The combination and interaction of histone marks and DNA-associated proteins
are critical in the regulation of gene transcription. Individual histone marks
have been associated with different gene expression states - histone H3 lysine
4 trimethylation (H3K4me3) is associated with “open” chromatin and active
transcription while H3K27me3 is associated with “closed” chromatin and a
repressive transcriptional state. In certain cases, these marks have been shown to
co-localise at genomic loci, and on the same nucleosome. The co-localisation of
active H3K4me3 and repressive H3K27me3 marks at CpG promoters is a hallmark
of bivalent domains.
Bivalent domains have been implicated in priming developmental genes for timely
activation. However, the complex network of proteins that bind to these domains
to regulate and mediate their influence on transcription is unknown. This
study has developed tools to enable the characterisation of the protein networks
bound to bivalent domains and other specifically modified nucleosomes. In vitro
synthesised specifically modified nucleosomes were utilised in pulldown assays
with embryonic stem cell (ESC) nuclear extract to isolate the specific protein
binders for different combinations of histone marks. This assay was validated
by comparison of proteins bound to symmetrically modified nucleosomes with
previously identified protein binders.
A comparison of symmetrically and asymmetrically modified nucleosomes has
elucidated new binding preferences for known proteins. Analysis of proteins
bound to asymmetrically modified nucleosomes showed previously unknown
binding affinities and conformational preferences. TAF3, a known H3K4me3
mark binder, prefers to bind symmetrically rather than asymmetrically modified
nucleosomes, even when the same amount of the mark is present. Therefore, this
preference is not solely dependent on the amount of modification, but also due to
the conformation of the marks on the nucleosomes. We have identified multiple
key proteins that prefer binding to this specific mark (H3K4me3/K27me3)
conformation, including the acetyltransferase KAT6B. Work in mouse ESCs
confirmed KAT6B binding to bivalent domains and showed a pronounced
differentiation defect in KAT6B-/- cells due to mis-regulation of genes important
in development. Further characterisation of these proteins and their interactions
will help to clarify bivalent domain function
