Voigt, Philipp

Bird, Adrian

Bryan, Elana Faye Hadassah

Wellcome Trust

2020-02-03T09:58:44Z

2020-02-03T09:58:44Z

2020-07-06

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.

https://hdl.handle.net/1842/36725

http://dx.doi.org/10.7488/era/32

en

The University of Edinburgh

bivalent domains

H3K4me3

H3K27me3

mouse embryonic stem cells

KAT6B binding

Proteomic analysis of histone mark crosstalk at bivalent domains

Thesis or Dissertation

Doctoral

PhD Doctor of Philosophy