From the Département de Biologie et Génomique
Structurales, Institut de Génétique et de Biologie
Moléculaire et Cellulaire,
CNRS/INSERM/Université Louis Pasteur, 1 Rue
Laurent Fries, B.P. 10142, 67404 Illkirch Cedex, France and
Dipartimento di Biologia Animale, Università di
Modena e Reggio Emilia, Via Campi 213/d, 41100 Modena, Italy
Received for publication, September 19, 2002, and in revised form, October 18, 2002
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ABSTRACT |
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The heterotrimeric transcription factor NF-Y
recognizes with high specificity and affinity the CCAAT regulatory
element that is widely represented in promoters and enhancer regions.
The CCAAT box acts in concert with neighboring elements, and its
bending by NF-Y is thought to be a major mechanism required for
transcription activation. We have solved the structure of the
NF-YC/NF-YB subcomplex of NF-Y, which shows that the core domains of
both proteins interact through histone fold motifs. This histone-like
pair is closely related to the H2A/H2B and NC2 Transcription initiation by RNA polymerase II at class II gene
promoters is a finely regulated process requiring the interplay of many
different transcription factors (1). General transcription factors
(GTFs),1 namely TFIIA, TFIIB,
TFIID, TFIIE, TFIIF, and TFIIH, recognize specifically the core
promoters, recruit the RNA polymerase, and help melt the DNA, thus
enabling the initiation of transcription at the correct start site (2).
Assembly of this preinitiation complex is controlled by a large set of
transcriptional activators and repressors that recognize, in a
sequence-specific way, DNA sequences located on proximal or distal
enhancer regions of the promoters and function by contacting either
directly or indirectly, through co-activators and co-repressors, the
GTFs (1).
The eukaryotic transcription factor NF-Y (also termed CBF) specifically
recognizes the regulatory CCAAT element found in either orientation in
the proximal and distal enhancer regions of many genes (3-4). In
higher eukaryotes, this element is found in about 30% of the
promoters, preferentially in the NF-YC and NF-YB core regions are homologous in sequence to histones H2A
and H2B, respectively, and are required for heterodimerization, a
prerequisite for NF-YA association and CCAAT binding (8, 15-16). NF-YC
and NF-YB show an even higher sequence similarity with subunits The NF-YA core domain is less than 60 amino acids long and
is sufficient for DNA binding when complexed with NF-YC/NF-YB (9, 20-21). Contrary to NF-YC and NF-YB, careful examination of available data bases failed to reveal homologues of NF-YA. Several studies have
divided the NF-YA core domain into two segments: an N-terminal domain
responsible for NF-YC/NF-YB binding, and a C-terminal domain implicated
in specific recognition of the CCAAT element (20-24).
Once the trimeric complex is formed, it binds DNA with very
high specificity and affinity (6, 25). Specific recognition of the
bases seems to involve both minor and major groove interactions, and
circular permutation assays indicated that, upon binding, the DNA is
bent by about 60-80° (26-27). Footprinting and photocross-linking experiments have shown that the DNA is contacted by a subset of the
three subunits at three different locations, spanning about 24-26
nucleotides on each strand (6, 28). In agreement with these results, it
was shown that two CCAAT boxes cannot be occupied simultaneously,
unless they are separated by at least 22-24 bp (27, 29).
A major role of NF-Y is to act synergistically with other transcription
factors for activation. The CCAAT box is generally found in the close
vicinity of other promoter elements, and in many cases a precise
distance is required for proper transcription. Evidence that this
process requires CCAAT box bending and/or direct protein/protein
interactions has been reported repeatedly (4). Several lines of
evidence also indicate that NF-Y interacts directly with GTFs,
especially TFIID (30-32). Additionally, NF-Y has been shown to be the
target of regulatory proteins such as c-Myc (33) and
p53.2
We have started the structural characterization of
transcription factor NF-Y and have solved the structure of the complex between the conserved regions of human NF-YB and NF-YC by x-ray crystallography. The structure was refined at 1.6 Å resolution and
shows that both proteins interact through histone fold motifs in a
head-to-tail fashion. The structure is very close to that of H2A/H2B
and especially of NC2 Cloning, Expression, and Purification--
The various
constructs used for the co-expression study were amplified by standard
PCR procedures. All NF-YB constructs were inserted in the pACYC184-11b
vector (34), whereas all NF-YC constructs were inserted in the pET15b
(Novagen) and pGEX4T-2 (Amersham Biosciences) vectors, using
NdeI and BamHI restriction sites.
Co-expression tests were carried out using a standard procedure described previously (34). For large scale expression, 6× 1-liter cultures were grown, either in 2× LB medium for native complexes or in
M9 medium supplemented with seleno-methionine (Sigma) for seleno-methionylated complexes. Cells were grown at 37 °C to an absorbance of 0.3 at 600 nm, and the temperature was then switched to
25 °C. Growth was then carried on until cells reached an absorbance of 0.8-1.0 at 600 nm. At this point, co-expression of the complex was
induced by adding a final concentration of 1 mM
isopropyl- Crystallization--
For crystallization of the
NF-YC3/NF-YB3 complex, 2 µl of protein complex solution were mixed
with an equal volume of the reservoir solution containing 0.1 M NaHepes (Sigma), pH 7.5, 0.2 M
Mg(OAc)2 (Merck), and 10-14% PEG 4000 (Fluka). Crystals
appeared after a few hours and continued to grow for a few days or
weeks to reach a size of approximately 0.5 × 0.1 × 0.1 mm
(3). For the NF-YC2/NF-YB3 complex, the percentage of PEG 4000 had to
be raised to 20-24% to obtain crystals that were smaller and more clustered than for the NF-YC3/NF-YB3 complex. Only the latter dimer was
used for solving the phase problem with seleno-methionylated proteins.
For data collection, crystals were briefly transferred in a
cryoprotectant solution of 0.05 M NaHepes, 0.1 M Mg(OAc)2, 0.2 M NaCl, 13 or 22%
PEG 4000, 20% glycerol, and then quickly frozen in liquid ethane.
Structure Determination--
Data collection on
native and seleno-methionylated crystals was carried out on beamline
BM30A at the European Synchrotron Radiation Facility. A
three-wavelength multiwavelength anomalous diffraction experiment with
collection of data up to 1.8 Å resolution was first carried out, and
native data sets for the NF-YC3/NF-YB3 and NF-YC2/NF-YB3 complexes were
then collected at 1.57 and 1.67 Å resolution, respectively. All data
were processed and scaled using Denzo/Scalepack (35). Location of 5 of
the 6 selenium atoms was done using Shake and Bake (see Ref. 36). Their
positions were refined within the phasing program SHARP (37) and the
phases further improved with the solvent flattening program SOLOMON
(38).
Model Building, Refinement, and Modeling--
Model
building was carried out using program TURBO-FRODO (39). The model
built in the initial 1.9-Å resolution multiwavelength anomalous
diffraction electron density map was further refined independently
against both native data sets by several cycles of manual building and
refinement using standard protocols within the CNS (40). B-factor
restraints for bonded main chain and side chain atoms were 1.5 and 2.0, respectively. B-factor restraints for angle main chain and side chain
were 2.0 and 2.5, respectively. The coordinates of the NF-YC2/NF-YB3
complex have been deposited in the Protein Data Bank with code
1N1J.
Superimposition of NF-YC/NF-YB, H2A/H2B, H3/H4, and NC2
Modeling of the NF-YC/NF-YB/DNA complex was made by extracting a DNA
fragment from the structure of the nucleosome core particle once
superimposed as described above. Replacement of the bases and the
modeling of the interaction between NF-YA Production of NF-YC Mutants and EMSA
Experiments--
NF-YC mutants were produced by PCR mutagenesis with
the appropriate oligonucleotides in the backbone of the YC5 mutant
(41). The recombinant His-tagged YC5 mutants were obtained in inclusion bodies from BL21 bacteria, renatured with equimolar amounts of NF-YB,
and purified over nitrilotriacetic acid columns (29, 42). The resulting
dimers were assayed in immunoprecipitations and EMSA experiments with
recombinant NF-YA and the monoclonal antibody 7 monoclonal antibody
(42). Production and purifications of NF-Y and off-rate EMSA
experiments were done under conditions described previously (29,
43).
Structure Determination of the NF-YC/NF-YB
Complex--
All three subunits of NF-Y contain a core region that has
been highly conserved throughout evolution and, in the case of NF-YC and NF-YB, that displays sequence homology to the histone fold motifs
of H2A/NC2
For the subsequent crystallization trials, four of the six
soluble complexes obtained were used: NF-YC2/NF-YB3, NF-YC2/NF-YB4, NF-YC3/NF-YB3, and NF-YC3/NF-YB4 (see Table I). Small crystals were
initially obtained with the NF-YC3/NF-YB3 pair. Further refinement of
the crystallization conditions showed that crystals of NF-YC2/NF-YB3 could also be obtained. Both crystals belong to the same space group
with the same cell parameters (Table
II). Crystals of the seleno-methionylated NF-YC3/NF-YB3 complex were also grown and used for
solving the phase problem by multiwavelength anomalous diffraction
(44). An initial model was built manually into the experimental
electron density map at 1.9 Å resolution and was further refined
independently against native NF-YC3/NF-YB3 and NF-YC2/NF-YB3 data sets
at 1.57 and 1.67 Å resolution, respectively. The final models include
87 residues of NF-YB, 78 residues of NF-YC, about 300 water molecules,
and have R factors around 18% and R-free factors around 20%, with
very good deviations from ideal geometry (Table II).
In NF-YB3, no density was observed for the first seven residues. Mass
spectrometry revealed that all these residues except for the initial
methionine were present in the protein used for crystallization and
also in the crystals (data not shown). Thus, the N-terminal
residues of NF-YB3, which point toward a solvent channel, are probably
disordered. In NF-YC3, only the residual thrombin site residues Gly-Ser
at the N terminus were not unambiguously found in density. In the case
of the NF-YC2 construct, which is 16 residues longer than NF-YC3, no
additional residues could be built at the N terminus either. Once
again, mass spectrometry revealed that all the unobserved residues are
present in the crystals (data not shown). Because the initial
experimental phases were obtained for the NF-YC3/NF-YB3 complex, it
could be assumed that the initial model was not good enough to provide
phases for these residues. However, several loops in other parts of the
structure, which could not be seen in the initial electron density map,
appeared during refinement, whereas density at the N terminus of NF-YC2 never improved. Because a large solvent channel was found where the
residues should be located, it seems reasonable to assume that these
residues are disordered.
NF-YC/NF-YB Forms a Histone-like
Pair--
As expected, the core domains of NF-YB and NF-YC adopt a
histone-like fold and interact in a head-to-tail fashion, forming a
histone-like pair (Fig. 2A).
Interestingly, comparison of the NF-YC/NF-YB, NC2
One feature concerns the presence in both NF-YB and NF-YC
of an intra-chain arginine-aspartate bidentate pair which is found in
histones H3 and H4 but not in H2A and H2B (45) (Fig. 2A). In
NF-YC this pair is formed by residues Arg-93 (loop L2) and Asp-100
(helix
Another specific feature is the presence in NF-YC of an
absolutely conserved tryptophan at position 85, at the end of helix
Characteristic of the H2B family, a long Minimal DNA Fragment Required for Proper Binding by
NF-Y--
Previous footprinting experiments have shown that three
regions of the CCAAT boxes of the pro- Mutational Analysis on NF-YC DNA Binding by NF-YC/NF-YB--
The structure described
here confirms that the NF-YC/NF-YB histone pair is structurally closely
related to the H2A/H2B and NC2
The DNA fragments recognized by H2A/H2B (within the nucleosome core
particle) and NC2
Such an interaction was modeled by superimposing the NF-YC/NF-YB
structure onto a H2A/H2B dimer from the nucleosome core particle (see
"Experimental Procedures"). Upon modeling, no steric clashes between the NF-YC/NF-YB dimer and the DNA are observed (Fig.
4C). As in the nucleosome structure (45-46), both the DNA
interactions sites L1L2 (formed by loops L1 and L2 at both extremities
of the dimer) and
The interaction between NF-Y and the CCAAT box was further
modeled by replacing the DNA bases found in the nucleosome structure by
those of the pro-
The model further provides a good explanation for the results of our
EMSA experiments. Indeed, both strands are contacted by the
dimer at all the protected sites. Partial removal, on one strand only,
of one of the external sites would lead to smaller effects in terms of
binding, as is seen experimentally (Table III). On the other hand,
complete removal of one of these sites on both strands should have a
much drastic effect, which is the case when considering oligonucleotide
Ea-10. It is interesting to note that in the case of NC2 where the
histone pair recognizes a preformed TBP/TATA element complex, the
requirement for three interaction sites seems to be less stringent
(19).
Recognition of NF-YA and CCAAT Box Binding by
NF-Y--
Many biochemical studies have attempted to decipher the set
of interactions between yeast and mammalian NF-YA, NF-YC/NF-YB, and the
CCAAT box (15-16, 20-24, 29, 42). Most of these studies were
mutational analyses, performed either by point or deletion mutations.
Recollection of all these data in the light of our structure and of the
proposed model reveals that most of the mutants described can affect
dimer and/or dimer/CCAAT interaction as follows: (i) interfering with
the packing of the NF-YC/NF-YB dimer; (ii) destroying the dipole effect
of
Two regions of the core domain of NF-YA have been identified as
follows: an N-terminal region (NF-YA1; residues 234-257) recognizing the NF-YC/NF-YB dimer, and a C-terminal region (NF-YA2; residues 269-289) responsible for specific recognition of the DNA (20-24) (Fig. 1A). The N terminus of NF-YA1 forms an
From secondary structure prediction analysis, the NF-YA2 domain can be
divided into an
Several questions still remain. First, the orientation of the NF-YA2
helix is not known because the linker connecting NF-YA1 to NF-YA2 could
possibly either go through the space left between the NF-YC/NF-YB dimer
and the DNA or cross over to the DNA (Fig. 4D). Second, NF-Y
was shown to bind into the minor groove (6, 26), a fact that could
possibly be explained by having the NF-YA linker region crossing over
the DNA, although it cannot be excluded that the supposed coiled
C-terminal region of NF-YA2 could also play such a role. Third, the
flanking regions of the CCAAT pentanucleotide are crucial for efficient
binding of NF-Y. It is not known whether these bases can be recognized
specifically by NF-Y, or are necessary for proper distortion of the
DNA, or both. Finally, another unresolved question concerns NF-YA and
NF-YC having been shown by photocross-linking studies to interact more
extensively with CCAAT elements than the model would explain (28). We
suspect that this might be because of the interaction between the
activation domains of these two proteins and the DNA. Such open
questions, and possibly others, will only be answered with the
determination of the structure of the quaternary complex.
Structural and Functional Differences with
H2A/H2B and NC2
Next, NF-YC/NF-YB has also been shown to interact in
vitro with TBP but not with a preformed TBP/TATA element (30, 42). In the NC2/TBP/TATA structure, NC2 makes relatively few protein-DNA contacts, which could also be formed by NF-YC/NF-YB, and it recognizes TBP on both sides at two locations, thus encircling the DNA with TBP
(19). The strongest interaction corresponds to numerous contacts
between the
The previous examples suggest the existence of specific
determinants implicated in the functionality of each pair. Another aspect in which the NF-YC/NF-YB dimer might play a role independently from NF-YA association is related to the positive transcription function of NC2, recently unmasked on distal promoter elements, for
which the histone folds are sufficient (18). The mechanistic details
are poorly understood at present but clearly independent from TBP
binding, and are possibly related to facilitation of correct
connections between the DNA and the H3/H4-like
TAFIIs within TFIID. Because NF-YC/NF-YB is known to
interact with histone-like TAFs (32), it would be interesting to
investigate whether NF-YC/NF-YB might, in this case, play an
essentially identical positive role than NC2 at the distal promoter
element or whether other determinants make this process once again
NC2-specific.
The NF-YC /NC2
families, with
features that are both common to this class of proteins and unique to
NF-Y. The structure together with the modeling of the nonspecific
interaction of NF-YC/NF-YB with DNA and the full NF-Y/CCAAT box complex
highlight important structural features that account for different and
possibly similar biological functions of the transcriptional regulators NF-Y and NC2. In particular, it emphasizes the role of the newly described
C helix of NF-YC, which is both important for NF-Y trimerization and a target for regulatory proteins, such as MYC and p53.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
60/
100 region, and analysis of
various CCAAT boxes showed that specific flanking sequences are
required for efficient binding (5-7). NF-Y is a heterotrimeric complex
composed of NF-YA, NF-YB, and NF-YC which are all required for CCAAT
binding (8). Each subunit contains a core region that has been highly
conserved throughout evolution and that is sufficient for subunit
interactions and CCAAT binding, whereas the flanking regions, which
include the activation domains, are much less conserved (8-13). In
yeast, the activation function is encoded by a fourth subunit with no
apparent homologues in other species (14).
and
of NC2 (16), a protein that represses TATA
box-dependent transcription, while increasing the activity of the distal promoter element (17-18). The recent structure of a
NC2/TBP/TATA element complex confirmed that NC2
and -
subunits interact through histone fold motifs and that NC2 recognizes the preformed TBP/TATA complex (19).
/NC2
, but changes at the sequence and
secondary structure level provide explanations for various functional
roles played by these different complexes. Based on this overall
structural homology, which extends up to the electrostatic properties,
the interaction between the NF-YC/NF-YB dimer and DNA was modeled and
further extended to the full NF-Y/CCAAT element complex, in agreement
with several biochemical studies performed on NF-Y, including
footprinting experiments and mutational analyses. EMSA experiments were
also carried out which emphasized the importance of the NF-YC/NF-YB
histone-like pair in DNA binding and bending. Finally, the structure
reveals an important element of secondary structure, the
C helix of
NF-YC, which is not only involved in NF-YA binding but plays also a
role in the regulatory pathway of important growth regulators such as
MYC and p53.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-D-thiogalactopyranoside (Euromedex), and
cells were further grown overnight at 25 °C. Cells were then
collected by low speed centrifugation, resuspended in buffer A (10 mM Tris, pH 8.0; 400 mM NaCl), and lysed by
sonication. The soluble fraction recovered by high speed centrifugation
was mixed with 1 ml of Talon resin (Clontech) in
the case of a His-tagged complex or 1 ml of glutathione-Sepharose 4B
resin (Amersham Biosciences) in the case of a GST-tagged complex. After
1 h of incubation, the supernatant was removed and the resin
washed extensively with buffer A. The resin was then resuspended in 2 ml of buffer A, and 5 units of bovine thrombin (Sigma) were added
overnight at 4 °C for cleaving off the tag. The supernatant
containing the soluble dimer was recovered and applied onto a gel
filtration column Hiload 16/60 Superdex 75 (Amersham Biosciences)
equilibrated with buffer B (buffer A + 2 mM
1.4-dithiothreitol, Roche Molecular Biochemicals). The purified
complexes were concentrated on Microsep 10K Omega (Pall Filtron) to a
final concentration of ~10-14 mg/ml as assayed with Bio-Rad protein
assay (Bio-Rad).
/NC2
complexes was carried out using polyglycine models with our in-house
program Superpose,3 and the
transformations were applied onto the full models of the nucleosome
core particle (Protein Data Bank code 1AOI) and of the NC2/TBP/TATA
element complex (Protein Data Bank code 1JFI). The root mean square
differences were obtained from the superimposition of the polyglycine
model, removing additional residues but also helix
1-loop L1 in the
case of superimpositions with H2A and H3, because these elements
clearly have a different trajectory with respect to those of NF-YC or
NC2
.
-helices and the
NF-YC/NF-YB/DNA complex was carried out manually in TURBO-FRODO. The
coordinates of the model are available upon request.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and H2B/NC2
, respectively (Fig.
1). The core domains of NF-YB and NF-YC
have been shown to be necessary and sufficient for DNA binding in the
context of the trimeric complex (15-16, 20, 41). However, less
conserved stretches at their N and C termini seem to influence this
process (29). The majority of histone fold proteins are produced in
bacteria as insoluble material in inclusion bodies. We have studied the
formation of the NF-YC/NF-YB pair with protein constructs of different
lengths, by testing protein solubilization using the technique of
co-expression in Escherichia coli (34). The results
summarized in Table I indicate that only
the evolutionary conserved domains of NF-YC and NF-YB, but not the less
conserved regions, are necessary for complex formation.
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Fig. 1.
NF-Y subunits sequence
alignments. Sequence alignments of the core regions of NF-YA
(A), NF-YB (B), and NF-YC (C) from
human, Xenopus laevis, Drosophila melanogaster,
Caenorhabditis elegans, Arabidopsis thaliana,
Schizosaccharomyces pombe, and Saccharomyces
cerevisiae. In the case of A. thaliana, one sequence
was included for each subunit, but actually several different genes
coding for each subunit are found in its genome (13). For NF-YB and
NF-YC, the alignments also include the sequences from human H2B/NC2
and H2A/NC2
, respectively, and are based on the superposition of the
three structures. Human NF-Y subunits numbering is used. A,
fully and almost totally conserved residues of NF-YA core domain are
colored red and blue, respectively. Domains
implicated in NF-YC/NF-YB (NF-YA1) and DNA (NF-YA2) binding are
indicated, with important residues for each function boxed.
B and C, fully conserved residues in all, in the
NF-Y/NC2, and only in the NF-Y sequences are colored red,
green and blue, respectively. Secondary structure
elements (bars for
-helices and solid lines
for coils), as observed in the structure, are colored orange
above the alignments. Black solid lines indicate
regions present in the crystals that are not seen in density.
Intra-chain arginine-aspartate pairs have been schematically
represented in red. Boxed residues indicate amino
acids of H2A/H2B and NC2
/NC2
pairs that hydrogen-bond directly
the DNA backbone with at least main chain atoms (red boxes)
or only their side chains (blue boxes).
Summary of co-expression experiments
Data collection and refinement statistics
1, 0.979650; Seleko-Met
2, 0.979407; and
Seleko-Met
3, 0.977775.
/NC2
, H2A/H2B,
but also H3/H4 histone pairs, reveals relatively little differences
between their core histone motifs (helix
1-loop L1-helix
2-loop
L2-helix
3; see Fig. 2B) both in terms of sequence
identity (ranging from 10 to 20%) or pairwise main chain root mean
square differences (ranging from 1.5 to 1.1). Actually, that
NF-YC/NF-YB belongs to the H2A/H2B family is confirmed by the presence
of additional elements of secondary structure, at the C termini of both
proteins, characteristic of H2A and H2B, although H3/H4 features are
also observed (see below). The interactions between the various
elements of secondary structure of NC2
/NC2
and the comparison of
this pair with the H2A/H2B dimer have already been described at length
(19). The conclusions mostly apply to NF-YC/NF-YB and will not be
discussed further. Rather, we will focus on the differences and the
specificities we observe.
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Fig. 2.
Comparison of NF-YC/NF-YB, H2A/H2B,
NC2 /NC2
, and H3/H4
histone pairs. A, ribbon representations of the NF-YC/NF-YB
dimer (47), NF-YC and NF-YB, have been colored orange and
green, respectively, with elements of secondary structure
indicated. Arginine-aspartate pairs are displayed, together with Trp-85
of NF-YC that may play an important role in the specific interaction
between NF-YB and NF-YC, in comparison to NC2. B, stereo
C
traces of the superimposition of the NF-YC/NF-YB
(orange), H2A/H2B (gray), NC2
/NC2
(blue), and H3/H4 (green) histone pairs. The
tails of the histone proteins and H3
N helix have been removed for
clarity. C, superimposition of NF-YC/NF-YB
(orange) and H2A/H2B (gray) histone pairs. The
tails of H2A and H2B have been removed for clarity. The elements of
secondary structure showing major differences have been labeled.
D, superimposition of NF-YC/NF-YB (orange) and
NC2
/NC2
(blue) histone pairs. Helix
5 of NC2
has
been removed for clarity. NF-YC Trp-85 is displayed.
3), and both are absolutely conserved in the NF-YC but
neither in H2A nor in NC2
families (Fig. 1C). In NF-YB,
residues Arg-108 (loop L2) and Asp-115 (helix
3) form an identical
pair and are also absolutely conserved throughout evolution. Once
again, this pair is not conserved in NC2 and is replaced by an
arginine/lysine-glutamate pair in H2B (Fig. 1B). In this
latter case, however, the pair is not formed, and the arginine contacts
the DNA; interestingly, this is not seen in H3 and H4, where the pairs
are formed even if the arginines are in the vicinity of the DNA
backbone (45).
2, sandwiched between loop L2 of NF-YC and loop L1 of NF-YB (Fig. 2). Such a bulky residue at this position clearly influences the overall structure of this region. Interestingly, this amino acid is not
conserved in NC2
and the conformation of L1 of NC2
is different
from that of NF-YB. Because L2 of NC2
was not seen, it is impossible
to compare it to that of NF-YC. The difference in L1 loop conformation
is certainly dependent on their lengths; the length of NC2
is in
general one residue shorter, although in yeast it has the same length.
The hydrophobic cores organizing these regions are rather different,
with little difference observed in the rest of the structures. These
cores may be the reason why NF-YB/NC2
and NF-YC/NC2
pairs cannot
be formed (42). Notably, when both structures are superimposed, it is
clear that steric clashes would occur between residues of NC2
L1 and
Trp-85 which are not likely to be accommodated by conformational changes.
C helix is
found in NF-YB (Fig. 2). This helix is shorter than those of H2B and NC2
, but because additional residues at its C terminus seem to prevent crystallization, it is possible that it extends further. In
NF-YC, a loop-short helix-loop motif is found C-terminal to the core
histone fold (Figs. 2 and 3). A short
C helix is also found in H2A but is positioned rather differently;
it packs against the C terminus of helix
3 of H2A on one side, and
loop LC/start of helix
C of H2B on the other side, making few other
interactions with the rest of the dimer (Fig. 2C). The
packing is totally different in the case of NF-YC, where the
C folds
back onto
3 and participates in a large hydrophobic core formed by
residues of
2 and
3 of NF-YC and
2 of NF-YB. The interactions
between loop LC/helix
C of NF-YC and loop LC/helix
C of NF-YB are
fewer than in H2A/H2B, where loop LC/start of helix
C of H2B is
closer and interacts strongly with helix
C of H2A (Fig.
2C), especially with an absolutely conserved glutamate of
H2A being fixed by the dipole effect of helix
C of H2B.
Interestingly, the differences between these short
C helices extend
further, and as in the case of NF-YC this region does not fold as an
-helix, but a 310-helix (however, for clarity, the term
C has been kept). For technical reasons, the sequence spanning this
region in NC2
was replaced by unrelated residues, and this chimera
was subsequently used during crystallization studies of the
NC2/TBP/TATA element complex (19). Based upon the strong sequence
homology between NF-YC and NC2
in the
C region (residues
109-114) and in the rest of the secondary structure elements
participating in the hydrophobic core stabilizing it, we anticipate
that a helix is also present in NC2
at the corresponding position.
Whether additional residues at the C terminus of this helix adopt the
same loop conformation seen in NF-YC is not clear, as NF-YC and NC2
sequences tend to diverge from this point.
View larger version (39K):
[in a new window]
Fig. 3.
View of helix C of
NF-YC. A, ribbon representation of the NF-YC/NF-YB dimer
with a close-up view of the NF-YC
C region. Residues that have been
mutated in our study are shown and labeled (see also Table IV).
B, stereo figure showing the 2Fo
Fc electron density map contoured at 1.2
around
helices
3 and
C of NF-YC. For clarity, the orientation has been
slightly changed compared with A.
2(I) and pro-
1(I)
collagen promoters are protected upon NF-Y binding (6). We have
performed EMSA experiments on the Ea promoter to assess the minimal DNA fragment required for proper NF-Y binding. The results of dose-response and off-rate experiments performed with full-length as well as with a
mutant containing the conserved domains of the three subunits are
summarized in Table III. Essentially, all
the protected DNA stretches are important for proper recognition by
NF-Y. Partial removal of one of these regions generally leads to a
decrease in binding (oligonucleotides Ea-6, 12-Ea, and 8-Ea-2). The
effect observed is rather weak when considering the region the farthest from the CCAAT pentanucleotide (see oligonucleotides Ea-4, Ea-6, and
8-Ea-2), but deletion of this site results in an almost complete loss
of binding (oligonucleotide Ea-10). Besides, the comparison between
oligonucleotides of identical length (Ea-10 and 8-Ea-2) clearly shows
the asymmetry in position of the CCAAT pentanucleotide on the minimal
DNA fragment required for NF-Y binding.
EMSA experiments
C Residues--
NF-YC
helix
C has been shown to be important for NF-YA binding (15). We
have mutated certain residues of this helix (Fig. 3 and Table
IV), either solvent-exposed or buried in
hydrophobic cores, to further characterize its role in dimer and trimer
formation and DNA binding, using EMSA experiments. None of the
mutations affected dimerization, showing that the helix does not play
an important role in the interaction between NF-YB and NF-YC. The mutation of the solvent-exposed aspartate 112 into asparagine leads to
a decrease in trimerization but not in DNA binding (Table IV), showing
that this residue most probably plays a role in NF-YA binding but that
in the presence of DNA the trimeric interaction is stabilized. Two
mutations, F111S and L114T, were supposed to destabilize the
hydrophobic core in which the
C helix participates. From their
positions, the F111S mutation should destabilize the overall
hydrophobic core, whereas the L114T mutation should weaken the
anchoring of helix
C to the rest of the dimer. Both F111S and L114T
are indeed highly reduced in association with NF-YA. However, as for
the D112N mutant, DNA binding was not affected, confirming that in
presence of DNA the interaction with NF-YA is stabilized.
Two more radical mutations were performed on solvent-exposed isoleucine
residues, I115P and I117P, that are outside helix
C. Interestingly,
both mutations prevent trimerization but also DNA binding (Table IV).
On the other hand, we mutated isoleucine 115 into a lysine, the only
residue of human NC2
that is deviant within this conserved stretch.
Contrary to the I115P mutant, the I115K behaves like wild-type NF-YC in
dimerization, trimerization, and DNA-binding assays (Table IV), showing
that the conformation of the loop following helix
C is also
important for NF-YA binding.
Mutational analysis on NF-YC C helix
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/NC2
dimers and suggests that DNA
binding by NF-YC/NF-YB might also be similar. Both H2A/H2B and
NC2
/NC2
interact directly and non-specifically with DNA in a
multiprotein context, within the histone octamer and with TBP,
respectively. In these complexes, few direct protein/DNA contacts are
made by the core histones motifs (Fig. 1). A stable interaction between
these histone pairs and the DNA seems to require other protein
stretches, e.g. the histone tails and helix
5 of NC2
.
Such additional regions do not seem to exist in NF-YB and NF-YC, and it
is clear that to obtain the remarkable specificity and affinity for the
CCAAT sequence, NF-YA must stabilize the complex, although
photocross-linking experiments have confirmed that all three subunits
of NF-Y interact with DNA directly (28).
/NC2
(in complex with TBP and the TATA element)
have rather different conformations (in the latter case the DNA is
strongly distorted upon TBP binding). However, both complexes display
similar DNA binding properties (19), and the trajectory of these
fragments on the surface of these complexes is extremely similar (Fig.
4A). The electrostatic
properties of NF-YC/NF-YB are almost identical to those of H2A/H2B and
NC2
/NC2
; thus, it is tempting to postulate that the CCAAT box
would also follow an identical trajectory onto NF-YC/NF-YB (Fig.
4B).
View larger version (41K):
[in a new window]
Fig. 4.
Comparison of DNA binding by
H2A/H2B and NC2 and models of NF-YC/NF-YB/DNA and NF-Y/CCAAT complexes.
A, GRASP representation (48) of the electrostatic potential
at the surface of the NC2 dimer missing the 5 helix of NC2
. The
electrostatic potentials
8 and +8 kB T
(kB, Boltzmann constant; T, temperature)
are colored red and blue, respectively. The DNA
fragment of the NC2/TBP/TATA structure (19) is shown as ribbons colored
yellow/green. The DNA fragment spanning a H2A/H2B
dimer of the nucleosome (45) is shown as ribbons colored
red/blue. The figure was made after
superimposition of both histone pairs (see text). B,
electrostatic potential at the surface of the NF-YC/NF-YB dimer. The
modeled DNA is shown as ribbons colored red/blue.
C, model of the complex of NF-YC/NF-YB with the CCAAT
element from the pro-
2(I) collagen promoter. The DNA backbone is
shown as ribbons (purple) with the bases displayed. The two
possible locations of the CCAAT box, according to the modeling, have
been colored cyan. D, model of the NF-Y/CCAAT
complex. NF-YC, NF-YB and DNA are colored as in C, whereas
NF-YA is colored blue. The two alternative positions for the
linker connecting NF-YA1 and NF-YA2 sub-domains are shown as blue
dotted lines. Secondary structure elements of the histone pair
that are implicated in NF-YA1 and NF-YA2 recognition (see text) are
labeled and colored in red and gray,
respectively. For clarity, only the bases for the CCAAT pentanucleotide
are shown and labeled.
1
1 (formed by both
1-helices) are able to
make contacts with the DNA, and the dipoles of helices
1 and
2
also point toward phosphate groups. On the other hand, the two
arginines of H2A and H2B penetrating into the minor groove have not
been conserved. The same observation is true for Lys/Arg-29 of NC2
, the only residue contacting directly a base of the TATA element, which
is replaced by an absolutely conserved methionine in NF-YC. In fact,
careful inspection of the model could not identify any residue from the
core domains of NF-YB and NF-YC which would be able to make specific
contacts with a base of the CCAAT box. Besides, in this model the
histone dimer spans about 24-26 bp, which is in excellent agreement
with biochemical data (7, 27, 29).
2(I) collagen promoter. Two locations for the CCAAT
box are possible (depending on the strand chosen as the plus strand)
that are related by the pseudo 2-fold axis of the histone dimer (Fig.
4C). One of them agrees better with NF-YA binding (see
below). This model is in good agreement with the footprinting and
methylation interference experiments made on several promoters (6, 26).
In particular, the L1L2 sites would be responsible for interacting with
the protected sites at both extremities of the footprinted region and
are actually sufficient to explain the protection by hydroxyl radical
cleavage at these sites. Because these protein regions are not supposed to make specific contacts with the DNA, it would also explain why no
interference by methylation has ever been observed at these locations.
As for the
1
1 site, it only partially accounts for the central
region footprint; specifically, the CCAAT element itself, the only
region of the DNA where methylation interference occurs (6, 26), is not
protected by any region of the dimer (Fig. 4C). This clearly
suggests that this protection is brought by the third subunit,
NF-YA.
-helices
1 and
2 supposed to fix phosphate groups; and
(iii) abolishing interactions, or causing steric or electrostatic
hindrance, between the histone dimer and the DNA. Essentially, the vast
majority of the mutations, particularly those falling in the last two
classes, favor the NF-YC/NF-YB/CCAAT element model. The remaining
mutations that do not fall into these three classes have been further
considered to model the interaction of NF-YA with both the histone
dimer and the DNA.
-helix in
solution and only residues on one side of this helix are functionally
important (23). In NF-YB, mutations on helix
2 (E90R and S97R) were
shown to influence NF-YA binding (16, 22). In NF-YC, both helices
1
and
C have been shown to be important for NF-YA binding (15, 42)
(our mutational analyses). These three elements of secondary structure
are on one side of the NF-YC/NF-YB dimer and form a groove where NF-YA1
N-terminal
-helix could bind (Fig. 4D). Such an
interaction was modeled, showing that functionally important residues
of NF-YA1, such as Arg-245 and Arg-249, could contact residues at the
surface of the dimer, including Glu-90 and Ser-97. Interestingly, in
this model the conserved NF-YA Ile-246 would pack against Ile-117,
which is solvent-exposed in the loop following NF-YC helix
C. We
have shown that a mutation of the latter isoleucine into proline
completely abolishes NF-YA binding (Table IV), a result that further
highlights the importance of NF-YC C-terminal region in trimer
formation. Intriguingly, the model does not provide an explanation to
the fact that the D112N mutant is impaired in NF-YA recognition (Table
IV), showing that NF-YA1 binding possibly requires other interactions
than the ones mentioned above.
-helical N-terminal region (residues 269-281) and a
small coiled C-terminal region (residues 282-289), as for NF-YA1 (data
not shown). Several mutational experiments indicate that helix
1 of
NF-YB influences DNA recognition by NF-YA2 (16, 22, 42) and suggest
that these two regions interact directly, with NF-YB helix
1
possibly positioning NF-YA2
-helix in a correct orientation. We have
already mentioned that modeling of the CCAAT box left two possible
locations for this DNA sequence. Interestingly, NF-YB helix
1 is
positioned exactly below one of these two locations, on its major
groove side. Modeling of the interaction between the helical part of
NF-YA2 with both NF-YB helix
1 and the CCAAT pentanucleotide was
rendered difficult by the fact that essential residues of NF-YA2 might
be involved either in specific base recognition, in phosphate backbone
recognition, or might interact with NF-YB. Besides, one
cannot exclude that NF-YA binding distorts DNA into a conformation that
would be preferred for proper recognition by NF-YC/NF-YB, a fact that
could not be accounted for by our model. However, as it stands, the
model could fully explain the footprinting pattern observed for NF-Y on
the collagen promoter and our EMSA experiments.
/NC2
--
A lot of
controversy arises from the fact that the NF-YC/NF-YB, H2A/H2B, and
NC2
/NC2
histone pairs share sequence and structural similarity
but have different functional roles inside the nucleus. This is clearly
reinforced by the fact that their DNA binding characteristics are
strongly conserved and that the NF-YC/NF-YB pair has been shown to
interact with protein partners of the two other dimers. Indeed,
NF-YC/NF-YB can associate with H3/H4, but not with H2A/H2B, to form
higher order structures (43). After superposition of our dimer onto a
H2A/H2B dimer of the core nucleosome particle, we looked at the
possibility of forming (H3/H4)2(NF-YC/NF-YB)2 octamers reminiscent of the histone octamer. Clearly, such an hypothesis is not valid, as many steric clashes occur at the different interfaces between the pairs (data not shown). This result is in
agreement with previous data and with the fact that NF-YC/NF-YB can
also associate with formed nucleosomes, suggesting indeed that the
interactions between these dimers are rather different (43).
5 helix of NC2
and the C-terminal domain of TBP,
whereas at the other location an arginine side chain from TBP contacts
a main chain carbonyl and is stacked against other side chains of
NC2
. From sequence alignments, it is not clear whether NF-YB
contains a fifth helix which might contact TBP as NC2
does. If there
is any interaction between NF-YB and TBP, it would most probably be
different. In the case of NF-YC, the replacement of the absolutely
conserved Gly-28 in NC2
by Lys/Arg-59 would cause a
strong steric hindrance, preventing interaction with TBP. In
conclusion, and in agreement with experimental data, although the
determinants for DNA binding in the core histone regions of
NC2
/NC2
are conserved in NF-YC/NF-YB, recognition of TBP by this
latter complex must be rather different and cannot be achieved in the
context of a preformed TBP/TATA element, as observed with NC2.
C Region Is a Target for Regulatory
Proteins--
The distortion of the DNA by NF-Y, as modeled here,
possibly coupled to the recruitment operated by NF-YA and NF-YC
activation domains would make it possible for other gene-specific
regulatory factors (e.g. RFX, SREBPs, Sp1, and C/EBP) to
come in close vicinity of the GTFs, thus facilitating transcription
activation. More globally, the emergence in highly regulated promoters
of several CCAAT elements located 30-40 bases apart clearly raises the
question of the precise three-dimensional arrangement mediated by NF-Y, and of the requirement of such large DNA distortions for the
recruitment of incoming positive as well as negative cofactors. Recent
studies reveal that this process might be influenced by different
regulatory proteins in a promoter-dependent way and that
the nearly invariant NF-YC
C region, besides its role in NF-YA
binding, is a target element for these proteins. First, it has been
shown that the C terminus of the NF-YC core region is the docking site
for c-MYC and that this interaction is absolutely necessary for
transcriptional repression by c-MYC on the platelet-derived growth
factor
-receptor (33). Second, p53 transcriptional repression on
promoters having multiple CCAAT boxes has been shown to be dependent on
NF-Y, and once again, the
C region of NF-YC is required for this
process.2 The different location of NF-YC helix
C,
compared with that of H2A, reveals a unique specificity of the NF-Y and
also most probably of the NC2 sub-family. The overall strong
evolutionary sequence conservation between these both histone pairs
raises the question whether NF-Y and NC2, contrary to their different functional roles, could share common regulatory pathways. In this respect, it would be interesting to study whether c-MYC and p53 would
have any effects on NC2 known functions.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank the BM30A scientific staff, especially Richard Kahn, for help during data collection and initial processing. We also thank Noëlle Pottier, Sarah Sanglier, and Isabelle Billas for mass spectrometry analyses by electrospray.
![]() |
FOOTNOTES |
---|
* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The atomic coordinates and the structure factors (code 1N1J) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).
§ Supported by grants from MIUR-COFIN (Chromatin dynamics in transcription) and AIRC.
¶ To whom correspondence should be addressed. Tel.: 33-3-88-65-32-20; Fax: 33-3-88-65-32-76; E-mail: moras@igbmc.u-strasbg.fr.
Published, JBC Papers in Press, October 24, 2002, DOI 10.1074/jbc.M209635200
2 C. Imbriano, A. Gurtner, F. Cocchiarella, M. Gostissa, G. del Sal, G. Piaggio, and R. Mantovani, manuscript in preparation.
3 B. Rees, unpublished data.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: GTFs, general transcription factors; EMSA, electrophoretic mobility shift assay.
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