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The hematopoietic transcription factor RUNX1: a structural view
Umeå University, Faculty of Science and Technology, Umeå Centre for Molecular Pathogenesis (UCMP).
2004 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The malfunction of the transcriptional regulator RUNX1 is the major cause of several variants of acute human leukemias and its normal function is to regulate the development of the blood system in concert with other transcriptional co-regulators. RUNX1 belongs to a conserved family of heterodimeric transcription factors that share a conserved DNA binding domain, the Runt domain (RD), named after the first member of this group – Runt - found in Drosophila melanogaster. The binding partner CBFβ serves as a regulator of RUNX by enhancing its DNA binding affinity through an allosteric mechanism.

The main focus ofo my thesis work has been the crystallization and structural analysis of the RUNX1 RD and involved also more technical methodological aspects that can be applied to X-ray crystallography in general.

The high resolution crystal structure of the free RD shows that this immunoglobulin-like molecule undergoes significant structural changes upon binding to both CBFβ and DNA. This involves a large flip of the L11 loop from a closed conformation in the free protein to an open conformation when CBFβ and/or DNA are bound. We refer to this transition as the “S-switch”. Smaller but significant conformational changes in other parts of the RD accompany the “S-switch”. We suggest that CBFβ triggers and stabilizes the “S-switch” which leads to the conversion of the RD into a conformation enhanced for DNA binding.

During the structural analysis of the RD we identified two chloride ions that are coordinated by residues otherwise involved in DNA binding. In electrophoretic mobility-shift analyses (EMSA) we demonstrated a chloride ion concentration dependent stimulation of the DNA binding affinity of RUNX1. We further showed by NMR line width broadening experiments that the chloride binding occurred within the physiological range. A comparable DNA binding stimulation of RUNX1 was seen in the presence of negative amino acids. This suggests a regulation of the DNA binding activity of RUNX1 proteins through acidic amino acid residues possibly provided by activation domains of transcriptional co-regulators that interact with RUNX1.

The use of the anomalous signal from halide ions has become a powerful technique for obtaining phase information. By replacing the sodium chloride with potassium bromide in the crystallisation conditions of the RD, we could demonstrate in a single wavelength anomalous diffraction (SAD) experiment that the anomalous signal from 2 bromide ions were sufficient to phase a 16 kDa protein. Due to lack of completeness in the low-resolution shells caused by overloaded intensities, density modification schemes failed and the resulting electron density maps were not interpretable. By combining the highresolution

synchrotron data with low-resolution data from a native data set collected on a home X-ray source, the density modified bromide phases gave easily traceable maps.

Place, publisher, year, edition, pages
Umeå: Umeå centrum för molekylär patogenes (UCMP) (Teknisk-naturvetenskaplig fakultet) , 2004. , 29 p.
Keyword [en]
Molecular medicine, RUNX1, Runt domain, CBFβ, transcription factor, leukaemia, protein crystallography, anomalous diffraction
Keyword [sv]
Molekylärmedicin
National Category
Medical Genetics
Research subject
Molecular Biology
Identifiers
URN: urn:nbn:se:umu:diva-192ISBN: 91-7305-476-3 (print)OAI: oai:DiVA.org:umu-192DiVA: diva2:142429
Public defence
2003-09-05, Umeå, 10:00
Available from: 2004-02-06 Created: 2004-02-06 Last updated: 2012-06-28Bibliographically approved
List of papers
1. Crystallization and preliminary studies of the DNA-binding runt domain of AML1.
Open this publication in new window or tab >>Crystallization and preliminary studies of the DNA-binding runt domain of AML1.
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2001 (English)In: Acta Crystallogr D Biol Crystallogr, ISSN 0907-4449, Vol. 57, no Pt 2, 269-71 p.Article in journal (Refereed) Published
Abstract [en]

The acute myeloid leukaemia 1 (AML1) protein belongs to the Runx family of transcription factors and is crucial for haematopoietic development. The genes encoding Runx1 and its associated factor CBF beta are the most frequent targets for chromosomal rearrangements in acute human leukaemias. In addition, point mutations of Runx1 in acute leukaemias and in the familial platelet disorder FPD/AML cluster within the evolutionary conserved runt domain that binds both DNA and CBF beta. Here, the crystallization of the Runx1 runt domain is reported. Crystals belong to space groups C2 and R32 and diffract to 1.7 and 2.0 A resolution, respectively.

Keyword
acute myeloid leukaemia 1 protein, Runx1, runt domain, transcription factors
Identifiers
urn:nbn:se:umu:diva-17921 (URN)doi:10.1107/S0907444900015791 (DOI)11173476 (PubMedID)
Available from: 2008-05-05 Created: 2008-05-05 Last updated: 2012-05-11Bibliographically approved
2. The RUNX1 Runt domain at 1.25 Å resolution: a structural switch and specifically bound chloride ions modulate DNA binding.
Open this publication in new window or tab >>The RUNX1 Runt domain at 1.25 Å resolution: a structural switch and specifically bound chloride ions modulate DNA binding.
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2002 In: J Mol Biol, Vol. 322, no 2, 259-272 p.Article in journal (Refereed) Published
Identifiers
urn:nbn:se:umu:diva-3634 (URN)
Available from: 2004-02-06 Created: 2004-02-06Bibliographically approved
3. Chloride binding by the AML1/Runx1 transcription factor studied by NMR
Open this publication in new window or tab >>Chloride binding by the AML1/Runx1 transcription factor studied by NMR
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2001 (English)In: FEBS Letters, ISSN 0014-5793, Vol. 488, no 1-2, 81-4 p.Article in journal (Refereed) Published
Abstract [en]

It is known that the DNA binding Runt domain of the AML1/Runx1 transcription factor coordinates Cl(-) ions. In this paper we have determined Cl(-) binding affinities of AML1 by (35)Cl nuclear magnetic resonance (NMR) linewidth analysis. The Runt domain binds Cl(-) with a dissociation constant (K(d,Cl)) of 34 mM. If CBFbeta is added to form a 1:1 complex, the K(d,Cl) value increases to 56 mM. Homology modeling suggests that a high occupancy Cl(-) binding site overlaps with the DNA binding surface. NMR data show that DNA displaces this Cl(-) ion. Possible biological roles of Cl(-) binding are discussed based on these findings.

Keyword
AML1, Runt domain, 35Cl, Nuclear magnetic resonance spectroscopy, Anion binding to protein
National Category
Structural Biology
Identifiers
urn:nbn:se:umu:diva-17923 (URN)doi:10.1016/S0014-5793(00)02390-5 (DOI)11163800 (PubMedID)
Available from: 2008-05-05 Created: 2008-05-05 Last updated: 2012-06-28Bibliographically approved
4. The importance of low resolution data: A SAD bromide example
Open this publication in new window or tab >>The importance of low resolution data: A SAD bromide example
Manuscript (Other academic)
Identifiers
urn:nbn:se:umu:diva-3636 (URN)
Available from: 2004-02-06 Created: 2004-02-06 Last updated: 2010-01-13Bibliographically approved

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