CCAAT-box binding protein NF-Y (CBF, CP1)recognizes the minor groove and distorts DNA

Ronchi, Antonella; Bellorini, Marianna; Mongelli, Nicola; Mantovani, Roberto

doi:10.1093/nar/23.22.4565

Cited by 87 publications

(90 citation statements)

References 77 publications

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“…Plasmid Constructions-The starting plasmid for the circular permutation assays was pBend2 (14). The proximal and distal CCAAT box oligos described in Table I were inserted by blunt end ligation into the XbaI site.…”

Section: Methodsmentioning

confidence: 99%

“…Calculations of Bending Angles-Location of the points of flexure and amplitudes of the bending angles were described previously (14,40). Briefly, the mobilities of the NF-Y-DNA complexes were normalized to the mobilities of the corresponding free DNA fragments; bending angles were calculated considering the ratio between the fastest and the slowest migrating complexes in EMSA, according to the formula m M /m E ϭ cos a/2, where m M is the relative mobility of the complex exactly in the middle, m E is the relative mobility of the complex at the end of the fragment, and a is the angle of deviation.…”

Section: Methodsmentioning

confidence: 99%

“…4B). With the double mutant, both the slower and the faster complexes were also crippled (lanes [13][14][15][16]. These data suggest that the faster band corresponds to NF-Y binding to either the proximal or the distal site, whereas the upper one results from double occupancy of the two CCAAT boxes.…”

Section: Fig 3 In Vitro Transcription Analysis Of the ␥-Globin Prommentioning

confidence: 99%

“…Shortening the distance between the two CCAAT boxes progressively prevents simultaneous The CCAAT box is a widespread regulatory sequence found in promoters and enhancers of several genes (1), whose functional importance has been well established in different systems (2-12). NF-Y (also termed CBF) has an almost absolute requirement for these five nucleotides and a strong preference for additional flanking sequences (13,14). Based on supershift experiments with anti-NF-Y antibodies, on competition analysis with the original Ea Y box oligo, 1 or on the heteromeric nature of the DNA-binding complex, NF-Y has been identified as the CCAAT box activator in over 100 promoters (7-11, 14, 15).…”

mentioning

confidence: 99%

“…NF-Y (also termed CBF) has an almost absolute requirement for these five nucleotides and a strong preference for additional flanking sequences (13,14). Based on supershift experiments with anti-NF-Y antibodies, on competition analysis with the original Ea Y box oligo, 1 or on the heteromeric nature of the DNA-binding complex, NF-Y has been identified as the CCAAT box activator in over 100 promoters (7-11, 14, 15).…”

mentioning

confidence: 99%

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NF-Y Organizes the γ-Globin CCAAT Boxes Region

Liberati

Ronchi²,

Lievens

et al. 1998

Journal of Biological Chemistry

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The CCAAT-binding activator NF-Y is formed by three evolutionary conserved subunits, two of which contain putative histone-like domains. We investigated NF-Y binding to all CCAAT boxes of globin promoters in direct binding, competition, and supershift electrophoretic mobility shift assay; we found that the ␣, , and proximal ␥ CCAAT boxes of human and the prosimian Galago bind avidly, and distal ␥ CCAAT boxes have intermediate affinity, whereas the ⑀ and ␤ sequences bind NF-Y very poorly. We developed an efficient in vitro transcription system from erythroid K562 cells and established that both the distal and the proximal CCAAT boxes are important for optimal ␥-globin promoter activity. Surprisingly, NF-Y binding to a mutated distal CCAAT box (a C to T at position ؊114) is remarkably increased upon occupancy of the high affinity proximal element, located 27 base pairs away. Shortening the distance between the two CCAAT boxes progressively prevents simultaneous The CCAAT box is a widespread regulatory sequence found in promoters and enhancers of several genes (1), whose functional importance has been well established in different systems (2-12). NF-Y (also termed CBF) has an almost absolute requirement for these five nucleotides and a strong preference for additional flanking sequences (13,14). Based on supershift experiments with anti-NF-Y antibodies, on competition analysis with the original Ea Y box oligo, 1 or on the heteromeric nature of the DNA-binding complex, NF-Y has been identified as the CCAAT box activator in over 100 promoters (7-11, 14, 15). The CCAAT consensus derived statistically by Bucher (1) (RRCCAAT(C/G)(A/G)) fits well with the optimal NF-Y-binding site.NF-Y is a ubiquitous heteromeric protein composed of three subunits, NF-YA, NF-YB, and NF-YC, all necessary for DNA binding (16,17). The cloning of NF-Y genes from several species including yeast, maize, lamprey, and sea urchin, evidenced highly conserved domains (16 -22). The NF-YA homology domain can be divided into subunit association and DNA-contacting subdomains (20). The N-terminal contains a hydrophobic and Gln-rich activation surface (18). The NF-YC gene has been recently cloned and is specular with respect to NF-YA, since the homology domain is at the N terminus, whereas the Cterminal 180 amino acids are rich in glutamines and hydrophobic residues. NF-YB and NF-YC tightly interact with each other, and their association is a prerequisite for NF-YA binding and sequence-specific DNA interactions (16,22). Both the NF-YB-and NF-YC-conserved domains contain putative histone fold motifs. This motif, common to all core histones, is responsible for the formation of the histone octamer (24) and is composed of three ␣-helices, separated by short loops/strand regions, enabling histones to dimerize with companion subunits (24,25). Recent experiments on the yeast HAP3 (26), NF-YB/ CBF-A (27), and NF-YC/CBF-C (28) indicate that this 65 amino acid long motif is necessary for subunit interactions and DNA binding. NF-Y has additional interesting featur...

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Fig 3 In Vitro Transcription Analysis Of the ␥-Globin Prommentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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NF-Y Organizes the γ-Globin CCAAT Boxes Region

Liberati

Ronchi²,

Lievens

et al. 1998

Journal of Biological Chemistry

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Design of a Hairpin Polyamide, ZT65B, for Targeting the Inverted CCAAT Box (ICB) Site in the Multidrug Resistant (MDR1) Gene

et al. 2005

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A novel hairpin polyamide, ZT65B, containing a 3-methylpicolinate moiety was designed to target the inverted CCAAT box (ICB) of the human multidrug resistance 1 gene (MDR1) promoter. Binding of nuclear factor-Y (NF-Y) to the ICB site upregulates MDR1 gene expression and is, therefore, a good target for anticancer therapeutic agents. However, it is important to distinguish amongst different promoter ICB sites so that only specific genes will be affected. All ICB sites have the same sequence but they differ in the sequence of the flanking base pairs, which can be exploited in the design of sequence-specific polyamides. To test this hypothesis, ten ICB-containing DNA hairpins were designed with different flanking base pairs; the sequences ICBa and ICBb were similar to the 3'-ICB site of MDR1 (TGGCT). Thermal-denaturation studies showed that ZT65B effectively targeted ICBa and ICBb (DeltaTM=6.5 and 7.0 degrees C) in preference to the other DNA hairpins (<3.5 degrees C), with the exception of ICBc (5.0 degrees C). DNase I-footprinting assays were carried out with the topoisomerase IIalpha-promoter sequence, which contains five ICB sites; of these, ICB1 and ICB5 are similar to the ICB site of MDR1. ZT65B was found to selectively bind ICB1 and ICB5; footprints were not observed with ICB2, ICB3, or ICB4. A strong, positive induced ligand band at 325 nm in CD studies confirmed that ZT65B binds in the DNA minor groove. The selectivity of ZT65B binding to hairpins that contained the MDR1 ICB site compared to one that did not (ICBd) was confirmed by surface-plasmon studies, and equilibrium constants of 5x10(6)-1x10(7) and 4.6x10(5) M-1 were obtained with ICB1, ICB5,and ICB2 respectively. ZT65B and the previously published JH37 (J. A. Henry, et al. Biochemistry 2004, 43, 12 249-12 257) serve as prototypes for the design of novel polyamides. These can be used to specifically target the subset of ubiquitous gene elements known as ICBs, and thereby affect the expression of one or a few proteins.

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Synthesis and Evaluation of an Intercalator–Polyamide Hairpin Designed to Target the Inverted CCAAT Box 2 in the Topoisomerase IIα Promoter

et al. 2006

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The synthesis and DNA-binding properties of a novel naphthalimide-polyamide hairpin (3) designed to target the inverted CCAAT box 2 (ICB2) site on the topoisomerase IIalpha (topoIIalpha) promoter are described. The polyamide component of 3 was derived from the minor-groove binder, 2, and tailored to bind to the 5'-TTGGT sequence found in and flanking ICB2. The propensity of mitonafide 4 to intercalate between G-C base pairs was exploited by the incorporation of a naphthalimide moiety at the N terminus of 2. Hybrid 3 targeted 5'-CGATTGGT and covered eight contiguous base pairs, which included the underlined ICB2 site. DNase I footprinting analysis with the topoIIalpha promoter sequence demonstrated that 3 bound selectively to the ICB2 and ICB3 sites. Thermal-denaturation studies confirmed these results, and the highest degree of stabilization was found for ICB2 and -3 in preference to ICB1 (4.1, 4.6, and 0.6 degrees C, respectively). CD studies confirmed minor-groove binding and suggested a 1:1 binding stoichiometry. Emission-titration experiments established intercalative binding. Surface plasmon resonance results showed strong binding to ICB2 (2.5x10(7) M(-1)) with no observable binding to ICB1. Furthermore, the binding constant of 3 to ICB2 was larger than that of the parent polyamide 2. The increased binding affinity was primarily due to a reduction in the dissociation-rate constant of the polyamide-DNA complex, which can be attributed to the N-terminal naphthalimide moiety. In addition, the binding site of 3 was larger than that of 2, which innately improved sequence selectivity. We conclude that the polyamide-naphthalimide 3 selectively binds to the ICB2 site by simultaneous intercalation and minor-groove binding, and warrants further investigation as a model compound for the regulation of topoIIalpha gene expression.

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CCAAT-box binding protein NF-Y (CBF, CP1)recognizes the minor groove and distorts DNA

Cited by 87 publications

References 77 publications

NF-Y Organizes the γ-Globin CCAAT Boxes Region

NF-Y Organizes the γ-Globin CCAAT Boxes Region

Design of a Hairpin Polyamide, ZT65B, for Targeting the Inverted CCAAT Box (ICB) Site in the Multidrug Resistant (MDR1) Gene

Synthesis and Evaluation of an Intercalator–Polyamide Hairpin Designed to Target the Inverted CCAAT Box 2 in the Topoisomerase IIα Promoter

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