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. 2020 May 12;9:e53885. doi: 10.7554/eLife.53885

Figure 3. Condensin extrudes DNA loops in metaphase and cohesin extrudes loops in interphase.

(A) DNA loop extrusion probability—the frequency at which looping occurs on a DNA strand with sufficient slack—in metaphase and interphase under different depletion conditions. In metaphase, co-depleting condensin I, condensin II, and H3-H4 (using anti-XCAP-C/E and anti-H4K12Ac) significantly (** represents p<0.01, Binomial test) reduced loop extrusion probability, whereas the same depletion condition in interphase had no effect on loop extrusion probability compared to the control H3-H4-depleted extract. However, co-depleting cohesin and H3-H4 (using anti-XRAD21/XSMC1 and anti-H4K12Ac) had no effect in metaphase, though significantly (p<0.01) decreased loop extrusion probability in interphase compared to H3-H4-depleted extract. (B) Snapshots of antibody stainings of representative loops in metaphase and interphase. (Top) In metaphase, Alexa488-labeled anti-XRad21 bound to cohesin does not localize to the DNA loop, whereas in interphase (right panels), the anti-XRad21 co-localizes to the loop. (Bottom) Alexa488-labeled anti-XCAP-C bound to condensin localizes to the DNA loop in metaphase, but does not localize to the loop in interphase.

Figure 3.

Figure 3—figure supplement 1. Co-immunodepletions of Xenopus egg extracts using antibodies targeting H3-H4, cohesin, and condensin I and II.

Figure 3—figure supplement 1.

(A) Co-immunodepletion of H3-H4 using anti-H4K12Ac antibodies and cohesin using anti-XRAD21 and anti-XSMC1 antibodies. H3 protein levels are detected using anti-H3 and exhibits a ~ 90–95% depletion of soluble H3-H4 heterodimers compared to IgG-depleted extract. XSMC1 protein levels are detected using anti-XSMC1, which exhibits a ~ 90% depletion. We used anti-DM1a that detects tubulin as a loading control. (B) Co-immunodepletion of H3-H4 using anti-H4K12Ac antibodies and condensin using anti-XCAP-C and anti-XCAP-E antibodies. H3 protein levels are detected using anti-H3 and exhibits a ~ 90% depletion of soluble H3-H4 heterodimers compared to IgG-depleted extract. XCAP-C protein levels are detected using anti-XCAP-C, which displays a ~ 85% reduction, although the signal is rather weak and made it challenging to quantify. We used anti-DM1a that detects tubulin as a loading control. (C) Enzymatic depletion of ATP by apyrase (left) in H3H4-depleted interphase extract greatly reduces loop formation probability compared to control H3H4 depleted interphase extract (right).