Elucidation of the interactions between the transcription factor E2A and the transcriptional co-activator CBP/p300

E2A is a transcription factor that plays a particularly critical role in lymphopoiesis. The chromosomal translocation 1;19, disrupts the E2A gene and results in the expression of the fusion oncoprotein E2A-PBX1, which is implicated in acute lymphoblastic leukemia. Both E2A and E2A-PBX1 contain two activation domains, AD1 and AD2, which comprise conserved ΦxxΦΦ motifs where Φ denotes a hydrophobic amino acid. These domains function to recruit transcriptional co-activators and repressors, including the histone acetyl transferase CREB binding protein (CBP) and its paralog p300. The PCET motif within E2A AD1 interacts with the KIX domain of CBP/p300, the disruption of which abrogates the transcriptional activation by E2A and the transformative properties of E2A-PBX1. The generation of a peptide-based inhibitor targeting the PCET:KIX interaction would serve useful in further assessing the role of E2A and E2A-PBX1 in lymphopoiesis and leukemogenesis. An interaction between E2A AD2 and the KIX domain has also been recently identified, and the TAZ domains of CBP/p300 have been shown to interact with several transcription factors that contain ΦxxΦΦ motifs. Thus the design of an inhibitor of the E2A:CBP/p300 interaction requires the full complement of interactions between E2A and the various domains of CBP/p300 to be elucidated. Here, we have used nuclear magnetic resonance (NMR) spectroscopy to determine that AD2 interacts with KIX at the same site as PCET, which indicates that the E2A:KIX interaction can be disrupted by targeting a single binding site. Using an iterative synthetic peptide microarray approach, a peptide with the sequence DKELQDLLDFSLQY was derived from PCET to interact with KIX with higher affinity than the wild type sequence. This peptide now serves as a lead molecule for further development as an inhibitor of the E2A:CBP/p300 interaction. Fluorescence anisotropy, peptide microarray technology, and isothermal titration calorimetry were employed to characterize interactions between both TAZ domains of CBP/p300 and the PCET motif and AD2 of E2A. Alanine substitution of residues within PCET demonstrated that the ΦxxΦΦ motif is a key mediator of these interactions, analogous to the PCET:KIX interaction. These findings now inform future work to establish possible physiological roles for the E2A:TAZ1 and E2A:TAZ2 interactions.

Between Principle and Practicality: A Dynamic Realist Examination of Independence in the Canadian Justice System

This work examines independence in the Canadian justice system using an approach adapted from new legal realist scholarship called ‘dynamic realism’. This approach proposes that issues in law must be considered in relation to their recursive and simultaneous development with historic, social and political events. Such events describe ‘law in action’ and more holistically demonstrate principles like independence, rule of law and access to justice. My dynamic realist analysis of independence in the justice system employs a range methodological tools and approaches from the social sciences, including: historical and historiographical study; public administrative; policy and institutional analysis; an empirical component; as well as constitutional, statutory interpretation and jurisprudential analysis. In my view, principles like independence represent aspirational ideals in law which can be better understood by examining how they manifest in legal culture and in the legal system. This examination focuses on the principle and practice of independence for both lawyers and judges in the justice system, but highlights the independence of the Bar. It considers the inter-relation between lawyer independence and the ongoing refinement of judicial independence in Canadian law. It also considers both independence of the Bar and the Judiciary in the context of the administration of justice, and practically illustrates the interaction between these principles through a case study of a specific aspect of the court system. This work also focuses on recent developments in the principle of Bar independence and its relation to an emerging school of professionalism scholarship in Canada. The work concludes by describing the principle of independence as both conditional and dynamic, but rooted in a unitary concept for both lawyers and judges. In short, independence can be defined as impartiality, neutrality and autonomy of legal decision-makers in the justice system to apply, protect and improve the law for what has become its primary normative purpose: facilitating access to justice. While both independence of the Bar and the Judiciary are required to support access to independent courts, some recent developments suggest the practical interactions between independence and access need to be the subject of further research, to better account for both the principles and the practicalities of the Canadian justice system.