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. 2019 Jun 6;74(5):1020–1036.e8. doi: 10.1016/j.molcel.2019.03.024

Figure 4.

Figure 4

Pervasive PCGF3/5-Dependent H2AK119ub1 Is Dispensable for Classical Polycomb Target Gene Repression but Is Associated with Xist-Mediated Chromosome Silencing

(A) Western blots of PCGF proteins and RING1B in Pcgf3/5fl/fl ESCs before (UNT) and after OHT treatment. BRG1 is shown as a loading control.

(B) Western blot for H2AK119ub1 in Pcgf3/5fl/fl ESCs (UNT and OHT) together with quantification of H2AK119ub1 levels relative to histone H3. Error bars represent SEM (n = 3).

(C) A genomic snapshot of a typical Polycomb target gene illustrating H2AK119ub1 and RING1B cChIP-seq and cnRNA-seq in Pcgf3/5fl/fl ESCs (UNT and OHT).

(D) A chromosome density plot showing H2AK119ub1 cChIP-seq across chromosome 18 in Pcgf3/5fl/fl ESCs (UNT and OHT).

(E) A metaplot of H2AK119ub1 cChIP-seq at PRC1-bound sites in Pcgf3/5fl/fl ESCs (UNT and OHT).

(F) An MA plot of log2-fold changes in gene expression (cnRNA-seq) in Pcgf3/5fl/fl ESCs following OHT treatment. Significant gene expression changes (p-adj < 0.05 and >1.5-fold) are shown in red.

(G) A schematic of the Mus domesticus (129S1) × Mus castaneus F1 ESCs, in which a doxycycline (DOX)-inducible Xist transgene is integrated into one allele of chromosome 3. DOX treatment causes Xist-mediated silencing of the allele harboring the Xist integration site (XIS) (silenced allele, from Mus castaneus), but not the other allele (active, from Mus domesticus).

(H) qRT-PCR gene expression analysis for Xist transgene and two neighboring genes before (UNT) and after DOX treatment. Expression is normalized to b-actin and shown relative to the average expression in UNT cells. Error bars show SEM (n = 3).

(I) Chromosome density plots of H2AK119ub1 cChIP-seq across two alleles of chromosome 3 in the system described in (G). Below is a heatmap of corresponding log2-fold changes in gene expression upon addition of DOX. Bins containing no expressed genes are shown in white.

(J) Allele-specific metaplots of H2AK119ub1 cChIP-seq for the genes on chromosome 3 in the system described in (G).

See also Figure S4.