Role of Histone Glycosylation on Gene Expression: Functional Consequences of N-Acetylglycosamine Attachment to Histone H2B

• Applicant: Dr. Felix Wojcik
• Subject Area: Biochemistry ; General Genetics and Functional Genome Biology ; Biological and Biomimetic Chemistry ; Organic Molecular Chemistry - Synthesis and Characterisation
• Term: from 2015 to 2017
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 268261783

Changes in gene expression patterns are associated with most if not all biological processes, as well as a range of human pathologies such as cancer and diabetes. How eukaryotic gene transcription is regulated in time and space remains a central question in molecular cellular biology. Understanding this complex process at the molecular level is an incredibly daunting problem, but one that promises to yield fundamental insights into the inner workings of metazoan organisms and, potentially, new paradigms for the treatment of diseases. Chemical modification of the DNA template as well as the proteins (histones) that package nuclear DNA lies at the heart of many gene regulatory processes, often referred to as the epigenetic code. Of the manifold posttranslational modifications (PTMs) known to occur on histones, the focus of this proposal will be on histone glycosylation, which has been recently recognized as playing a regulatory role in gene expression. In order to study these environment-driven epigenetic changes and the underlying molecular mechanisms, we here propose a semi-synthetic approach towards obtaining natural and mimetic glycosylated histones for the preparation of a novel designer chromatin.Special focus will be placed on understanding the biochemical crosstalk between GlcNAcylated serine 112 on histone H2B (herein referred to as gH2B) and monoubiquitylation of K120 on the same histone (uH2B). Importantly, modulation of uH2B directly influences lysine methylation of histone H3 (residues K4 and K79), modifications that are intimately tied to active transcription. Their misregulation can be directly linked to diseases such as human leukemia. Recent studies have shown that uH2B (and by extensive H3 methylation) is regulated by gH2B, however the mechanistic details of this regulation are unclear. Using chemically defined designer chromatin containing gH2B, uH2B or both, we will systematically explore how these PTMs regulate the methyltransferases responsible for H3K4 and H3K79 methylation, Set1 and Dot1 respectively. We will also investigate the effect of glycosylation on nucleosomal stability and chromatin compaction, in this case using biophysical assays recently developed in the Muir lab. Finally, we will develop an in vivo labeling approach, based on protein trans-splicing, in order to study the effects of GlcNAcylation on chromatin in living cells. This innovative strategy will allow us to corroborate the previously obtained in vitro results using chromatin arrays with in vivo data, and also provides the unique opportunity to identify novel binding partners of glycosylated histones.

• DFG Programme: Research Fellowships
• International Connection: USA
• Host: Professor Tom W. Muir, Ph.D.