control treated cells. Two-tailed student’s t-tests *P<0.05, t=2.538, df=6; **P<0.001, t=3.791, df=6; ***P<0.0001, shDDX5 t=12.6, df=6, shDDX17 t=19.35, df=6; n.s. non-significant. All error bars indicate s.d.

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