elucidate the mechanism of its activity [223, 224]. One long-standing experimental
observation is that PRC2 activity is markedly higher with native nucleosomes
substrates, naturally containing many chemical modifications, than with unmodified
recombinant nucleosomes [217]. This implies a role for the pre-existing marks in
regulating PRC2 activity, and this model was refined further when it was observed
that the complex binds to the H3K27me3 mark in vivo [225]. Thus, pre-existing
H3K27me3 marks recruit PRC2 to newly synthesized DNA, and this, in turn,
facilitates the H3K27 methylation of the nascent histones. The determination of
the EED β-propeller structure in complex with histone peptides has provided
an explanation for the role of pre-existing modifications in the recruitment and
activation of PRC2 [216].
An initial EED structure, in complex with a helical peptide derived from the
amino terminus of EZH2, confirmed that it is a seven-bladed β-propeller, which
has a helical insertion of currently unknown function into blade 3 [226]. Later,
EED was crystallized in the absence of binding partners, however, serendipitously
the conditions needed to produce well-diffracting crystals of the apo-form
contained 3-(ethyldimethylammonio)propane-1-sulfonate (NDSB-195), a nondetergent sulfobetaine additive [216]. Fluorescence competition assays were used
to assess the relative affinity of EED for histone peptides carrying epigenetically
relevant trimethyl marks. These experiments confirmed that EED does bind to
histone tails, but in addition, they revealed that the binding was restricted to
those associated with repressive chromatin – H1K26, H3K9, H3K27, and H4K20.
Conversely, peptides representing marks associated with active chromatin such
as H3K4, H3K36, and H3K79 did not bind. The authors of this study went on
to determine a series of crystal structures of EED bound to the four repressive
trimethylated peptides [216]. The four trimethyl peptides bound to EED
β-propeller, in fluorescence and isothermal calorimetry measurements, with dissociation constants in the range 10 μM for H3K36me3 to 45 μM for H3K27me3.
H3K27me2 peptide had a K D of only 114 μM, and H3K27me1 had a K D of only
434 μM, showing that recognition of H3K27 by EED is methylation state specific.
The structure also revealed the mechanism that allows EED to select for only histone
marks associated with repressive chromatin domains.
5.3 Small Molecules Targeting WD40 Domains
As mentioned above, in addition to being associated with disease gene networks,
many WDR domain-containing proteins are target candidates for therapy in cancer,
metabolic disorders, neurological diseases, and regenerative medicine. Although
these putative targets remain to be fully validated, the WDR domain is potentially
a common yet unexploited entry point for drug discovery in many disease areas.
Indeed, several compounds have been recently identified as inhibitors of WD40
domain-containing protein complexes.
Methyl-Readers and Inhibitors
375
observation is that PRC2 activity is markedly higher with native nucleosomes
substrates, naturally containing many chemical modifications, than with unmodified
recombinant nucleosomes [217]. This implies a role for the pre-existing marks in
regulating PRC2 activity, and this model was refined further when it was observed
that the complex binds to the H3K27me3 mark in vivo [225]. Thus, pre-existing
H3K27me3 marks recruit PRC2 to newly synthesized DNA, and this, in turn,
facilitates the H3K27 methylation of the nascent histones. The determination of
the EED β-propeller structure in complex with histone peptides has provided
an explanation for the role of pre-existing modifications in the recruitment and
activation of PRC2 [216].
An initial EED structure, in complex with a helical peptide derived from the
amino terminus of EZH2, confirmed that it is a seven-bladed β-propeller, which
has a helical insertion of currently unknown function into blade 3 [226]. Later,
EED was crystallized in the absence of binding partners, however, serendipitously
the conditions needed to produce well-diffracting crystals of the apo-form
contained 3-(ethyldimethylammonio)propane-1-sulfonate (NDSB-195), a nondetergent sulfobetaine additive [216]. Fluorescence competition assays were used
to assess the relative affinity of EED for histone peptides carrying epigenetically
relevant trimethyl marks. These experiments confirmed that EED does bind to
histone tails, but in addition, they revealed that the binding was restricted to
those associated with repressive chromatin – H1K26, H3K9, H3K27, and H4K20.
Conversely, peptides representing marks associated with active chromatin such
as H3K4, H3K36, and H3K79 did not bind. The authors of this study went on
to determine a series of crystal structures of EED bound to the four repressive
trimethylated peptides [216]. The four trimethyl peptides bound to EED
β-propeller, in fluorescence and isothermal calorimetry measurements, with dissociation constants in the range 10 μM for H3K36me3 to 45 μM for H3K27me3.
H3K27me2 peptide had a K D of only 114 μM, and H3K27me1 had a K D of only
434 μM, showing that recognition of H3K27 by EED is methylation state specific.
The structure also revealed the mechanism that allows EED to select for only histone
marks associated with repressive chromatin domains.
5.3 Small Molecules Targeting WD40 Domains
As mentioned above, in addition to being associated with disease gene networks,
many WDR domain-containing proteins are target candidates for therapy in cancer,
metabolic disorders, neurological diseases, and regenerative medicine. Although
these putative targets remain to be fully validated, the WDR domain is potentially
a common yet unexploited entry point for drug discovery in many disease areas.
Indeed, several compounds have been recently identified as inhibitors of WD40
domain-containing protein complexes.
Methyl-Readers and Inhibitors
375