Kinetic Analysis of the Interactions Between the Different ELL and EAF Proteins Involved in Acute Myeloid Leukemia
Authors: Sharma Nimisha , Solomaha Elena , Simone Federico , Thirman Michael 
Published: April 2020

The ELL gene was first cloned as a fusion partner of MLL in the (11;19)(q23;p13.1) translocation that occurs in acute myeloid leukemia. Subsequently, the ELL2 gene was cloned on the basis of its sequence homology to ELL. Both proteins stimulate the rate of transcript elongation by RNA polymerase II. Previously, we isolated two closely related proteins, EAF1 and EAF2, which interact with ELL and ELL2. Deletion mapping studies carried out to delineate the domain(s) of ELL involved in its interaction with either EAF1 or EAF2 showed that the N-terminus (amino acids 1–207) of ELL binds to both EAF1 and EAF2. In comparison, the middle region (207–411 amino acids) does not bind to either of the two EAF proteins and the C-terminus region (411–621 amino acids) binds only to the EAF1 protein. Biochemical studies have revealed that EAF1 and EAF2 enhance the rate of mRNA chain elongation by the ELL proteins in vitro. Although both ELL and ELL2 have similar roles in transcriptional elongation, ELL2 has not been shown to be involved in any hematological abnormality so far. In an attempt to gain a deeper understanding of the biology and functions of the interactions between these different proteins, we determined the kinetic properties of these interactions using the biophysical techniques of surface plasmon resonance (SPR) and isothermal calorimetry (ITC). SPR detects complex formation in real time and provides a better comprehension of the dynamics of association and dissociation of an interaction, and ITC is used to determine the thermodynamics of the interaction. Our SPR analysis has provided novel insights into the nature of the binding of the ELL proteins to the EAF proteins. We observed that both ELL and ELL2 bind to EAF1 and EAF2 with a high affinity, but the binding affinity of ELL2 for both EAF1 and EAF2 is almost twelve-fold greater than the affinity of ELL for both the EAF proteins. The higher affinity of ELL2 is due to much slower uptake and release kinetics reflected by the low association and dissociation rate constants of ELL2 compared to ELL. The stoichiometry of ELL, ELL2, EAF1 and EAF2 in the ELL-EAF1, ELL-EAF2, ELL2-EAF1 and ELL2-EAF2 complexes was estimated to be 1:1 after fitting the respective sensorgrams obtained by SPR analysis to the Langmuir’s bimolecular model. Interestingly, we did not observe any difference in the affinity of either ELL or ELL2 for binding to EAF1 or EAF2. We used SPR-based competition experiments to show that ELL and ELL2 bind to the same sites on the EAF proteins. We have also investigated the characteristics of binding of the various ELL domains to the EAF1/2 proteins. In the (11;19)(q23;p13.1) translocation, the C-terminus of ELL fuses to the N-terminus of MLL to generate a chimeric protein that interacts with EAF1 and this interaction is critical for the role of ELL in cell immortalization in vitro and leukemogenesis in vivo. In agreement with this observation, we found that the C-terminus of ELL binds EAF1 with a higher affinity than EAF2, while the N-terminus of ELL binds with similar affinities and displays similar kinetics of binding to both EAF1 and EAF2. We also found that the individual binding sites on the ELL N-terminus and the C-terminus exhibited a lower affinity for the EAF proteins, but the affinity increases when the two sites function together in the context of the full-length protein, suggesting that the two sites co-operate with each other to increase the affinity for the full-length ELL protein. Taken together, these observations suggest that although ELL and ELL2 share many similarities in terms of their sequence and function in transcription elongation, they bind to the EAF proteins with different affinities and kinetics. Alternative interaction dynamics and the interplay between the different ELL and EAF proteins permit distinct functional regulation of transcriptional elongation in normal and leukemic cells.