Uncovering the role of novel histone modifications in transcription and their mechanistic impact on chromatin function – CoreAc

The main methods used for this project are :
ChIP, biochemstry, complex purifications, chromatin reconsitutions, in vitro transcription

We want to demonstrate that small chemical modifications of the histone proteins can indeed regulate the expression of genes, or if they are merely bystanders. For this we focused on a modification called acetylation within the copact core of the nucleosome the structure compacts DNA. We showed that these modifications can ideed directly regulate gene expression and can hence be important in all cellular processes.

Our work will be the first systematic functional characterization of novel modifications within the core of the nucleosome and will therefore address key questions in biology. We will study these new lateral surface acetylations comprehensively by a unique combination of in vivo and in vitro approaches that will allow us to understand for the first time their function and the mechanism(s) how they regulate transcription and hence all cellular processes.

During the first 18mont the 2 partners published 12 peer reviewed international publications and 1 book chapter. Furthermore results from this project were presented at 8 international meetings

Histone proteins form the building blocks of eukaryotic chromatin. Covalent modifications of histones can regulate all DNA dependent processes. Multiple studies have highlighted histone N-terminal modifications as key regulators of chromatin function and the current model is that tail modifications act via the recruitment of specific binders that then regulate many different processes, such as transcription. However, fundamental questions are still unanswered: to date (i) the set of identified histone modifications is still incomplete and very little is known about modifications in the core of the nucleosome (ii) so far functional outcomes have not been directly attributable to a particular modification per se, but rather to the downstream recruitment of proteins with specialized binding domains (and their associated partner) and (iii) we still do not know whether histone modifications actually regulate transcription directly (i.e. have a causative role rather than being mere by-products of e.g. transcription).

We want to address these questions. For this we are in particular interested in novel lysine acetylations. that map to regions in close proximity to the DNA, the so-called lateral surface of the histone octamer. Most of the modified residues on this lateral surface make direct or indirect contacts with the DNA. Our working model is that acetylation of these lysines neutralizes the charge of the negatively charged side chain and has therefore the potential to alter DNA-histone interaction. This could in turn regulate nucleosomal mobility/dynamics and hence gene expression directly.

Our aim is to systematically and comprehensively study acetylation of lysines on the lateral surface of the histone octamer. Our preliminary mass spectrometry results identified several additional, previously undescribed acetylation sites on the lateral surface. These sites are: histone H3 lysine 64 (H3K64), H3 lysine 115 (H3K115) and H3 lysine 122 (H3K122).

With the support of ANR we want to unravel the biological function of these acetylations at the lateral surface of the histone octamer. We will a) systematically study the distribution and dynamics of H3K64, H3K115, H3K122 acetylation in vivo b) investigate their regulation by the identification of the modifying enzymes, c) study the mechanism how these modifications regulate chromatin dynamics and impact on transcription and d) determine the in vivo relevance of these modification. Together these tasks will allow us to understand the function of lateral surface acetylations and thus, it will constitute a significant advance in our understanding of how histone modifications can (directly) impact on important biological processes. Furthermore we will link specific acetylation sites with a defined process(es) (i.e. transcription) and will demonstrate the underlying mechanism(s).

This project combines the expertise of the Schneider lab in the functional characterisation of novel histone modifications and chromatin biochemistry with the expertise of the Tora lab in histone acetylation and characterization of the corresponding acetylating enzymes and complexes. Only the combined and complementary expertise will allow us to pursue this research program.

Overall the aim of project is (i) to functionally characterise novel histone modifications, to (ii) significantly advance our understanding of how histone acetylations can regulate transcription, (iii) to try to demonstrate that histone modifications can regulate DNA-dependent processes beyond the recruitment of specific binders, as the current model for tail modifications dictates, and (iv) to try to show that acetylation of histone lysines can be causative, rather than merely consequential, with respect to regulating DNA dependent processes, such as transcription.