Histone H3.3, a histone H3 variant, is evolutionarily conserved across species. H3.3 is loaded into chromatin by its chaperone ATRX (α-Thalassemia X-Linked Mental Retardation) and DAXX (Death Domain Associated Protein) at DNA repeats including the telomeres and pericentric DNA to form a compacted and transcriptionally silenced heterochromatin. In mammalian cells, loss of H3.3 or ATRX fails to form heterochromatin, accompanied by increased transcriptional activities at these repeats.
Unique to H3.3 is a serine residue at position 31 which is not found on the canonical histone H3. This serine residue is phosphorylated (H3.3S31ph) at the telomeres and pericentric region during mitosis. Given the role of H3.3 in telomeric repeat silencing, we hypothesise that H3.3S31ph is a histone mark associated with heterochromatin silencing and is essential for maintaining telomeric chromatin integrity. To determine this, we generated two endogenous H3.3-null H3.3S31 mutant embryonic stem cell lines expressing either an S31 phospho-null (Serine to Alanine, H3.3S31A) or a phospho-mimic (Serine to Glutamic acid, H3.3S31E) mutant. Consistent with our hypotheses, we find that the H3.3S31A mutation results in reduced levels of ATRX and H3K9me3 and aberrant transcription at the telomeres, while the H3.3S31E mutation leads to gains in ATRX and H3K9me3 levels, indicating a loss in heterochromatin silencing. In H3.3S31A mutant cells, we also detect an increased binding of KDM4B, a histone H3K9 and H3K36 demethylase at the telomeres, and RNAi knockdown experiments show that depletion of KDM4B expression restores levels of ATRX and H3K9me3 levels at the telomeres. Based on these findings, we propose a model of which H3.3S31ph regulates KDM4B binding and that it plays a key role in heterochromatin silencing at the telomere. We also propose H3.3S31ph as a vital component of a heterochromatin silencing pathway at other DNA repeats across the genome.