Determination of functional domains in the C subunit of the CCAAT- binding factor (CBF) necessary for formation of a CBF-DNA complex

CBF-B interacts simultaneously with both the CBF-A and CBF-C subunits to form a heterotrimeric CBF molecule

In-San Kim, Satrajit Sinha, Benoit De Crombrugghe, Sankar N. Maity

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Abstract

The mammalian CCAAT-binding factor (CBF; also called NF-Y and CP1) is a heterotrimeric protein consisting of three subunits, CBF-A, CBF-B, and CBF- C, all of which are required for DNA binding and all of which are present in the CBF-DNA complex. In this study using cross-linking and immunoprecipitation methods, we first established that CBF-B interacts simultaneously with both subunits of the CBF-A-CBF-C heterodimer to form a heterotrimeric CBF molecule. We then performed a mutational analysis of CBF- C to define functional interactions with the other two CBF subunits and with DNA using several in vitro assays and an in vivo yeast two-hybrid system. Our experiments established that the evolutionarily conserved segment of CBF-C, which shows similarities with the histone-fold motif of histone H2A, was necessary for formation of the CBF-DNA complex. The domain of CBF-C which interacts with CBF-A included a large portion of this segment, one that corresponds to the segment of the histone-fold motif in H2A used for interaction with H2B. Two classes of interactions involved in formation of the CBF-A-CBF-C heterodimer were detected; one class, provided by residues in the middle of the interaction domain, was needed for formation of the CBF-A- CBF-C heterodimer. The other, provided by sequences flanking those of the first class was needed for stabilization of the heterodimer. Two separate domains were identified in the conserved segment of CBF-C for interaction with CBF-B; these were located on each side of the CBF-A interaction domain. Since our previous experiments identified a single CBF-B interaction domain in the histone-fold motif of CBF-A, we propose that a tridentate interaction domain in the CBF-A-CBF-C heterodimer interacts with the 21-amino-acid-long subunit interaction domain of CBF-B. Together with our previous mutational analysis of CBF-A (S. Sinha, I.-S. Kim, K.-Y. Sohn, B. de Crombrugghe, and S. N. Maity, Mol. Cell. Biol. 16:328-337, 1996), this study demonstrates that the historic fold-motifs of CBF-A and CBF-C interact with each other to form the CBF-A-CBF-C heterodimer and generate a hybrid surface which then interacts with CBF-B to form the heterotrimeric CBF molecule.

Original languageEnglish
Pages (from-to)4003-4013
Number of pages11
JournalMolecular and Cellular Biology
Volume16
Issue number8
DOIs
Publication statusPublished - 1996 Jan 1
Externally publishedYes

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CCAAT-Binding Factor
DNA
Histones

ASJC Scopus subject areas

  • Molecular Biology
  • Cell Biology

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@article{b7ea4b52c23444ddbc1520ed26c2e923,
title = "Determination of functional domains in the C subunit of the CCAAT- binding factor (CBF) necessary for formation of a CBF-DNA complex: CBF-B interacts simultaneously with both the CBF-A and CBF-C subunits to form a heterotrimeric CBF molecule",
abstract = "The mammalian CCAAT-binding factor (CBF; also called NF-Y and CP1) is a heterotrimeric protein consisting of three subunits, CBF-A, CBF-B, and CBF- C, all of which are required for DNA binding and all of which are present in the CBF-DNA complex. In this study using cross-linking and immunoprecipitation methods, we first established that CBF-B interacts simultaneously with both subunits of the CBF-A-CBF-C heterodimer to form a heterotrimeric CBF molecule. We then performed a mutational analysis of CBF- C to define functional interactions with the other two CBF subunits and with DNA using several in vitro assays and an in vivo yeast two-hybrid system. Our experiments established that the evolutionarily conserved segment of CBF-C, which shows similarities with the histone-fold motif of histone H2A, was necessary for formation of the CBF-DNA complex. The domain of CBF-C which interacts with CBF-A included a large portion of this segment, one that corresponds to the segment of the histone-fold motif in H2A used for interaction with H2B. Two classes of interactions involved in formation of the CBF-A-CBF-C heterodimer were detected; one class, provided by residues in the middle of the interaction domain, was needed for formation of the CBF-A- CBF-C heterodimer. The other, provided by sequences flanking those of the first class was needed for stabilization of the heterodimer. Two separate domains were identified in the conserved segment of CBF-C for interaction with CBF-B; these were located on each side of the CBF-A interaction domain. Since our previous experiments identified a single CBF-B interaction domain in the histone-fold motif of CBF-A, we propose that a tridentate interaction domain in the CBF-A-CBF-C heterodimer interacts with the 21-amino-acid-long subunit interaction domain of CBF-B. Together with our previous mutational analysis of CBF-A (S. Sinha, I.-S. Kim, K.-Y. Sohn, B. de Crombrugghe, and S. N. Maity, Mol. Cell. Biol. 16:328-337, 1996), this study demonstrates that the historic fold-motifs of CBF-A and CBF-C interact with each other to form the CBF-A-CBF-C heterodimer and generate a hybrid surface which then interacts with CBF-B to form the heterotrimeric CBF molecule.",
author = "In-San Kim and Satrajit Sinha and {De Crombrugghe}, Benoit and Maity, {Sankar N.}",
year = "1996",
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T1 - Determination of functional domains in the C subunit of the CCAAT- binding factor (CBF) necessary for formation of a CBF-DNA complex

T2 - CBF-B interacts simultaneously with both the CBF-A and CBF-C subunits to form a heterotrimeric CBF molecule

AU - Kim, In-San

AU - Sinha, Satrajit

AU - De Crombrugghe, Benoit

AU - Maity, Sankar N.

PY - 1996/1/1

Y1 - 1996/1/1

N2 - The mammalian CCAAT-binding factor (CBF; also called NF-Y and CP1) is a heterotrimeric protein consisting of three subunits, CBF-A, CBF-B, and CBF- C, all of which are required for DNA binding and all of which are present in the CBF-DNA complex. In this study using cross-linking and immunoprecipitation methods, we first established that CBF-B interacts simultaneously with both subunits of the CBF-A-CBF-C heterodimer to form a heterotrimeric CBF molecule. We then performed a mutational analysis of CBF- C to define functional interactions with the other two CBF subunits and with DNA using several in vitro assays and an in vivo yeast two-hybrid system. Our experiments established that the evolutionarily conserved segment of CBF-C, which shows similarities with the histone-fold motif of histone H2A, was necessary for formation of the CBF-DNA complex. The domain of CBF-C which interacts with CBF-A included a large portion of this segment, one that corresponds to the segment of the histone-fold motif in H2A used for interaction with H2B. Two classes of interactions involved in formation of the CBF-A-CBF-C heterodimer were detected; one class, provided by residues in the middle of the interaction domain, was needed for formation of the CBF-A- CBF-C heterodimer. The other, provided by sequences flanking those of the first class was needed for stabilization of the heterodimer. Two separate domains were identified in the conserved segment of CBF-C for interaction with CBF-B; these were located on each side of the CBF-A interaction domain. Since our previous experiments identified a single CBF-B interaction domain in the histone-fold motif of CBF-A, we propose that a tridentate interaction domain in the CBF-A-CBF-C heterodimer interacts with the 21-amino-acid-long subunit interaction domain of CBF-B. Together with our previous mutational analysis of CBF-A (S. Sinha, I.-S. Kim, K.-Y. Sohn, B. de Crombrugghe, and S. N. Maity, Mol. Cell. Biol. 16:328-337, 1996), this study demonstrates that the historic fold-motifs of CBF-A and CBF-C interact with each other to form the CBF-A-CBF-C heterodimer and generate a hybrid surface which then interacts with CBF-B to form the heterotrimeric CBF molecule.

AB - The mammalian CCAAT-binding factor (CBF; also called NF-Y and CP1) is a heterotrimeric protein consisting of three subunits, CBF-A, CBF-B, and CBF- C, all of which are required for DNA binding and all of which are present in the CBF-DNA complex. In this study using cross-linking and immunoprecipitation methods, we first established that CBF-B interacts simultaneously with both subunits of the CBF-A-CBF-C heterodimer to form a heterotrimeric CBF molecule. We then performed a mutational analysis of CBF- C to define functional interactions with the other two CBF subunits and with DNA using several in vitro assays and an in vivo yeast two-hybrid system. Our experiments established that the evolutionarily conserved segment of CBF-C, which shows similarities with the histone-fold motif of histone H2A, was necessary for formation of the CBF-DNA complex. The domain of CBF-C which interacts with CBF-A included a large portion of this segment, one that corresponds to the segment of the histone-fold motif in H2A used for interaction with H2B. Two classes of interactions involved in formation of the CBF-A-CBF-C heterodimer were detected; one class, provided by residues in the middle of the interaction domain, was needed for formation of the CBF-A- CBF-C heterodimer. The other, provided by sequences flanking those of the first class was needed for stabilization of the heterodimer. Two separate domains were identified in the conserved segment of CBF-C for interaction with CBF-B; these were located on each side of the CBF-A interaction domain. Since our previous experiments identified a single CBF-B interaction domain in the histone-fold motif of CBF-A, we propose that a tridentate interaction domain in the CBF-A-CBF-C heterodimer interacts with the 21-amino-acid-long subunit interaction domain of CBF-B. Together with our previous mutational analysis of CBF-A (S. Sinha, I.-S. Kim, K.-Y. Sohn, B. de Crombrugghe, and S. N. Maity, Mol. Cell. Biol. 16:328-337, 1996), this study demonstrates that the historic fold-motifs of CBF-A and CBF-C interact with each other to form the CBF-A-CBF-C heterodimer and generate a hybrid surface which then interacts with CBF-B to form the heterotrimeric CBF molecule.

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