H3.3 Ser31 phosphorylation consolidates heterochromatin for retrotransposon silencing and X chromosome inactivation (PRJCA023903)

Heterochromatin is crucial for silencing repetitive elements and restraining gene expression during development. Dysfunctions of heterochromatic state closely associate with the pathogenesis of various human diseases. Increasing evidences show that the histone variant H3.3 plays important roles in heterochromatin formation and retrotransposon silencing. However, the molecular mechanism underlying H3.3-primed heterochromatin formation remains elusive. Here, we demonstrate that the phosphorylation of H3.3 unique residue Ser31, which is absent from canonical histones H3.1/H3.2, is essential to the instatement of H3K9me3-associated heterochromatin during both retrotransposon silencing and X chromosome inactivation. Mechanistically, Ser31-phosphorylated H3.3 nucleosomes facilitate H3.3K27me3 recognition by the Polycomb protein CBX2/7, which further recruits KAP1-SetDB1, enabling the formation of H3K9me3-associated heterochromatin across the genome. Expression of the nonphosphorylatable H3.3 (H3.3S31A) or the CBX7 mutant that is defective in H3.3 pSer31 recognition (CBX7R22A) leads to global reduction of H3K9me3, along with the derepression of multiple types of retrotransposons. Importantly, these mutants also lead to defective assembly of H3K9me2/3 heterochromatin at the inactivated X chromosome in differentiated female mouse cells, implying the significance of H3.3 Ser31 phosphorylation in the transition from facultative to constitutive heterochromatin during X chromosome inactivation. Taken together, our results reveal a mechanism of heterochromatin consolidation regulated by the histone variant H3.3 and its phosphorylation