(Received for publication, April 12, 1995; and in revised form, July 6, 1995)
From the
Naturally occurring nondeletional mutations affecting the distal
CCAAT box of the human -globin gene promoter result in hereditary
persistence of fetal hemoglobin in adult life. Although the distal
CCAAT box is the target of several factors, including CP1/NFY, CDP,
GATA-1 and NFE3, only NFE3 binding activity is consistently sensitive
to well characterized mutations in this region such as G
A, C
T, and
13 hereditary
persistence of fetal hemoglobin. We extensively characterized the
binding specificities of NFE3 and demonstrated that NFE3 has unique
properties with respect to other CCAAT box-binding proteins.
Affinity-purified NFE3 from erythroid K562 cells binds the distal but
not the proximal human
-globin CCAAT box, the single CCAAT box of
the human
-globin promoter, and the proximal CCAAT box of the
evolutionarily related Galago crassicaudatus
-globin
gene. Within the
-globin CCAAT box, NFE3 represents the major and
almost exclusive binding activity. Disruption of such a binding site
essentially inactivates the
-globin promoter, suggesting that NFE3
plays an important role in the embryonic expression of this gene.
In humans, different types of hemoglobins are produced during
the embryonic, fetal, and adult stages(1) . The first
hemoglobin switch occurs early in gestation and involves the
substitution of - and
-globin for
- and
-globin,
respectively. At birth,
-globin synthesis is almost completely
switched off and is replaced by the synthesis of the major
-globin
and the minor
-globin chains. Functional studies of the
-like
globin cluster highlighted the role of the upstream locus control
region in promoting high level erythroid-specific expression of the
globin genes(2) . However, temporal regulation of the
expression of various globin genes, in particular
- and
-globin, appears to be largely dependent on sequences comprised
within the genes themselves or in their immediate
vicinity(3, 4) .
Several different approaches have been taken to analyze functional elements in the promoters of the human globin genes. Evolutionarily conserved DNA motifs were identified and provided clues to the discovery of DNA binding motifs(5, 6, 7) . Deletion and site-directed mutagenesis also identified potentially important motifs(8, 9, 10) .
Finally, the existence
of natural mutants within the human population provided in vivo evidence for the role of specific sequences (reviewed in (11) and (12) ). In particular, -globin promoter
mutations (
-thalassemia) demonstrated an important role of two
conserved motifs, the TATA and the CACC box, in promoter function;
surprisingly, no mutations have so far been identified in another
highly conserved element, the CCAAT box. However, although these
mutations significantly decrease
-globin expression, they do not
appear to modify its temporal regulation.
On the other hand,
mutations within the promoter of the fetal -globin gene do
increase, often substantially, its postnatal and adult expression. Some
of these mutations have been suggested to create better binding sites
for transcription factors, such as GATA1 and Sp1 (13, 14, 15, 16, 17, 18) ;
others decrease or abolish the in vitro binding of nuclear
proteins to the mutated
-globin fragments and have been suggested
to prevent the binding of negatively acting proteins that might repress
transcription of the
-globin gene
postnatally(13, 19, 20, 21) . The in vitro binding of one protein identified in these studies
(NFE3) is consistently decreased with four different hereditary
persistence of fetal hemoglobin (HPFH) (
)mutations ( (13) and (20) and present paper). We show that
purified NFE3, in addition to binding to the distal
-globin CCAAT
box, also represents the major
-globin CCAAT box-binding protein.
In the supershift experiments shown in Fig. 2and Fig. 7, the samples derived from extracts chromatographed onto a Sephacryl-300HR column. Fractions containing CP1/NFY or NFE3 activities were used.
Figure 2:
NFE3 is not an alternative form of
CP1/NFY. Sephacryl-300HR CP1/NFY or NFE3 fractions were incubated with
labeled DH in the presence of increasing amounts of antisera
against the A (>YA) or the B (>YB) subunits of
NFY or with two control antisera (>GATA1 and >Sp1). CP1/NFY and NFE3 complexes are indicated with arrows. The asterisks indicate nonspecific complexes.
For other abbreviations, see Fig. 1.
Figure 7:
CP1/NFY does not bind the -globin
CCAAT box. The Sephacryl-300HR fraction containing CP1/NFY but not NFE3
(see Fig. 2) was incubated with labeled
probe in the
presence of increasing amounts of antisera against the A (>YA) or the B (>YB) subunits of CP1/NFY.
Although the mobility of band A is similar to that of CP1/NFY,
the band is not supershifted by the antibodies (compare with Fig. 2). The asterisk indicates the same complexes as
shown in Fig. 2.
Figure 1:
Purification of NFE3. Gel mobility
shift assays representing the degree of NFE3 fractionation are shown.
In all cases the oligonucleotide probe was from the human -globin
distal CCAAT box (D
H). The fractions eluted at 0.4-2 M NaCl from the heparin column were also assayed by competition with
unlabeled D
H. CP1/NFY and NFE3 complexes are indicated. The asterisk indicates a nonspecific complex (see text). The
protein eluted from a
-affinity column was also tested by
competition assays with CCAAT box region oligonucleotides (indicated above the figure) from the adenovirus type 2 origin of
replication (Ad), the human
-globin promoter distal CCAAT
box (D
H), the human
-globin promoter CCAAT box (
), a CP1/NFY canonical binding site from the E
major histocompatibility complex gene promoter (NFY) and the
-117 HPFH mutation (-117) of the
-globin
promoter (see Table 1for nucleotide
sequences).
Transfection experiments in erythroleukemic K562 cells were according to (14) . For each construct, at least two independent preparations were tested in triplicate. In some experiments, luciferase reporter constructs were added to normalize for transfection efficiency; the results were essentially identical to those obtained in the absence of a cotransfected plasmid.
Nuclear or whole cell
extracts from K562 cells were first processed by conventional
chromatography to fractionate the NFE3 from CP1/NFY activity. This was
achieved by salt step elution of proteins bound to heparin-Sepharose.
The NFE3 activity eluted in the 0.6 and 1 M salt fractions (Fig. 1). In the 1 M fraction a faster migrating
complex was observed that is competed by DH and is likely due to a
proteolytic product of NFE3. Additional complexes, comigrating with
this band, are present in 0.4 and 2 M fractions. Because they
are not competed by D
H, they represent nonspecific
complexes(13) .
CP1/NFY eluted in the 0.4 M fraction, though its binding activity could be restored only after
dialysis against 0.1 M TM buffer (data not shown). The
heparin-Sepharose 0.6 and 1 M fractions were further processed
by Superdex 200 chromatography, and NFE3 was purified by affinity
chromatography on a resin containing concatamerized DH
oligonucleotide (Fig. 1).
Figure 3:
Methylation interference with binding to
the distal -globin CCAAT box region. A, NFE3 binding. B, CP1/NFY binding. C, schematic representation of
bases essential for the binding of NFE3 and
CP1/NFY.
Figure 4:
Binding of affinity-purified NFE3 to wild
type (WT) and HPFH
-globin CCAAT box regions.
Labeled probes are indicated below the figure. -114,
13, and -117 indicate HPFH
-globin mutated
oligonucleotides (see Table 1); for other abbreviations, see Fig. 1. A, direct binding. B, competition of
the binding by the unlabeled oligonucleotides indicated on the top of the figure.
Figure 5:
Binding of NFE3 to -globin and
D
H CCAAT boxes. The labeled probes are indicated below the figure; unlabeled competitors are indicated above the figure. X, unrelated oligonucleotide. A,
crude K562 nuclear extract. B, NFE3 fractions from the
- (lanes 1 and 2) and
-affinity resin (lanes 3 and 4) tested by gel mobility shift assay with either
D
H or
probes. C, competition by unlabeled
oligonucleotides of the binding of NFE3 eluted from
-affinity
resin to the
probe. Ad,
adenovirus.
DMS
interference analysis shows that the interaction of NFE3 with the
-globin CCAAT box is similar to that observed on the core D
H
CCAAT box element (see Fig. 3) but extends an additional 7 bases
upstream to the core motif (Fig. 6), suggesting that these bases
might be responsible for the stronger binding to the
-globin
probe.
Figure 6:
Methylation interference with binding to
the -globin CCAAT box region. Filled and open
circles, strong and weak interference, respectively. A,
top strand; B, bottom strand.
As shown in Fig. 5A the weak band a has a
mobility similar to that of the CP1/NFY complex with DH. However,
using either a crude or a CP1/NFY Sephacryl-300HR fraction (Fig. 7A, lane 1), the complex (Fig. 5A, band a) was not significantly
supershifted by either antiserum against CP1/NFY subunits A or B (Fig. 7, lanes 2-7) at concentrations that are
able to completely supershift the CP1/NFY complex with D
H (compare
with Fig. 2). Thus, NFE3 seems to be the major factor
interacting with the
-globin CCAAT box.
Figure 8:
Binding of NFE3 to human -globin and G. crassicaudatus CCAAT boxes. A, crude nuclear
extract. B, affinity-purified NFE3. Labeled oligonucleotides
are indicated below the figure; unlabeled competitors are
indicated above the figure. The proximal and distal
-globin CCAAT box regions from G. crassicaudatus are
P
G and D
G; the human
and
-globin CCAAT box regions
are
H and
H.
We analyzed two types of constructs containing the human
-globin promoter from -299 to +35 (H
CATwt) or the
human
-globin promoter from -220 to +18 (H
CATwt)
linked to the CAT reporter gene(26) . When K562 cells were
electroporated with either H
CATwt or H
CATwt, the observed CAT
expression levels were comparable. A CC
AA mutation in the
single
-globin CCAAT box (H
CATmut) that abolished the binding
of NFE3 decreased CAT expression to the level of a control promoterless
plasmid. When the
-globin GAC triplet immediately downstream from
the CCAAT box (positions -77 to -75) was replaced by an AGT
triplet restoring the critical G (position -109) on the D
H
region necessary for CP1/NFY binding (Fig. 3), the binding of
NFE3 was abolished, as expected from the DMS interference data (Fig. 6), but was replaced by the binding of CP1/NFY (not
shown). This mutant (H
CATmutAGT) was equally efficient as the wild
type
-globin construct in K562 cells (Table 2).
This
result shows that either a NFE3 binding or a CP1/NFY binding site is
sufficient (and necessary) for driving -globin promoter activity.
It should further be noticed that replacing the distal
-globin
CCAAT box within the
-globin promoter with the
-globin CCAAT
box had no effect (H
CAT
) (Table 2). These results have
been confirmed using some of the same constructs linked to the strong
46-base pair erythroid enhancer (28) located in the human locus
control region hypersensitive site II (Table 2).
In the present work, we report a further characterization of
the nuclear protein NFE3, a factor whose in vitro binding to
the -globin distal CCAAT box was previously shown to be affected
by HPFH mutations of this region (G
A and
-115 to -102 deletion, see (13) and (20) ). NFE3 appears to differ from CP1/NFY on the basis of
both immunological and functional (in vitro DNA-protein
interactions) criteria. In particular, antibodies directed against the
A or B subunit of CP1/NFY do not affect the formation of the NFE3-DNA
complex, ruling out the possibility that NFE3 is a heterodimer of
either CP1/NFY subunit with an unidentified additional protein (Fig. 2). In addition, DMS interference experiments show that
NFE3 and CP1/NFY both interact with nucleotides -117, -115,
and -114 of the
-globin promoter, but only NFE3 extends its
contacts to nucleotides -107 and -108 (Fig. 3). This
result is in agreement with the inability of the -117 and
-114 HPFH oligonucleotides to bind NFE3 ( Fig. 4and (13) ). Finally, the intensity of NFE3 binding to different
oligonucleotides is inversely correlated to that of CP1/NFY (see Fig. 5A and 8A; compare Fig. 2and Fig. 7).
The interaction of NFE3 with bases downstream to the
CCAAT box is likely to be important for sequence recognition. The most
5` C immediately downstream from the CCAAT box (position -108 in
the human -globin promoter) is conserved in the sequences (P
G
and
) able to bind NFE3 but not in those (D
G and P
H)
unable to bind it (Table 1); the G (antisense) residue at
position -108 is critical in DMS interference experiments with
D
H,
( Fig. 3and Fig. 6) and P
G (not
shown) oligonucleotides.
Other sequence differences, upstream to the
CCAAT box, must be responsible for the stronger binding of NFE3 to the
human -globin and the G. crassicaudatus
-globin
CCAAT box regions, as indicated by DMS interference analysis of the
-globin-NFE3 complex (Fig. 6).
The strong binding of
NFE3 to the human -globin and G. crassicaudatus proximal
CCAAT box regions was unexpected. Previous binding studies (5, 6, 7, 37) have employed
different oligonucleotides to ours, and no experiments with purified
factors have been reported. Recently, Gumucio et al.(38) reported gel shift patterns for the P
G and
human CCAAT box regions (but not for D
H) that are similar to those
reported by us to represent NFE3 binding and are likely to correspond
to it. They also proposed a TGACCT motif as the element mediating the
binding to P
G and
-globin oligonucleotides; however, this
element (antisense -91-96) is only partially DMS-sensitive
in the
-globin promoter (Fig. 6) and is modified to TGACCA
and TGACAA in D
H (Table 1). A less extensive TGAC binding
motif would, however, be fully consistent with the results of our DMS
interference experiments.
The observation that both the fetal
-globin gene and the embryonic human
-globin and G.
crassicaudatus
-globin genes have binding sites for NFE3
raises questions as to their role. The two embryonic genes have strong
NFE3 binding sites, while showing little (G. crassicaudatus proximal CCAAT box) or no (human
-globin gene) CP1/NFY
binding activities; in contrast, the fetal
-globin gene has two
very efficient CP1/NFY sites, and a low affinity NFE3 site. In
agreement with these observations, genomic footprint analysis in K562
cells shows protection of nucleotides -117 (top strand) and
-115 and -114 (bottom strand) in the distal CCAAT box of
the human
-globin gene and of the equivalent positions in the
proximal CCAAT box(39, 40) ; these results are better
explained by predominant occupancy of the distal CCAAT box by CP1/NFY
rather than NFE3 (see also Fig. 3) in cells expressing the
-globin gene. A mutation (H
CATmut) in the
-globin CCAAT
box that completely abolishes NFE3 binding results in the almost
complete inactivation of the
-globin promoter in transfection
experiments in K562 cells (Table 2); however, a different
mutation (H
CATmut.AGT) that also abolishes NFE3 binding but allows
substantial CP1/NFY binding results in essentially normal activity of
the promoter. Therefore these data suggest that, in the K562
environment, both NFE3 and CP1/NFY may act as positive transcription
factors. The unique ability of the human
-globin and G.
crassicaudatus embryonically expressed
-globin genes to
strongly bind NFE3, tempts one to speculate that NFE3 may be involved
in the positive regulation of the embryonic globin genes. A similar
idea was previously put forward by Gumucio et
al.(6, 7) , who observed the differential binding
of unidentified proteins to the G. crassicaudatusversus the human proximal
-globin CCAAT box (for further discussion,
see also (38) ). A resolution of these issues must await
transgenic assays and an accurate evaluation of the levels of NFE3
during development.
The present investigation was prompted by
evidence provided by HPFH mutations in the CCAAT box region that the
loss of NFE3 binding is a common factor in these conditions, suggesting
that NFE3 may potentially act as a repressor of -globin gene
activity. However, recent experiments (
)show that specific
disruption of the NFE3 site is not sufficient to cause adult
overexpression (HPFH) of the mutated
-globin gene in transgenic
mice.
Our present evidence that NFE3 may be a positively acting
factor for transcription (of the -globin gene) is therefore not in
contrast with the available information about its role in globin gene
regulation, although alternative explanations for the HPFH phenotype
caused by point mutations in the CCAAT box region must now be sought.