The E-protein transcription factors guide immune cell differentiation, with E12 and E47 (hereafter called E2A) being essential for B-cell specification and maturation. E2A and the oncogenic chimera E2A-PBX1 contain three transactivation domains (ADs), with AD1 and AD2 having redundant, independent, and cooperative functions in a cell-dependent manner. AD1 and AD2 both mediate their functions by binding to the KIX domain of the histone acetyltransferase paralogues CREB-binding protein (CBP) and E1A-binding protein P300 (p300). This interaction is necessary for B-cell maturation and oncogenesis by E2A-PBX1 and occurs through conserved ΦXXΦΦ motifs (with Φ denoting a hydrophobic amino acid) in AD1 and AD2. However, disruption of this interaction via mutation of the KIX domain in CBP/p300 does not completely abrogate binding of E2A and E2A-PBX1. Here, we determined that E2A-AD1 and E2A-AD2 also interact with the TAZ2 domain of CBP/p300. Characterization of the TAZ2:E2A-AD1(1–37) complex indicated that E2A-AD1 adopts an α-helical structure and uses its ΦXXΦΦ motif to bind TAZ2. Whereas this region overlapped with the KIX recognition region, key KIX-interacting E2A-AD1 residues were exposed, suggesting that E2A-AD1 could simultaneously bind both the KIX and TAZ2 domains. However, we did not detect a ternary complex involving E2A-AD1, KIX, and TAZ2 and found that E2A containing both intact AD1 and AD2 is required to bind to CBP/p300. Our findings highlight the structural plasticity and promiscuity of E2A-AD1 and suggest that E2A binds both the TAZ2 and KIX domains of CBP/p300 through AD1 and AD2.

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The microphthalmia‐associated transcription factor (MITF) is a master regulator of development and differentiation within the melanocyte lineage. However, aberrant MITF activity can lead to multiple malignancies such as skin cancer, where it plays a key role in modulating the proliferation and invasiveness of melanoma. MITF is a basic helix‐loop‐helix leucine zipper transcription factor that binds to specific gene promoters via a central DNA‐binding domain. MITF also recruits transcriptional co‐activators, such as the histone acetyltransferase CREB‐binding protein and its homologue p300 (CBP/p300) through an N‐terminal acidic transactivation domain (TAD), however the details of these interactions are not yet fully understood. In order to gain insight into the mechanisms of gene regulation by MITF, we investigated the structure and functional interaction between MITF‐TAD and the transcription adapter putative zinc finger (TAZ2) domain of CBP/p300. In mammalian‐one‐hybrid assays MITF transcriptional activity was enhanced in the presence of co‐transfected CBP/p300 and abolished upon deletion of residues within the MITF‐TAD. Peptide microarrays indicated that no one residue of MITF is essential for TAZ2 binding, however, deletion of multiple residues in MITF‐TAD ablated its ability to bind TAZ2. NMR‐based chemical shift mapping experiments determined that MITF‐TAD interacts with the same surface of TAZ2 as the adenoviral protein E1A, which has been shown to inhibit MITF function. We determined that E1A and MITF‐TAD directly compete for CBP/p300 through the TAZ2 domain using NMR‐based titrations, pulldown, and mammalian‐hybrid assays. Furthermore, we used qPCR to measure the effect of disrupting these interactions on the transcription of MITF‐specific target genes in melanoma‐derived cell lines. Understanding mechanistic details regarding the interaction between MITF and its co‐activators is fundamental to our understanding of gene regulation by MITF and may outline a potential new strategy to inhibit MITF function. Support or Funding Information This research was funded by the Beatrice Hunter Cancer Research Institute, Nova Scotia Heath Research Foundation Scotia Scholars Award, a Canadian Institutes of Health Research studentship, Dalhousie Nova Scotia Graduate Scholarship, and Izaak Walton Killam Predoctoral Scholarship.

The microphthalmia-associated transcription factor (MITF) is a master regulator of the melanocyte cell lineage. Aberrant MITF activity can lead to multiple malignancies including skin cancer, where it modulates the proliferation and invasiveness of melanoma. MITF-dependent gene expression requires recruitment of transcriptional co-activators such as CBP/p300, but details of this process are not fully defined. Here, we investigate the structural and functional interaction between the MITF N-terminal transactivation domain (MITFTAD) and CBP/p300. A combination of pulldown assays and nuclear magnetic resonance spectroscopy determined that MITF binds both TAZ1 and TAZ2 domains of CBP/p300 with high affinity. The solution-state structure of the MITFTAD:TAZ2 complex reveals that MITF interacts with a hydrophobic surface of TAZ2, while remaining relatively dynamic. Peptide array and mutagenesis experiments determined that an acidic motif is integral to the MITFTAD:TAZ2 interaction and is necessary for transcriptional activity of MITF. Peptides that bind to the same surface of TAZ2 as MITFTAD, such as the adenoviral protein E1A, are capable of displacing MITF from TAZ2 and inhibiting transactivation. These results provide mechanistic insight into co-activator recruitment by MITF that are fundamental to our understanding of MITF targeted gene regulation and melanoma biology.

The transcriptional co-regulator β-catenin is a critical effector of the canonical Wnt-signalling pathway, which plays a crucial role in regulating cell fate and maintaining tissue homeostasis. Deregulation of the Wnt/β-catenin pathway is characteristic in the development of major types of cancer, where accumulation of β-catenin promotes cancer cell proliferation and renewal. β-catenin gene expression is facilitated through recruitment of co-activators such as histone acetyltransferases CBP/p300; however, the mechanism of their interaction is not fully understood. Here we investigate the interaction between the C-terminal transactivation domain of β-catenin and CBP/p300. Using a combination of pulldown assays, isothermal titration calorimetry, and nuclear resonance spectroscopy we determine the disordered C-terminal region of β-catenin binds promiscuously to the TAZ1 and TAZ2 domains of CBP/p300. We then map the interaction site of the C-terminal β-catenin transactivation domain onto TAZ1 and TAZ2 using chemical-shift perturbation studies. Luciferase-based gene reporter assays indicate Asp750-Leu781 is critical to β-catenin gene activation, and mutagenesis revealed that acidic and hydrophobic residues within this region are necessary to maintain TAZ1 binding. These results provide a mechanistic understanding of Wnt/β-catenin gene regulation that underlies cell development and provide a framework to develop methods to block β-catenin dependent signalling in the future.