Research Article: Thermodynamic investigation of DNA-binding affinity of wild-type and mutant transcription factor RUNX1

Date Published: May 2, 2019

Publisher: Public Library of Science

Author(s): Fangrui Wu, Tidie Song, Yuan Yao, Yongcheng Song, Fenfei Leng.

http://doi.org/10.1371/journal.pone.0216203

Abstract

Transcription factor RUNX1 and its binding partner CBFβ play a critical role in gene regulation for hematopoiesis. Mutations of RUNX1 cause ~10% of acute myeloid leukemia (AML) with a particularly poor prognosis. The current paradigm for the leukemogenesis is that insufficient activity of wild-type (WT) RUNX1 impairs hematopoietic differentiation. The majority of mutant RUNX1 proteins lose the DNA-binding affinity and inhibit WT RUNX1 by depletion of CBFβ. Here, isothermal titration calorimetry (ITC) was used to quantitatively study the interactions of WT and three clinical mutant RUNX1, CBFβ and DNA. Our data show that the binding of RUNX1 to DNA is enthalpy-driven, and the affinity decreases in the order of WT > S114L > R139Q >> K83E, which support previous observations and conclusion. To find potentially beneficial RUNX1 mutations that could increase the overall RUNX1 activity, K83R and H179K mutations of RUNX1 were designed, using structure-based computational modeling, and found to possess significantly higher DNA-binding affinities than does WT RUNX1. K83R and H179K mutant RUNX1 could therefore be protein-based RUNX1 activators.

Partial Text

Hematopoiesis, generating ~1011-12 differentiated blood cells daily in humans, requires exquisite gene expression regulation. Dysfunction of these controls could lead to initiation of a blood disease, or even leukemia. Transcription factor RUNX1 (also known as AML1) [1] is a master regulator for hematopoiesis [2,3]. It belongs to the core binding factor (CBF) transcription factor family, which consists of CBFα (including RUNX1, 2 and 3) and CBFβ proteins. RUNX1 contains an N-terminal RUNT domain (50–180), which is highly conserved from yeast to mammals, and C-terminal transactivation domains (Fig 1) [4,5]. The biological function of RUNX1 is that RUNT recognizes and binds to the promoter of its target genes with a consensus sequence of PyGPyGGTPy [6–9], with the transactivation domains recruiting other transcriptional proteins (e.g., p300) to activate or repress transcription of the gene [2,10–13]. While CBFβ does not interact with DNA, it forms a heterodimeric complex with RUNT, which can significantly increase the DNA-binding affinity [7] and is essential for hematopoiesis [14,15]. This suggests high affinity binding of RUNX1 to DNA is crucial: reduction of its DNA-binding affinity (e.g., loss of CBFβ) may block the RUNX1-mediated gene transcription and cause abnormal hematopoiesis.

Transcription factor RUNX1, together with its partner CBFβ, plays a critical role in gene expression regulation in hematopoiesis. RUNX1 gene mutations are frequently found in acute myeloid leukemia (AML) with a particularly poor prognosis. There have been no effective treatments for this subtype of AML. In addition, RUNX1 mutations are also commonly found in a number of pre-leukemic diseases, such as myelodysplastic syndrome (MDS) and family platelet disorders (FPD), with predisposition to AML, showing RUNX1 mutation is an early event and the driving force to the cancer. The current paradigm of the mechanism underlying the malignancy is due to insufficient overall biological activity of WT RUNX1, which stems from haploinsufficiency or a mutant RUNX1 that either reduces the amount of CBFβ (for N-terminal mutations) or lacks the transactivation activity (for C-terminal mutations). Insufficient activity of RUNX1 causes hampered hemopoietic differentiation, accumulation of undifferentiated blood cells, and eventually, upon acquisition of other mutations, onset of the leukemia.

 

Source:

http://doi.org/10.1371/journal.pone.0216203

 

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