Institute of Biological and Biomedical Sciences

Alan Thorne

Our current laboratory research

For further details and publication history please see my staff profile

Mapping Chromosomal Proteins

Core histone proteins have a critical structural role in organizing DNA into nucleosomes within the chromatin of all eukaryotic cells. Their biochemical modification e.g. by acetylation, methylation, phosphorylation and ubiquitination alongside histone sequence variants also has key functional roles and facilitates nuclear processes such as gene activation or repression.

Chromatin ImmunoPrecipitation (ChIP) assays are used to define ‘where’ these modifications lie in the genome for a given cell type. Specific antibodies that recognise and bind modified histones in chromatin fragments allow them to be precipitated and separated from the bulk chromatin.

Analysis of the sequence content of the DNA (by qPCR or next generation sequencing) reveals their genomic location allowing correlations with function to be made. For example experiments in this and other labs has established that H3 modified by mono-methylation at lysine 4 (H3K4Me­3) is a defining characteristic of gene enhancers.

Similar ChIP experiments using chromatin prepared from chicken embryo erythrocytes or cell lines representing different stages of differentiation of macrophages allow us to follow gene activation events and define ‘when’ the different modifications appear.

Mechanisms of regulation

Understanding how the cell ‘reads’ and interprets the various modified histones in gene regulation is of great interest. It is generally held that proteins/protein complexes bind particular modifications to effect downstream events.

Despite the identification of proteins that bind acetylated or methylated lysine residues identification of the proteins that read combinations of these modifications in a nucleosomal context has been slow. Our approach is to to use pull-down experiments using reconstituted nucleosomes carrying defined combinations of modified or variant histones as bait with nuclear protein extracts.

Use of stable heavy isotope labelling of amino acids in cell culture (SILAC) for extract preparation allows the isolation and identification of modification specific binding proteins by tryptic-MS analysis.