SUMOylated PRC1 controls histone H3.3 deposition and genome integrity of embryonic heterochromatin (2020)
Figure 6

Figure 6. DAXX and PRC1 control nucleosome density and chromatin stability at pat-PCH and chromosome segregation in early mouse embryos.

(A) Developmental progression of Daxxm+z+, Daxxm-z+ and Daxxm-z- early embryos.

(B) Examples of morula and blastocyst embryos with normal and large polyploid nuclei.

(C) Percentages of embryos with normal or polyploid nuclei.

(D) Still images of time-lapse imaging of the first cleavage division in wild-type and maternally deficient zygotes expressing H2B-mCherry. Time points represent minutes after prometaphase. Lagging chromosomes and micronuclei are indicated by yellow arrowheads.

(E) Percentage of zygotes with lagging chromosomes during 1st cleavage division. Data represent mean ± s.e.m. in n≥3 replicate experiments.

(F) IF of 5-methyl and 5-hydroxy-methyl cytosine staining of wild-type (n=42), Daxxm-z+ (n=14) and Ring1m-z+; Rnf2m-z+ (n=6) zygotes with lagging chromosomes. Yellow arrowheads indicate lagging chromosomes.

(G) DNA-FISH for major (green) and minor (red) satellites on cleavage chromosomes of a Daxxm-z+ zygote with a chromosome broken within the major satellite region.

(H) H3.3-EGFP live-imaging signal in pat- and mat-PCH of control Daxxm+z+ (n=18), Daxxm-z+ (n=8) and Ring1m-z+; Rnf2m-z+ (n=12) zygotes. Scale bar 10µm.

(I) Left: IF of endogenous H3 and ATRX at pat-PCH in control and Daxxm-z+ PN5 zygotes. Right: Fluorescence intensity profiles of H3 and ATRX along white line in left panel at pat-PCH of zygotes. Scale bars 5 µm.

(J) Boxplot displaying H3 signal intensity at pat-PCH of Daxxm+z+ and Daxxm-z+ PN5 zygotes. In boxplots, the enter lines show the medians; box limits indicate the 25th and 75th percentiles as determined by R software; whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles. Datapoints are indicated by circles. Experiments were replicated 2 times. ***P<0.001, t-test, n=12 for both groups.

(K) IP of DAXX-EGFP and DAXXR244A-EGFP proteins with an anti-GFP antibody from nuclear extract of HEK293 cells. Immuno-blot detection was performed with antibodies recognizing GFP, histone variant H3.3 and CBX2.

(L) Percentage of Daxxm-z+ zygotes with lagging chromosomes following injection of Tubulin-EGFP, Daxx-EGFP or DaxxR244A-EGFP mRNAs (20 or 100 ng/µl) at PN2/3 stage. Data represent mean ± s.e.m. (n ≥2 injection experiments). *** P<0.001 High Daxx-EGFP versus other 3 groups; Fisher's exact test (on pooled embryos).

(M) Left: IF of EGFP and endogenous H3 at pat-PCH in Daxxm-z+ PN5 zygotes expressing DAXX-EGFP or DAXXR244A-EGFP or nothing exogenously. Right: Fluorescence intensity profiles of H3 and EGFP along white lines in left panels at pat-PCH of zygotes. Scale bars 5 µm.

(N) Boxplot displaying 3D quantification of H3 signal intensity at pat-PCH in Daxxm-z+ zygotes expressing exogenous DAXX-EGFP (n=14) or DAXXR244A-EGFP (n=13). In boxplots, the enter lines show the medians; box limits indicate the 25th and 75th percentiles as determined by R software; whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles. Datapoints are indicated by circles. Experiments were replicated 2-3 times. ***P<0.001 Daxx-EGFP versus other 2 groups; t-test.

Data information: All scale bars 10µm.