Identification of distinct SET/TAF-Ibeta domains required for core histone binding and quantitative characterisation of the interaction.
BACKGROUND
The assembly of nucleosomes to higher-order chromatin structures is finely tuned by the relative affinities of histones for chaperones and nucleosomal binding sites. The myeloid leukaemia protein SET/TAF-Ibeta belongs to the NAP1 family of histone chaperones and participates in several chromatin-based mechanisms, such as chromatin assembly, nucleosome reorganisation and transcriptional activation. To better understand the histone chaperone function of SET/TAF-Ibeta, we designed several SET/TAF-Ibeta truncations, examined their structural integrity by circular Dichroism and assessed qualitatively and quantitatively the histone binding properties of wild-type protein and mutant forms using GST-pull down experiments and fluorescence spectroscopy-based binding assays.


RESULTS
Wild type SET/TAF-Ibeta binds to histones H2B and H3 with Kd values of 2.87 and 0.15 microM, respectively. The preferential binding of SET/TAF-Ibeta to histone H3 is mediated by its central region and the globular part of H3. On the contrary, the acidic C-terminal tail and the amino-terminal dimerisation domain of SET/TAF-Ibeta, as well as the H3 amino-terminal tail, are dispensable for this interaction.


CONCLUSIONS
This type of analysis allowed us to assess the relative affinities of SET/TAF-Ibeta for different histones and identify the domains of the protein required for effective histone recognition. Our findings are consistent with recent structural studies of SET/TAF-Ibeta and can be valuable to understand the role of SET/TAF-Ibeta in chromatin function.
Laboratory of Biological Chemistry, Medical School, University of Ioannina, 451 10 Ioannina, Greece. zkarets@cc.uoi.gr
Zoe
Z
Karetsou
Anastasia
A
Emmanouilidou
Ioannis
I
Sanidas
Stamatis
S
Liokatis
Eleni
E
Nikolakaki
Anastasia S
AS
Politou
Thomais
T
Papamarcaki
D000595:Amino Acid Sequence; D002843:Chromatin / Q000378:metabolism; D002868:Chromosomal Proteins, Non-Histone / Q000737:chemistry / Q000378:metabolism*; D002942:Circular Dichroism; D004268:DNA-Binding Proteins; D056488:Histone Chaperones; D006657:Histones / Q000378:metabolism*; D018832:Molecular Chaperones / Q000378:metabolism; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D014157:Transcription Factors / Q000737:chemistry / Q000378:metabolism*
D002843:Chromatin; D002868:Chromosomal Proteins, Non-Histone; D004268:DNA-Binding Proteins; D056488:Histone Chaperones; D006657:Histones; D018832:Molecular Chaperones; D050505:Mutant Proteins; D011994:Recombinant Proteins; C513473:SET protein, human; D014157:Transcription Factors