Biochemical characterization of the Chp1 chromodomain binding to the nucleosome core and its role in heterochromatin formation

Eukaryotic genomes are organized inside the cell nucleus in a
structured macromolecular DNA-protein polymer named chromatin,
formed by single discrete unites called Nucleosomes. The packing of
the genetic information into chromatin allows the efficient
regulation of several nuclear processes, such as gene expression
and transcription, DNA replication, cell cycle progression,
chromosome segregation and DNA damage repair. Chromatin comes in
two flavors: a transcriptionally active, more loosened state,
called euchromatin and a transcriptionally silent or low expressed,
more compact state, called heterochromatin. The assembly of silent
chromatin or heterochromatin is fundamental for the regulation of
every nuclear process and it is driven in most Eukaryotes by the
deposition and the read-out of the histone H3 lysine 9 methylation
(H3K9me) post-translational modification (PTM). H3K9me on the
nucleosome is specifically bound by chromatin readers called
chromodomains (CD) and this recognition is fundamental for the
downstream processes that lead to the formation of heterochromatin
and shut down the expression of single genes or entire gene
clusters. Despite several studies have been done on different
chromodomains binding to H3K9me histone tail peptides, to date
there was no structural information on how chromodomains interact
with their natural binding partners, the H3K9me3 Nucleosomes. In a
preliminary structural study carried out in our laboratory we
solved the cryo-electron microscopy (Cryo-EM) structure of the
chromodomain of the fission yeast Chp1 protein (Chp1CD) in complex
with an H3K9me nucleosome. The structure showed that the Chp1CD
interacts not only with the histone H3 tail but also with the
histone globular domains in the Nucleosome core, primarily with
histone H3. Mutations in the residues of Chp1CD that form the
binding interface with the Nucleosome core (two loops in the
β-sheet of the domain) caused a drop of the affinity in vitro for
the H3K9me Nucleosome, which was independent from the histone
H3K9me tail interaction. Cells harboring the same Chp1CD loop
mutations were defective in silencing centromeric transcripts and
maintain the deposition of the H3K9me mark for heterochromatin
formation. This indicated that Chp1CD-nucleosome core interaction
is fundamental for heterochromatin formation in fission yeast and
opened up to the possibility that chromodomains could read multiple
histone PTMs, on both the recruiting histone tail and on the
nucleosome core. This study substantially contributes to understand
how chromodomains interact with chromatin, how much the nucleosome
core interaction is conserved among different CDs and how different
chromodomain proteins are regulated at the same loci. Understanding
how chromodomain readers recognize nucleosomes is fundamental to
uncover the basics of gene silencing and heterochromatin formation.