INTRODUCTION

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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 C-terminal 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 features as follows: (i) CCAAT boxes are not able to activate alone even if multimerized, but they increase the activity of neighboring enhancer motifs. (ii) NF-Y appears to increase the affinity of transcription factors for their target sequence (29). However, the exact mechanisms of transcriptional activation by NF-Y are still elusive.

Globin genes are transcribed in a tissue-specific and developmentally regulated manner by means of various regulatory elements in their clusters (30). In addition to the locus control regions which have been characterized in transgenic mice, the promoters of each globin gene contain sequences that usually impart tissue-specific control in transfection experiments. Several lines of reasoning point to CCAAT sequences as important elements in globin gene expression as follows: (i) they are present in all globin promoters; (ii) they have been remarkably conserved in different species at a fixed distance from the cap site; (iii) genomic footprinting of all globin promoters in erythroid cells showed invariable protection of CCAAT sequences in vivo, indicating binding of activators (31); (iv) the functional importance has been documented in the -, -, -, and -globin promoters (2-5, 15); (v) the -globin duplicated CCAAT boxes are the target of mutations affecting developmental silencing of -globin expression in adult life. In particular, strong genetic evidence suggests that point mutations in the CCAAT box region are causative of the HPFH syndromes, characterized by increased fetal globin levels in adults (32-37).

Several studies investigated factors binding to CCAAT; CDP, NFE3, and c/EBP appear to recognize different globin promoters (3, 32-36). One CCAAT-binding protein, -CP1, was purified to homogeneity using an -globin CCAAT box affinity column and found to be a trimeric factor most likely identical to NF-Y (15). Moreover, competition and supershift EMSA experiments performed with nuclear extracts determined that a CCAAT binding activity similar to NF-Y (termed CP1 in the globin field) binds to the -globin promoters (32, 33). However, identification of CCAAT binding activities in other promoters is less certain; therefore, we wished to definitely determine which of the globin CCAAT boxes represents a bona fide NF-Y-binding site and establish a hierarchy of affinities. We then focused our attention on the highly regulated -globin duplicated CCAAT box region. Because of the histone-like nature of NF-Y and its ability to distort DNA, we examined the possibility that NF-Y organizes this region.

	MATERIALS AND METHODS

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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. The double CCAAT and the phasing analysis oligos were also cloned into the XbaI site.

Electrophoretic Mobility Shift Assay (EMSA)-- Labeled oligonucleotides (10,000 cpm) containing the different globin CCAAT boxes were incubated with K562 nuclear extracts (5 µg) for 30 min at 25 °C. Binding reactions for NF-Y were performed incubating labeled oligonucleotides for 20 min at 20 °C, in a buffer containing 5% glycerol, 50 mM NaCl, 20 mM Tris, pH 7.5, 0.5 mM EDTA, 5 mM MgCl₂, and 1 mM dithiothreitol, and run in 4% polyacrylamide gels (acrylamide/bis-acrylamide ratio of 29:1) at 4 °C. Supershift experiments were performed as described in Ref. 12.

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 = 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. To determine bending centers, the normalized mobility of each NF-Y-DNA complex was plotted as a function of the distance between the center of the CCAAT sequence and the end of the DNA fragment; the bend was determined as the position at which the NF-Y-DNA complex was at a minimum.

In Vitro Transcription-- Preparation of transcriptionally competent K562 nuclear extracts and in vitro transcription reactions were detailed in Ref. 38. Two independent CsCl plasmid preparations of the pAG vectors were used in different sets of experiments.

	RESULTS

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NF-Y Binds to CCAAT Boxes of All Globin Promoters-- To determine which of the globin CCAAT boxes is recognized by NF-Y, we labeled oligonucleotides (see Table I) for EMSA experiments with nuclear extracts, challenging the resulting complexes with anti-NF-YB- and anti-NF-YA-purified antibodies (12). Bands of different mobilities and intensities are generated with all oligos (Fig. 1), with the  (lanes 4-6),  (lanes 8-10), hP (human proximal, lanes 16-18), gP (prosimian Galago crassicaudatus proximal, see Ref. 36, lanes 28-30), hD (human  distal, lanes 20-22) and gD (G. crassicaudatus  distal, lanes 32-34), one predominant shifted band is visible; these complexes are supershifted by specific anti-YB and anti-YAc antibodies (12). In each case we ran parallel migrations of the Y box oligo incubated with recombinant NF-YA and purified NF-YB/NF-YC; as shown in Fig. 1, the bands have electrophoretic behaviors identical to the endogenous K562 bands shifted by the anti-NF-Y antibodies. On the other hand, with the  and  CCAAT multiple bands are visualized (lanes 12-14 and 24-26); for the  a weak band comigrating with NF-Y and supershifted by the antibodies is observed, and for the  the four major bands detected have different mobilities compared with the Y/NF-Y band, and no supershift is evident. These data show that NF-Y binding is readily visualized on all globin CCAAT boxes and is the most prominent binding protein, with the exception of  and , which show a much higher affinity for proteins unrelated to NF-Y.

View larger version (50K): [in this window] [in a new window]   Fig. 1.   EMSA analysis of nuclear proteins binding to globin CCAAT boxes. The labeled oligos indicated above are incubated with K562 nuclear extracts (5 µg) in the absence (lanes 1, 4, 8, 12, 16, 20, 24, 28, and 32) or in the presence of purified anti-YAC or anti-YB antibodies. Purified NF-Y was run in parallel with the MHC Ea Y box oligo (lanes 7, 11, 15, 19, 23, 27, 31, and 35). Asterisks refer to the NF-E3 band visible on the -hD and gP CCAAT boxes, as described previously (32, 36)

View larger version (66K): [in this window] [in a new window]   View larger version (30K): [in this window] [in a new window]   Fig. 2.   A, cross-competition analysis of globin CCAAT oligos. The indicated labeled oligos were incubated with purified NF-Y in the absence (lanes 1) or presence of a 20- (even numbers) or a 100-fold (uneven numbers) molar excess of the competing oligos (lanes 2-19). For the  and  oligos, long exposures of 60 h and 7 days, respectively, were necessary to visualize the NF-Y bands. B, quantification of the data in A were obtained by densitometric scanning of the gels.

Both -Globin CCAAT Boxes Contribute to Promoter Activity in Vitro-- We next focused our attention on the developmentally regulated -globin promoter. We fused the minimal tissue-specific -globin promoter (299 to +35) to a rabbit -globin reporter gene (plasmid pAG1) and generated mutants in the proximal or in the distal CCAAT boxes (see scheme in Fig. 3A). The wt and mutated constructs were tested in a functional in vitro assay with transcriptionally competent erythroid K562 extracts. RNA was purified and hybridized to a single-stranded end-labeled DNA probe; subsequent S1 mapping allowed the determination of qualitative and quantitative changes in the transcription rate. As an internal control we added a plasmid containing the adenovirus major late promoter TATA box devoid of any activating sequences and fused to the same reporter gene. Fig. 3B shows that our system efficiently transcribes the -globin promoter and faithfully reproduces the correct start site used in vivo. We tested the different mutants: alteration of the proximal CCAAT box or destruction of the distal by a 13-base pair deletion (pAG3 and pAG4) decreases transcription 3-4-fold (Fig. 3C, lanes 3 and 4). Swapping the weak -globin CCAAT box into the distal CCAAT box position partially restores transcription when the proximal CCAAT is mutated, while having minor effects when the proximal CCAAT is intact (pAG2 and pAG5, respectively; Fig. 3C, lanes 1, 2 and 5). A mutant promoter containing mutations in both CCAAT boxes (pAG6) was also compared with wt pAG1 and resulted in the lowest transcriptional rate (6-fold down, compare Fig. 3C, lanes 6 and 7). Note that the signals in Fig. 3C result from a 4-h exposure. These data indicate that both CCAAT elements contribute to the optimal promoter activity. However, other important activators are probably operating, since mutations in the CCAAT boxes do not abolish transcription completely.

View larger version (49K): [in this window] [in a new window]   Fig. 3.   In vitro transcription analysis of the -globin promoter. A, a scheme representing the different constructs used is depicted. All contained the -globin promoter (299/+35) fused to a rabbit -globin reporter. B, mapping of -globin promoter-driven RNA by S1 analysis. In vitro transcriptions with 100, 200, and 400 ng of pAG1 plasmid were run in parallel with a sequencing reaction, to spot the transcriptional start site in our in vitro system. The signal corresponding to the probe is indicated. C, analysis of CCAAT box -globin mutants. 200 ng of each plasmid and 4 µl of transcription extract (40 µg) was used for in vitro transcriptions, together with 50 ng of the pAL5 internal control, generating the correct -globin +1 signal and the shorter -globin signal. Given 100 in arbitrary units the activity of pAG1 and pAG2 was 60, pAG3 was 33, pAG4 was 26, pAG5 was 24, and pAG6 was 16.

Binding of NF-Y to the -Globin Double CCAAT Boxes Region-- By having shown that both -globin CCAAT boxes bind NF-Y and are important for in vitro promoter activity, we investigated their interplay; we labeled a long oligo encompassing the two CCAAT boxes, incubating increasing concentrations of recombinant NF-Y, containing wt NF-YA and NF-YB and the homology domain of NF-YC. Fig. 4 shows that two bands of different electrophoretic mobility are generated (lanes 1-4); to ascertain whether the slow migrating complex corresponds to DNA fragments bound by two NF-Y molecules, we used fragments mutated in the distal CCAAT (C¹¹⁴), in the proximal CCAAT (the corresponding C to T mutation at 87), or in both. We have deliberately chosen this mutation because genetic evidence strongly associates it with HPFH syndromes in humans (34). Mutations in such position are known to essentially abolish NF-Y binding to all CCAAT boxes tested so far, including the -globin (13-15, 40).² The faster complex is only modestly affected by mutations in the distal CCAAT; the slower complex is greatly diminished in the 87 CCAAT mutant and in the double mutant (compare lanes 4, 12 and 16). Surprisingly, the 114 mutant exhibited a considerable level of the upper complex (compare lanes 4 and 8 and see the calculated ratios in Fig. 4B). With the double mutant, both the slower and the faster complexes were also crippled (lanes 13-16). 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. Interestingly, consistent with the cross-competition experiments, binding of NF-Y to the proximal CCAAT is predominant and compensates for a crippling mutation in the distal CCAAT, whereas the reverse is not true.

View larger version (34K): [in this window] [in a new window]   Fig. 4.   A, EMSA analysis of the double CCAAT box -globin oligos. Dose-response of NF-Y with the wild type (lanes 1-4, 0.1, 0.3, 1, and 5 ng) or oligos with mutations (C to T in the second C of CCAAT) in the distal (lanes 5-8), proximal (lanes 9-12), or both CCAAT boxes. The bands corresponding to single and double occupation of the CCAAT boxes are indicated. B, quantification of the experiments in A. The upper/lower complex ratios are indicated above each mutant, except for DM, in which the upper complex was too weak to be quantitated. , upper band; , lower band.

View larger version (70K): [in this window] [in a new window]   Fig. 5.   EMSA analysis of short deletions between the CCAAT boxes. Dose-response of NF-YA/NF-YB/NF-YC5 was performed with either the wt oligo (lanes 1-4, 0.1, 0.3, 1 and 5 ng), or with oligos containing deletions of 3 (lanes 5-8), 5 (lanes 9-12) and 8 (lanes 13-16) nucleotides in the region between the CCAAT boxes.

View larger version (36K): [in this window] [in a new window]   Fig. 6.   EMSA competition analysis on oligos with CCAAT deletions. A, a 5-, 20-, 100-, and 500-fold molar excess of competing wt (lanes 2-5 and 12-15) or mutated Y box oligos (lanes 7-10 and 17-20) were incubated with NF-YA/NF-YB/NF-YC5 (5 ng) before addition of the wt and 3 labeled oligos (see scheme). B, same preincubation as in A, except that labeled 5 and 8 oligos were used. C, same as A except that the labeled wt and 3 oligos were first incubated with NF-YA/NF-YB/NF-YC5 (22) and then challenged with the competing oligos (see scheme). D, same as B, except that the same order of addition as in C was used. For the 5 and 8 experiments a 10-fold higher amount of NF-Y was necessary to visualize the upper NF-Y bands. E, quantification of the data in A-D.

NF-Y Induces Bending of -Globin CCAAT Boxes-- We have recently shown that NF-Y is able to induce distortions in the double helix, with angles that vary depending on the surrounding sequences (14). By using the circular permutation assay we checked the degree of distortion of the proximal and distal CCAAT boxes separately. We cloned the two oligos of identical length in the pBend2 vector (14); we then cut with different enzymes so that the CCAAT boxes were at different distances from the extremities of the fragments, and we performed EMSA with NF-Y. To calculate precisely the angles, we maximized the differences in mobilities of the protein-DNA complexes, using the small NF-YA9 mutant and purified endogenous NF-YB/NF-YC. We have shown that such combination does not alter significantly the distortion angles of four NF-Y sites (14). Clear indication that NF-Y induces distortions on both -globin CCAAT boxes was evident from the different electrophoretic mobilities of the fragments (Fig. 7). Calculations of the angles gave similar results for both sites, and the values, 66° and 72° for distal and proximal, respectively, are similar to those observed for the murine sarcoma virus and -globin and slightly lower than for the Ea Y box and HSP70 NF-Y-binding sites. As expected, the flexures are indeed centered on the CCAAT sequences (Ref. 14 and data not shown).

View larger version (40K): [in this window] [in a new window]   Fig. 7.   Circular permutation assay of NF-Y CCAAT complexes. The pBend2 -CCAAT plasmids were digested with the indicated enzymes, generating the fragments outlined in the underlying cartoon. They were incubated with NF-YA9/NF-YB/NF-YC (14). Lanes 1-5, proximal CCAAT; lanes 6-10, distal CCAAT; lanes 11-15, double CCAAT.

Phasing Analysis of CCAAT Boxes-- To verify whether NF-Y induces directed bends, as suggested by the previous experiment, and to seek further information about the relative distortions of the CCAAT region, we performed phasing analysis. This assay is usually tackled with appropriate vectors containing a fixed angle of known curvature, obtained exploiting the distortions caused by short AT-rich sequences spaced by one turn of the helix (Ref. 40 and references therein). Having fixed one angle, the binding site is rotationally moved on different sides of the double helix, by increasing the distance from the fixed angle, taking into account that one turn corresponds to 10.5 base pairs. The relative mobilities of the protein-bound complexes give clues about the orientation of the angles. Rather than adopting this strategy, which would give information about one isolated CCAAT, we took advantage of the two -globin-binding sites with their previously calculated distortions. We cloned in pBend2 the 3, 5, 8 oligos, and a +3 oligo, in which we added 3 bp between the two CCAAT. Cutting the resulting plasmids with any of the enzymes generates nearly identical fragments with NF-Y-binding sites rotationally displaced with respect to the wt situation. EMSA analysis of the central XhoI fragments is shown in Fig. 8, and comparable amounts of protein generated the single occupancy band with identical electrophoretic mobility for all fragments. The double occupancy band is visible with the +3, wt, and 3 fragments, with the 5 only after prolonged exposures, and is absent in the 8. The mobilities of the latter bands are different, a clear indication that indeed the DNA is oriented upon NF-Y binding (40). The slowest fragment should be the one in which the two bends are in phase with respect to the helical turn of DNA, whereas the fastest is the one in which the two bends are facing opposite sides and counteract each other; the +3, in which the two CCAAT boxes are only 1.5 bp away from perfect alignment on the same side of the helix, is the slowest, and the 3 and 5 are the fastest. This is consistent with overall distortion angles of about 100° among the two CCAAT in the wt configuration (see Fig. 9). These results are indeed confirmed by experiments with a "mini" NF-Y protein (22) that is able to generate double bands in the 8 as well, which give a phasing "period" of about 8 bp, corresponding to a rotation of about 100°.³

View larger version (29K): [in this window] [in a new window]   Fig. 8.   Phasing analysis of NF-Y binding to the double CCAAT box. The wt, +3, 3, 5, and 8 pBend2 vectors were digested with XhoI and the fragments incubated with NF-Y9/NF-YB/NF-YC. The band corresponding to double occupancy in the 5 was visible after prolonged exposures. The mobility of the upper bands of the +3, 3, and 5 was plotted against that of the wt fragment as indicated in the underlying scheme.

View larger version (19K): [in this window] [in a new window]   Fig. 9.   The promoter targets of NF-Y in the globin clusters and the NF-Y-induced distortions in the -globin CCAAT box region.

	DISCUSSION

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In this study we undertook a systematic analysis of NF-Y binding to the CCAAT boxes of globin gene promoters and established that most, but not all, contain bona fide high affinity sites. We focused then on the human -globin, and we developed an efficient in vitro functional assay with K562 extracts and provided for the first time evidence that both CCAAT boxes add to the overall level of expression. NF-Y binds better to the proximal than to the distal site, and DNA binding is neither cooperative nor mutually exclusive. The bending angles of the isolated CCAAT boxes are similar, and double binding induces compensatory alterations; the combined twisting angles are about 100°.

NF-Y and the Globin CCAAT Boxes-- Previous experiments with nuclear extracts identified several proteins binding to the CCAAT boxes of various globin promoters. CDP binds to the duplicated -globin CCAAT box region (32, 33), NF-E3 to the  distal CCAAT box and to the  box (32, 36), and c/EBP to the  CCAAT (3). The  CCAAT box region is bound by a number of factors (36). Binding of NF-Y has been more than suspected; the -globin CCAAT box has been shown to recognize a multimeric factor (-CP1) which by several criteria is NF-Y (15). The  CCAAT boxes were also shown to be cross-competed by the Ea Y box (32, 33). By performing a systematic EMSA analysis on all globin CCAAT boxes with NF-Y, we found that the , , and proximal  CCAAT bind NF-Y efficiently, the distal CCAAT well, whereas the affinity for  and  is so low that we consider it unlikely that they are in vivo targets for NF-Y. A good binding site for NF-Y requires two additional conserved nucleotides at the 5' and three at the 3' (13, 41). As in all high affinity sites, two purines are present in all globin sites at the 5' end, indicating that sequences at the 3' end are responsible for the dramatic variations in affinities; the T at second position of the 3' end of  deviates from the consensus and is severely underrepresented in over 178 bona fide NF-Y-binding sites (41). Within the -globin promoters, both in human and in the prosimian G. crassicaudatus, we note that the third position at the 3' end is the only different nucleotide among the distal and proximal CCAAT of human and Galago (see Table I), surprisingly pointing to this position, relatively distant to the core pentanucleotide, as important in affinity determination.

114

87

Role of CCAAT Boxes in -Globin Transcription-- The efficient in vitro transcription system for the -globin promoter allowed us to determine the role of the CCAAT boxes. Our data are consistent with the idea that both NF-Y-binding sites contribute to the optimal activity, and mutations in both CCAAT resulted in transcription levels that were lower (6-fold) than alterations in either CCAAT alone. A point mutation in the proximal and removal of the distal CCAAT box led to a comparable 4-fold decrease in transcription; and swapping an -like CCAAT box, which is a poor NF-Y-binding site, in the place of the distal  CCAAT weakens the promoter both when the proximal CCAAT is intact (2-fold) and even more if it is mutated, as in pAG5. Thus, from the functional point of view, the twin CCAAT boxes seem to have an additive rather than a multiplicative effect, a result that is in line with the lack of cooperativity between the two proteins observed in the binding assays. Either CCAAT is nevertheless sufficient to impart rather efficient levels of transcription. This might be entirely due to strong TATA box and basal elements or to the activity of additional upstream factors, as suggested by challenging transcription with anti-GATA antibodies.⁵

NF-Y Binding to Duplicated CCAAT Boxes-- CCAAT boxes show a strong position bias within promoters being usually positioned between 60 and 100 (1). Functional experiments indicated that a single CCAAT box is not able to increase transcription over basal levels, and multimerized CCAAT boxes also fail to do so. NF-Y can greatly improve the activity of diverse upstream transcription factors, and in some cases, it has been proved that it does so by dramatically improving the affinity of neighboring factors for their target DNA sequence (29, 42). Since no data were available on the binding of NF-Y in promoters harboring more than one CCAAT box, the -globin represents an excellent model to study their relationship. Several findings in our study bear implications for other systems as follows: (i) bringing two CCAAT sequences closer than 24 base pairs essentially abrogates double binding, and (ii) NF-Y complexes are more stable on the DNA only with the 5 mutant, a deletion bringing the two CCAAT boxes on the same side of the helix. This suggests that cooperativity is possible, provided that the correct rotational position is respected. From the long list of CCAAT-containing promoters activated by NF-Y, a growing number contains two or more sites (see Table II); we note that in all, bar the gp91^phox, the distance between the two NF-Y sites is greater than 27 nucleotides; in three cases, cdc2, H2b, and TK, it is of 30-32 bp, so that the two CCAAT face the same side of the helix. Most promoters have more distant sites (40/80 bp apart), and it is not known at present whether NF-Y complexes could influence each other at such distances. The only apparent exception is the gp91^phox promoter, in which two CCAAT boxes are separated by only 14 nucleotides; however, while the proximal CCAAT box has been formally proven to be a bona fide NF-Y site, the distal CCAAT box appears to bind NF-Y poorly and is possibly targeted by other proteins. In conclusion, wherever CCAAT boxes closer than 24 base pairs are found, binding of NF-Y should be considered as mutually exclusive, or one of them is presumably activated by proteins other than NF-Y. Simultaneous binding of NF-Y to sites with multiples of 10.5 bp might be considered as more stable. Experiments aimed at verifying such hypotheses with our EMSA systems are currently underway. The assays described here, binding, bending, and phasing, will also be used in testing the effect of HPFH mutations in the distal CCAAT region on NF-Y binding.