Heterochromatin silencing is critical for genomic integrity
and cell survival. It is orchestrated by chromodomain
(CD)-containing proteins that bind to
methylated histone H3 lysine 9 (H3K9me), a hallmark
of heterochromatin. Here, we show that phosphorylation
of tyrosine 41 (H3Y41p)––a novel histone
H3 modification––participates in the regulation
of heterochromatin in fission yeast. We show that a
loss-of-function mutant of H3Y41 can suppress heterochromatin
de-silencing in the centromere and subtelomere
repeat regions, suggesting a de-silencing
role forH3Y41p on heterochromatin. Furthermore,we
show both in vitro and in vivo that H3Y41p differentially
regulates two CD-containing proteins without
the change in the level of H3K9 methylation: it promotes
the binding of Chp1 to histone H3 and the
exclusion of Swi6. H3Y41p is preferentially enriched
on centromeric heterochromatin during M- to early S
phase, which coincides with the localization switch
of Swi6/Chp1. The loss-of-function H3Y41 mutant
could suppress the hypersensitivity of the RNAi mutants
towards hydroxyurea (HU), which arrests replication
in S phase. Overall, we describe H3Y41p as
a novel histone modification that differentially regulates
heterochromatin silencing in fission yeast via
the binding of CD-containing proteins.