Histone H3.3 phosphorylation facilitates H3K9me3-heterochromatin formation during retrotransposon silencing and X-chromosome inactivation via H3.3K27me3–CBX7–KAP1 axis

The conserved histone variant H3.3 plays pivotal roles in heterochromatin formation and retrotransposon silencing. However, the molecular mechanism underlying H3.3-primed heterochromatin regulation remains elusive. Here, we demonstrate that H3.3-specific Ser31 phosphorylation and Lys27 trimethylation synergistically promote H3K9me3-heterochromatin formation. Mechanistically, polycomb protein chromobox homolog 7 (CBX7) preferentially binds Ser31-phosphorylated H3.3K27me3 nucleosomes and then recruits KRAB-associated protein 1 (KAP1), which may further engage the histone lysine 9 methyltransferase to establish H3K9me3-associated heterochromatin. Remarkably, H3K9me3 is significantly impaired when the H3.3-CBX7 interaction is disrupted, accompanied by the activation of retrotransposons. Moreover, during X-chromosome inactivation (XCI), H3K9me2/3 fails to accumulate at the inactive X (Xi) when blocking the H3.3-CBX7-KAP1 axis. Taken together, our results reveal a novel molecular mechanism by which H3.3 Ser31 phosphorylation (H3.3Ser31p) facilitates H3K9me3-heterochromatin formation during retrotransposon silencing and XCI via the H3.3K27me3-CBX7-KAP1 axis.