Antibody development by using monoubiquitinated peptides as antigens.

24 April 2019

Antibody development by using monoubiquitinated peptides as antigens

Conserved from yeast to human, the histone methyltransferase Dot1 methylates lysine 79 of histone H3 (H3K79) on the nucleosome core. This methylation affects gene expression and DNA damage response, and is enhanced by monoubiquitination of the C-terminus of histone H2B [1 – 4]. Given the promising clinical development of Dot1 inhibitors (e.g. for the treatment of acute leukemia in children [5]), it is important to elucidate the regulatory mechanisms of Dot1 [1 – 4].

A recent paper in Nucleic Acid Research [6], describes a collaborative effort between scientists at the Netherlands Cancer Institute, Leiden University Medical Center, University Medical Center Utrecht, University of California and UbiQ, with the aim to gain more insight into the functions of Dot1 and its crosstalk with H2B. In order to directly demonstrate the site of ubiquitination on H2B, a monoclonal antibody was raised against yeast  lysine123 monoubiquitinated H2B [6 – 8] by using a synthetic lysine123 monoubiquitinated yeast H2B(115-130) peptide as antigen [9]. 

By using genetic interaction maps of increased-dosage alleles of Dot1, a functional relationship was identified between increased Dot1 dosage and loss of the deubiquitinating module of the SAGA co-activator complex, a deubiquitinating enzyme of monoubiquitinated H2B and as such negative regulator of H3K79 methylation (Figure 1). Increased Dot1 dosage was found to promote monoubiquitination of H2B in a dose-dependent manner and this was exacerbated by the loss of SAGA-deubiquitinating activity. Importantly, the stimulatory effect on H2B ubiquitination was mediated by the N-terminus of Dot1, independent of its methyltransferase activity. Thus, Dot1 and monoubiquitinated H2B are subject to bi-directional crosstalk and Dot1 has chromatin regulatory functions that are independent of its methyltransferase activity.
Figure 1. Model for the mutual crosstalk between Dot1 and monoubiquitinated H2B [6]. 
[1] Worden et al. Cell 2019176, 1490.
[2] Jang et al. Genes Dev 2019, doi: 10.1101/gad.323790.118.
[3] Valencia-Sánchez et al. Mol Cell 2019, doi: 10.1016/j.molcel.2019.03.029.
[4] Anderson et al. Cell Rep 2019, doi: 10.1016/j.celrep.2019.01.058.
[5] Shukla et al. Blood 2016, 128, 2780.
[6] Van Welsem et al. Nucl Acid Res 201846, 11251.
[7] Vlaming et al. eLife 20165, e18919.

If you are interested in the antibody, please see the link in reference 8. If you want to know more about our monoubiquitinated peptides (for antibody development), please click on the UbiQPEP button or contact us.
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