Development 115, 1149-1164 (1992)
Printed in Great Britain © The Company of Biologists Limited 1992
1149
The developmental switch in embryonic r-globin expression is correlated
with erythroid lineage-specific differences in transcription factor levels
MARK E. MINIE, TAKESHI KIMURA1 and GARY FELSENFELD*
Laboratory of Molecular Biology, Physical Chemistry Section, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of
Health, Bethesda, Maryland 20892, USA
1Present
Address: Ajinomoto Co., Inc., 15-1, Kyobashi 1-Chome, Chuo-Ku, Tokyo, 104 Japan
*Corresponding Author
Summary
During chicken embryogenesis, the r-globin gene is
expressed only in the early developmental stages. We
have examined the mechanisms that are responsible for
this behavior. The transcription of the r-globin gene is
strongly correlated with the presence during development of primitive erythroid lineage cells, consistent with
the idea that the expression of the r-globin gene is
restricted to that lineage. The “switching off” of r-globin
during development thus reflects the change from primitive to definitive cell lineages which occurs during erythropoiesis in chicken. We use transient expression
assays in primary erythroid and other cells to show that
the information for lineage- and tissue-specific
expression of the r-globin gene is contained in a 456 bp
region upstream of the gene’s translational start site.
DNA-binding studies, coupled with analysis of the effect
on expression of deletions and binding site mutations,
were used to identify important control elements within
this 456 bp region. We find that binding sites for the
ubiquitous transcription factor Sp1, and the specific
hematopoietic factor GATA-1, are crucial for
expression of the gene in primitive erythroid cells.
Quantitative analysis shows that nuclei of the primitive
erythroid lineage contain 10-fold more of these factors
than do the nuclei of definitive cells. We show that in
principle these differences in factor concentration are
sufficient to explain the lineage-specific behavior that
we observe in our assays. We suggest that this may be
an important part of the mechanism for lineagerestricted r-globin expression during chicken erythroid
development. Similar mechanisms may be involved in
regulation of other (but not all) members of the globin
family.
Introduction
each programmed to express exclusively one or the other
set of globin genes. Chickens have two erythroid cell types:
the primitive lineage cells, which predominate in the early
stages of development (3-6 days), and the definitive lineage
cells which become the majority cell type at later stages
(day 7 on) of embryogenesis.
Although erythroid stem cells are committed to one of
these two cell lineages quite early in hematopoiesis (Beaupain, 1985; Dieterlen-Lievre, 1988), some of the mechanisms for lineage-specific globin expression apparently
remain active throughout the lifetime of the red cell. Transient expression studies in primary erythroid cells have
shown that the promoters of the bA- and e-globin genes
carry sufficient information to confer stage-specific
expression (Choi and Engel, 1988; Hesse et al., 1986;
Lieber et al., 1987; Nickol and Felsenfeld, 1988). By implication, the mechanism necessary for this behavior must persist in these cells. The chicken bA-globin promoter, and the
b/e enhancer lying between the two genes, have been particularly well characterized. They contain regulatory ele-
The chicken a- and b-globin loci constitute two families
of highly conserved genes whose transcription is tightly
regulated during embryonic development. While the aAand aD-globin genes are constitutively expressed throughout development, the other members of the two globin gene
families exhibit differential activity as embryogenesis proceeds. In the early stages of development, the ap-, r- and
e-globin genes (the embryonic globin genes) are active,
while the bH- and bA-globin genes (the adult globin genes)
are silent. Later in chick embryogenesis, this pattern is
inverted, with the bH- and bA-globin genes active and the
embryonic genes silent (for a review, see Bruns and Ingram,
1973). The underlying cellular and molecular basis for this
‘globin switch’ is not well understood.
Considerable evidence supports a clonal model of
switching in chickens (Ingram, 1972; for review, see Nikinmaa, 1990). According to this model, cells expressing
embryonic or adult genes derive from distinct cell lineages,
Key words: hemoglobin switching, erythroid lineages, Sp1,
GATA-1, chicken erythropoiesis, r-globin, transcription factors.
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M. E. Minie and others
ments common to all globin genes, as well as some unique
to the bA-globin gene. In particular, expression of the bA
gene in definitive lineage cells seems to be dependent not
only on the presence of the enhancer, but also upon a stage
selector element (SSE; Choi and Engel, 1988) located
within the gene’s promoter (−39 bp, AAGAGGAGGGG).
The SSE appears to interact with either the ubiquitous transcription factor Sp1 or a definitive lineage specific factor,
NF-E4 (Evans, et al., 1990; Gallarda et al., 1989), which
may play a critical role in stage-specific expression of the
bA-globin gene. Other factors that bind to bA sequences
common to all globin genes may also play roles in modulating transcriptional activities (Lewis et al., 1988; Clark et
al., 1990; Jackson et al., 1989; Emerson et al., 1989).
Despite the presence of several common regulatory
motifs, there is no reason to think that the mode of globin
gene regulation in primitive lineage cells will necessarily
resemble that found in the definitive lineage. In order to
investigate the mechanism underlying the primitive lineagespecific expression of embryonic globins, we have focused
on the r-globin gene, a member of the b-cluster of genes.
We find that constructs containing 456 bp of sequence 5′
of the r gene translational start site mirror the lineage specificity of endogenous r-globin gene expression, and thus
contain the necessary information to direct lineage, as well
as tissue-specific expression. Deletion and site mutagenesis
reveal that both Sp1 and GATA-1 binding sites are essential to this behavior. Finally, we show by direct binding
studies that the amounts of Sp1 and GATA-1 factors vary
substantially between primitive and definitive cell nuclei,
with primitive lineage cell nuclei containing as much as 10fold more of these factors than definitive nuclei. These data
suggest that the lineage-specific expression of the r-globin
gene may depend, at least in part, upon lineage-specific differences in transcription factor abundance.
Materials and methods
Plasmids
The rCAT vector (Fig. 1A) was built in two steps. First, the region
between the NcoI site adjacent to the r-globin gene translational
start site and the SmaI site just 5′ of the cap site was synthesized
(66 bp), and inserted into the SmaI polylinker site of
pUC18CAT(#7) (gift from J. Hesse; Hesse et al., 1986). The 390
bp upstream of that region was isolated as a SmaI subfragment
from pB2H2 (Dolan et al., 1981), and subcloned into the first construct. The DNA sequence of this 456 bp region (see Fig. 3) was
determined by standard Sequenase (USB) protocols using appropriate oligonucleotide primers derived from the known 5′ sequence
of the r globin gene (Dodgson et al., 1983). The bACAT,
bACATE (pACAT and pACATE respectively in Hesse et al.,
1986) and RSVCAT constructs were provided by J. Hesse (Hesse
et al., 1986), and the aACATI construct (A′:J in Knezetic and
Felsenfeld, 1989) was obtained from J. Knezetic.
Deletion mutants of the rCAT construct were made by first iso lating the 390 bp SmaI fragment from the r insert. This was cut
with the appropriate restriction enzymes (Fig. 2) and the desired
subfragments were gel purified, filled in with Klenow enzyme,
and recloned into the SmaI cleaved vector employing standard
methods (Maniatis et al.,1983). Cluster mutants were generated
with an Amersham in vitro mutagenesis kit, using the synthetic
oligonucleotides described below and in the text. Each rCAT
mutant was given a numerical designation reflecting the oligomer
used to generate the mutant site, and the integrity of each mutant
was confirmed by sequencing. All plasmids were purified by either
standard CsCl gradient centrifugation (Maniatis et al.,1983) or
with a Qiagen plasmid Maxi kit (Qiagen). Plasmids isolated by
either method gave similar results.
Cells
Circulating red cells were isolated in bulk from appropriately aged
White Leghorn chicken embryos (Truslow Farms, Chesterton,
MD). Briefly, cells were isolated from early eggs (days 4-7, stages
24-31 at 37°C according to Hamburger and Hamilton, 1951) by
collection of 150 embryos into PBS at room temperature. Periodically, these early embryos were gently lacerated with sharp
scissors to promote bleeding. Once collection was completed, the
embryo bodies were removed by filtration through gauze, and the
cells were pelleted at 1000 revs/minute for 5 minutes at 5°C in a
Sorvall RT6000B centrifuge. Cells were further purified on Lymphocyte Separation Medium (LSM, Organon Teknika) according
to the manufacturer’s instructions. Cells were isolated from later
eggs (days 8-12, stages 35-38 at 37°C according to Hamburger
and Hamilton, 1951) by venous puncture of 48 embryos and collection of blood into PBS at room temperature, and purified as
described above. Typical yields per preparation for 4.5-5-day
embryos were ~1.5 3 109 cells; 9-12-day preparations produced
~3 3 109 cells. Smears were made of every preparation of cells
and stained by the May-Grünwald Giemsa method (Lucas and
Jamroz, 1961). It should be stressed that staging of embryos
according to the photographic series of Hamburger and Hamilton
(1951) was critical, and that simple chronological age was inadequate for estimating developmental age.
Chick embryo fibroblasts (CEFs) were prepared from 12-day
embryos according to standard protocols (Groudine and Weintraub, 1982), and grown in 8% fetal calf serum (FCS; GIBCO),
2% chick serum (CKS; GIBCO) in Dulbecco’s Modified Eagles
Medium (DMEM; GIBCO).
RNA isolation and primer extension analysis
Total cellular RNA from circulating red cells of each day of
embryonic development was prepared from frozen cell pellets
using the guanidine thiocyanate method (Chirgwin et al., 1979).
Primer extension (G. Felsenfeld and C. Trainor, unpublished; Reitman et al. 1990; Townes et al., 1985) was used to analyse the
RNA. Each reaction used RNA from ~5 3 106 cells. Synthetic
Table 1. Primer extension probes
Name
rEND1B
aA1B
GUP1B
Size
Sequence
21 bases
24 bases
21 bases
5′-ACT GTG TCC TGC TCT GGG AGC-3′
5′-GTT GTC AGC AGC GGA CAG CAC CAT-3′
5′-GGT GAT GAG CTG CTT CTC CTC-3′
Extension
product
r = 495 bases
aA = 64 bases
r and e = 84 bases
bH = 91 bases
bA = 116 bases
r-globin switching
1151
Fig. 1. (A) Schematic of the rCAT
expression vector. (B) Activities of
rCAT, aACATI, and bACATE
following transient transfection into
primitive (5-day) and definitive (12day) cells. Values are normalized to
the mean of RSVCAT (as a control
for transfection efficiency) ± s.e.m.
(n>10).
oligonucleotide probes for the globin gene RNAs used as primers,
and their respective extension products are described in Table 1.
The 20 nucleotide primer used to detect GATA-1 mRNA has been
described (Hannon et al., 1991). Products were quantitatively analyzed on a Molecular Dynamics PhosphorImager. Overall RNA
integrity in each preparation was indicated by the presence of
undegraded 18S and 28S RNA species in ethidium bromide
stained 1% agarose/MOPS-formaldehyde gels.
Transient transfection of primitive and definitive red cells
Mature primitive (from 4.5-5-day embryos, stages 24-27) and
definitive (from 9- or 12-day embryos, stages 35 and 38, respectively) erythroid cells were transfected with various plasmids as
described previously (Hesse et al., 1986; Lieber et al., 1987),
except that 13 A412 units of cells were used rather than 26. In
both cell types, 13 A412 units of cells were equivalent to about 3
3 107 cells. Following transfection, cells were maintained in
Liebovitz’s L15 media (GIBCO) + serum (see below) for 48 hours,
harvested in 0.1 M NH4HCO3 (containing 0.1 mM phenylmethyl-
sulfonylfluoride, PMSF) and frozen according to standard procedures. Transfected cell extracts were assayed for chloramphenicol
acetyl transferase (CAT) activity as previously described (Hesse
et al., 1986; Lieber et al., 1987). CEFs were transfected by the
DEAE-dextran method (Hesse et al.,1986), harvested by scraping
into 0.1 M NH4HCO3 (+ PMSF) and assayed according to standard methods (Hesse et al., 1986; Lieber et al., 1987). Acetylated
products of [14C]chloramphenicol were assayed either by liquid
scintillation counting or by PhosphorImager. All CAT activities
were normalized to that of RSVCAT, which was transfected in
parallel in every experiment to control for differences in transfection in the different cell types.
Initial results showed only low activity with standard post-transfection media (5% FCS, 2% CKS, and antibiotic-antimycotic mix),
but it was found that increasing the amount of chick serum to 30%
dramatically increased the rCAT activity in primitive cells, while
leaving RSVCAT controls virtually unchanged. The activity of the
endogenous r-globin gene in mock-transfected primitive cells, as
assayed by RNA dot blot (Blackman et al., 1986), showed a sim-
Fig. 2. Activities of rCAT deletion
constructs transfected into primitive
(5-day) and definitive (12-day) cells
(normalized to the mean of wild-type
rCAT) ± s.e.m. (n>10). BanI is
located at –246 bp upstream of the
cap site. Similarly, the BspMII site is
at –181 bp, the HpaII site at –98 bp,
and the SmaI site is at –23 bp.
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M. E. Minie and others
ilar dependence upon serum concentration. In contrast, FCS did
not have any stimulatory effect, indicating that factors specific to
CKS were responsible. The CKS did not, however, have a significant effect upon definitive cells. All subsequent erythroid transfections were carried out using media containing 30% chick
serum. This dependence on factors present in chick serum is not
entirely surprising in the light of previous reports of erythropoiesis-inducing factors in chick serum (Coll and Ingram, 1978;
Samarut, 1978, 1979; Samarut and Nigon, 1976), and a more
recent report of in vitro erythropoiesis induction with purified
TGFa, erythropoietin and insulin (Pain et al., 1991).
Nuclear extracts, DNAase I footprints and electrophoretic
mobility shift assays
Crude nuclear extracts were prepared essentially as described previously (Jackson et al. 1989; Evans et al. 1988), with the following modifications: (1) pellets of either fresh or frozen cells were
used, with no apparent differences between the two and (2) no
dialysis step was used, and samples were instead frozen (–70°C)
at this point. Buffer concentrations were adjusted prior to use by
addition of 1 3 binding buffer (Lewis et al., 1988). These two
changes resulted in lower levels of degradation and a 5-fold
increase in yield compared to previous methods. The integrity of
extracts was tested on 15-20% SDS-polyacrylamide gels (run on
a Phamacia PhastSystem apparatus), which revealed no substantial general degradation upon staining with Coomassie Blue, as
judged by the presence of intact globin and other bands (all nuclear
extracts unavoidably contained some contaminating hemoglobins,
attributable to their tremendous abundance in the red cells).
DNAase I footprinting on a 298 bp BanI/BamHI subfragment of
rCAT endlabeled at the Bam H1 site was performed according to
methods described by Kadonaga (1990).
Electrophoretic mobility shift assays followed methods previously described (Minie, 1986; Singh et al., 1986), except that 30
cm long gels were used and both poly d[I.C] and poly [d(A-T)]
served as non-specific competitors. In assays for abundances of
various factors, both Sp1 and BGP1 were measured using the
dsRGP2.0 probe (see Table 2), GATA-1 using the dsRGP3.0
probe, and PAL using oligomers containing either an ap PAL site
or bA PAL site (provided by G. Felsenfeld and J. Knezetic, unpublished results, and Jackson et al., 1989), which gave comparable
results. Sp1 was also measured using an oligonucleotide containing a canonical SV40 Sp1 binding site (Lewis et al., 1988), and
gave essentially the same results as those for the dsRGP2.0 probe.
Quantitation was done with a Molecular Dynamics PhosphorImager.
Measuring relative factor concentration
The relative amounts of a DNA-binding factor P in different
extracts can be obtained from gel mobility shift titrations, even
when a DNA competitor such as poly d[I.C] is used to suppress
binding by non-specific factors present in the extract. In typical
measurements, the specific DNA fragment is in large excess, so
that the free DNA concentration is nearly equal to the total DNA
concentration, i.e. (D)≈D0. Under such circumstances, we observe
that the fraction of DNA in complex is directly proportional to
the amount of specific protein factor added. In one set of experiments (see insert, Fig. 7C), we added increasing amounts of
nuclear extract to fixed amounts of DNA probe, and showed that
the fraction of the probe migrating as complex in the gel shift
assay was linearly dependent on the amount of extract added. In
other experiments, we used a pure synthetic peptide containing a
single GATA-1 finger as a test molecule (G. Felsenfeld, unpublished data). Again, the fraction of gel-shifted complex was proportional to the amount of peptide added.
Analysis of the equilibrium binding equations shows why this
method works. Let P0 be the total concentration of protein factor
P and N0 the concentration of non-specific competitor DNA (in
sufficient amount so that its concentration is not depleted by binding). The constant for specific binding is K1 = (DP)/(D)(P), and
for non-specific binding KN = (NP)/(N)(P), where (DP) is the concentration of specific complex and (NP) is the concentration of
non-specific complex. It is easy to show that
P0 = (DP)(1 + KNN0 + K 1D0)/(K1D0).
Under these conditions, the value of P0 is proportional to the
amount of complex whether or not most of the factor P is bound
non-specifically, and it is possible to compare relative amounts of
a given factor in different extracts. Provided the total probe concentration D0 is the same in all experiments, the ratio of band
intensity in the complex to unshifted band intensity is a measure
of P0, consistent with the experimental results.
Oligonucleotides
All oligonucleotides were synthesized on an Applied Biosystems
Synthesizer and gel purified following standard protocols.
Sequences of oligomers used for primer extension experiments are
given in Table 1 along with the expected extension products
(derived from Dodgson and Engel, 1983; Dodgson et al., 1983).
Sequences of oligomers used in mobility shift experiments are
given in Table 2. All oligomers for these experiments were endlabeled as single strands with T4 kinase as described by Lewis et
al. (1988). Those used as mobility shift probes were made double
stranded by annealing with their complementary strands using
conditions described in Lewis et al. (1988).
Results
The 456 bp 5′ of the r-globin gene contain the sequences
necessary for lineage and tissue specific regulation
A segment of DNA containing the 456 bp 5′ of the r-globin
gene translational start site was subcloned into a
pUC18CAT reporter vector (Hesse et al., 1986, and Materials and methods). This region is associated with a DNAase
I hypersensitive site specific to the primitive erythroid lineage (Stalder et al., 1980; Reitman and Felsenfeld, 1990).
This construct, designated rCAT (see Fig. 1A), was used
to explore whether elements in this region of the sequence
were involved in the regulation of r-globin gene transcription. The rCAT construct was transiently transfected into
primitive lineage red cells (Hesse et al., 1986; Lieber et al.,
1987), and CAT activity was assayed following 48 hours
of culture in optimal media (30% v/v chick serum; see
Materials and methods). rCAT was active in the primitive
lineage cells, and showed 25% of the activity seen with the
RSVCAT control (Fig. 1B). Transient transfection of the
rCAT vector into definitive lineage cells showed it was
nearly 10-fold less active in the definitive cells, with activity
only slightly in excess of a pUC18CAT control. Thus, the
rCAT construct mimicked the lineage specificity shown by
the endogenous r-globin gene (Figs 1B and 7B). Similarly,
the pattern of expression from aACATI (A:J′ in Knezetic
and Felsenfeld, 1990) and bACATE (a bA-globin
expression vector, pACATE in Hesse et al., 1986) paralleled the levels of their endogenous counterparts in primitive and definitive lineage cells (see Figs 1B and 7B). Furthermore, rCAT failed to show activity over pUC18CAT
r-globin switching
Table 2. Mobility shift probes, upper strands only
1153
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M. E. Minie and others
levels in CEFs, even though an RSVCAT control showed
high levels of CAT activity in those cells (data not shown).
We conclude that the r-gene promoter elements contained
in rCAT must carry the necessary control elements for both
the red cell lineage- and tissue-specific regulation of rglobin gene expression.
Deletions in rCAT define regions containing regulatory
elements
In order to analyze this promoter, a series of plasmids
having various deletions in the r promoter but otherwise
identical to rCAT were constructed (see Materials and
methods) and transiently transfected into both primitive and
definitive red cells (Fig. 2). Truncation of the promoter from
its 5′ side to the BanI site (–246 bp) resulted in only a 30%
loss of activity in the primitive cell background compared
to wild type. However, further truncation to the BspMII site
(–181 bp) resulted in a 70% loss of activity relative to the
wild type, and removal of sequence to the HpaII site (–98
bp) left merely 6%. These results suggested that sequences
located within the region spanning the BanI to HpaII sites
are critical to the regulation of the r-globin gene’s transcriptional activity. Site mutation experiments described
below confirm this view. None of the deletion constructions
gave activity greater than that of the pUC18CAT background control in definitive lineage cells.
Sequence analysis of the 5′ upstream region of the r-globin
gene
Examination of the 456 bp of 5′ sequence of the r-globin
gene indicated the presence of several regulatory elements
common to other globin genes (see Fig. 3). First, there are
two minimal BGP1 sites (GGGGGGG) (Lewis et al., 1988;
Clark et al., 1990) in opposite orientation to one another,
located at –279 and –125 bp, referred to here as BGP1distal
and BGP1proximal respectively. The proximal BGP1 site is
in opposite orientation to the distal one. Additionally, there
is a single GATA-1 site (AGATAAG) (Kemper et al., 1987;
Evans et al. 1988) located at –221 bp. There are also a
single, non-canonical Sp1 site (GGGGTGGG) (Li et al.,
1991) located at –50 bp and a TATA box located at −25
bp.
DNAase I footprinting of the region between –246 bp
and the translational start site indicated a complex pattern
of protection of these sequences by binding factors from
extracts of both primitive and definitive nuclei (data not
shown). Large regions of protection were seen between the
BanI site and the cap site of the gene, and it was not possible clearly to correlate specific protected regions with discrete binding sites. The complexity of the binding necessitated further dissection of the region using specific
double-stranded oligonucleotide probes in an electrophoretic mobility shift assay.
Definition of factor binding sites by the electrophoretic
mobility shift assay
The 456 bp of r-globin gene sequence was scanned for
specific binding activities by an electrophoretic mobility
shift assay using a series of double-stranded synthetic
oligonucleotide probes. These double-stranded r-globin
promoter (dsRGP) probes covered each region of the rglobin gene upstream sequence, and made it possible to dissect out and identify the various binding species (see Table
2 for list of probes). As shown in Fig. 4A-D, these
oligomers produced discrete binding species whose conjugate DNA-binding sites could be readily identified by competition with both wild-type and mutant probes. In typical
experiments, labeled probes containing putative binding
sites were competed with unlabeled oligomers containing
either mutated sites or binding sites for other factors (see
Fig. 4 for details).
As shown in Fig. 4A, the probe dsRGP4.0 forms a complex, in agreement with the sequence analysis suggesting
that this region contains a BGP1 site. The low levels of
binding activity seen, however, indicate that this site is
weak, consistent with previous observations of BGP1 binding to a minimal seven Gs (Clark et al., 1990), and consistent with the deletion of this region having little effect
upon activity (Fig. 2). No other high affinity binding complexes were observed with either primitive or definitive red
cell nuclear extracts, indicating that there are no other binding sites in this region. A mutant in which the BGP1 binding site was disrupted, dsRGP4.1, was unable to compete
for binding in a cold competition assay, further identifying
the distal G-string as a BGP1 binding site. A random duplex
oligomer, dsRandom (Lewis et al., 1988; Clark et al., 1990),
also failed to compete.
The single GATA site in the dsRGP3.0 (see Fig. 4B)
probe clearly binds to GATA-1 factor. No other specific
binding complexes are apparent, indicating no other binding sites exist in this region. This complex could be competed with a number of GATA probes (data not shown),
Fig. 3. Sequence of 456 bp r-globin gene insert in rCAT vector.
Sites for defined factor binding are highlighted.
r-globin switching
1155
Fig. 4. Electrophoretic mobility
shift assays of various regions of
the r-globin upstream region.
Assays were as described in
Materials and methods with
duplex oligonucleotides described
in Table 2 and in the text. The
amounts of extracts used were
optimized to allow easy resolution
of the two Sp1 complexes. All
cold competitions were done with
1003 molar excess relative to
labeled probe. Note that the open
arrow indicates a degradation
product of BGP1 which has been
reported elswhere (Clark et al.
1990). (A) dsRGP4.0 (B)
dsRGP3.0 (C) dsRGP2.0 (D)
dsRGP1.0.
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M. E. Minie and others
but was not competed with a dsRGP3.0 analogue
(dsRGP3.1) in which the GATA-1 site has been mutated to
a non-binding form.
Two distinct sets of binding complexes (Fig. 4C) are
associated with the region defined by the dsRGP2.0 probe:
(1) an upper pair of complexes, which could be competed
with oligomers containing Sp1 sites, as well as with an
oligomer containing a minimal BGP1 site (rhoG1; Clark et
al., 1990) and (2) a lower complex, which is competed only
with the oligomer containing the minimal BGP1 site. This
suggests that both Sp1 and BGP1 factors bind to sites within
this region. No canonical Sp1 sites appear in this region,
but it was possible to show with mutant oligomers that Sp1
binding indeed overlapped with the BGP1 binding site. As
shown in Fig. 4C, mutant oligomers dsRGP2.1 and 2.2 are
both incapable of competing out the upper bands, while
only dsRGP2.1 affects the BGP1 complex. The dsRGP2.1
oligomer contains mutations at the 5′ border of the proximal BGP1 binding site, while dsRGP2.2 contains a disabled
proximal BGP1 binding site. Conversely, dsRGP2.1 complexed with BGP1 but not Sp1, while dsRGP2.2 bound neither factor (data not shown).
The dsRGP1.0 oligomer (see Fig. 4D) also appeared to
generate two complexes similar to the upper complex bands
seen with dsRGP2.0, and the binding site of these factors
was shown to be the GGGGTGGG sequence by the lack
of competition with a cold mutant oligomer in which that
site was destroyed (dsRGP1.1). Furthermore, both
dsRGP1.0 and dsRGP2.0 oligomers mutually compete for
these two complexes (data not shown), indicating that the
same factor, Sp1, probably binds to both sites. This is the
only observable factor binding within this region. The site
within the dsRGP1.0 region is designated the Sp1proximal
site, while the site in the dsRGP2.0 region is referred to as
the Sp1distal. A TATA box is also present in this region,
but no TATA site-dependent binding complexes are
observed under the conditions used, consistent with the
instability of higher vertebrate TFIID reported by others
(Buratowski et al., 1988).
Both dsRGP1.0 and dsRGP2.0 Sp1 complexes were competed with a series of Sp1 sites derived from the bA-globin
gene promoter (a gift from T. Evans) as seen in Fig. 5A
(data shown for dsRGP2.0 only). The identity of the factors causing the upper complexes seen with both probes
was further verified by adding antisera specific for Sp1 (the
kind gift of Drs R. Tjian and S. Jackson). The amount of
the upper complexes was depleted by the anti-Sp1 antisera
due to hypershifting and binding interference (Fig. 5B).
These antisera had no effect upon the BGP1 complex.
Preimmune control antisera had no effect upon the band
shift patterns. Similar results were obtained for the complexes formed with the dsRGP1.0 probe (data not shown).
Site mutagenesis of the GATA-1, proximal Sp1, and TATA
sites
Having identified factor binding sites, we proceeded to
demonstrate that they are essential for r-gene expression.
Cluster mutants were introduced into the GATA, Sp1proximal
and TATA sites by in vitro mutagenesis (Materials and
methods), rendering each site unable to bind to its cognate
factor. A series of rCAT derivatives, each containing one
of the sites in its altered form, were tested for CAT
expression in primitive and definitive cells (Fig. 6). A
defective binding site for any one of these three factors
results in at least a 10-fold reduction in the activity of
rCAT. These observations are consistent with data obtained
from deletion mutants and strongly suggest a critical role
for each of these factors in the regulation of r-globin gene
activity.
Primitive red cell nuclei contain larger amounts of Sp1 and
GATA-1 than do definitive red cell nuclei
We wished to determine the relative nuclear abundance of
various erythroid cell components, and especially regulatory factors, at various developmental stages. To identify
erythroid cell types, smears of blood from each day of chick
development were made, stained by the May-Grünwald
Giemsa method (Lucas and Jamroz, 1961), and scored for
the presence of primitive and definitive cells. There are two
distinct types of red cell lineages within chick embryonic
development; their abundance in the embryonic circulation
as a function of the number of days of development is
shown in Fig. 7A.
We next analyzed the expression of the endogenous rglobin gene, as well as bA- and aA-globin as a function of
chick development. A primer extension assay was used to
measure the accumulated mRNAs of a variety of globin
genes. The globin genes are highly conserved, making
probe cross reaction a serious problem. The use of primer
extension allowed the development of probes which were
highly specific for each globin transcript (Dodgson and
Engel, 1983; Dodgson et al., 1983; see Materials and methods), thus eliminating this problem.
The expression of r-globin, bA-globin and aA-globin
mRNAs was assessed on a cellular basis at various stages
of chick development. r-globin mRNA accumulation
peaked at day 5 of chick embryo development, and then
decreased 10-fold by day 9 (Fig. 7B). The pattern of
expression of bA mRNA was nearly the inverse of the rglobin pattern, while the aA-globin pattern showed a significant level of expression for that gene at all stages of
development (Fig. 7B). These patterns of globin gene
expression agree well with earlier globin protein and RNA
measurements (Bruns and Ingram, 1973; Dodgson and
Engel, 1983; Dodgson et al., 1983; Landes et al., 1982; Lois
and Martinson, 1989).
We compared the relative abundance of individual DNAbinding factors using the electrophoretic mobility shift
assay (see Materials and methods). Nuclear extracts from
the circulating red cells of 4-12 day (inclusive) embryos
were prepared by methods that reduced the opportunity for
differential losses of factors and the extracts were assayed
for each of several binding species by gel mobility shift
assays (see Materials and methods and Jackson et al., 1989).
The amounts of Sp1 and GATA-1 per cell vary dramatically as the erythroid cell lineage switch proceeds in embryonic chick development (Fig. 7C). The amounts of Sp1 and
GATA-1 BGP1 in primitive cells are 10-fold greater than
the amounts found in definitive lineage cells. In contrast,
the amount of PAL site binding factor remains relatively
r-globin switching
1157
Fig. 5. (A) Cold competition of
complexes formed with dsRGP2.0
using various oligomers. All
competitions used a 1003 molar
excess of cold oligonucleotides,
which were added to reactions prior to
extracts. The open arrow indicates a
degradation product of BGP1. (B)
Depletion of upper dsRGP2.0
complexes by Sp1 antisera. 2 ml of
sera were added to each reaction prior
to the addition of extracts. After
extracts were added, reactions were
incubated for 5 minutes at room
temperature, and then loaded onto
gels. For convenience of presentation,
the top of the gel is not shown, but a
new complex of much slower
mobility was seen in the anti-Sp1
sera-treated samples, suggesting
hypershifting.
stable in embryonic development, consistent with previous
observations (Jackson et al., 1989; see Discussion), suggesting that the lower amounts of the other factors are not
due to differential extraction. Quantitation of the extracts
used in Fig. 7C was repeated three times, with similar
results (the ratio of 5-day to 11-day abundance was 9 ± 0.5
for Sp1 and 11.6 ± 0.9 for GATA-1). The extraction process
itself was repeated on other cell preparations; the 5-day:11day ratio was 12.7 ± 3 and 15.2 ± 4 for Sp1 and GATA1, respectively. The abudance of factors was determined by
mobility shift assays using extract from 1.4 3 106 erythroid
cells for each day of development. As a further control, the
mobility shift experiments were also normalized to the total
nuclear protein concentration in the preparation, with results
essentially identical to those obtained by using cell equivalents. It should be noted that this assay measures the
nuclear concentration of factor molecules capable of binding to DNA. It obviously will not detect modified factors
that cannot function in binding or transactivation.
We also carried out primer extension assays to measure
the abundance of GATA-1 mRNA in these cells. As shown
in Fig. 7B, this message undergoes a 4-fold decrease in cellular abundance as development proceeds, consistent with
the decrease in GATA-1 mRNA seen in avian virus transformed erythroblasts upon globin induction reported by
Yamamoto et al.(1990). The quite different abundance profiles of the globin mRNAs (Fig. 7B) suggest that the
decrease in GATA-1 mRNA in definitive lineage cells does
not reflect general RNA loss or degradation. (Because
chicken Sp1 has not been cloned, it is not yet possible to
monitor the abundance of Sp1 mRNA in the same way.)
These results imply that the nuclear concentrations of
1158
M. E. Minie and others
Fig. 6. Activities of rCAT cluster
mutants (normalized to the mean of
wild-type rCAT) ± s.e.m. (n>10).
Transfections were into primitive (5day) and definitive (9-day) cells.
Sp1 and GATA-1 factors, both critical to the activity of the
r-globin gene, are very different in primitive and definitive
erythroid cells. The observed decrease in GATA-1 mRNA
parallels the decrease in factor concentration.
Discussion
We ultimately wish to understand the ‘switching’ mechanism responsible for the difference between the primitive
lineage cells present in the circulation early in development,
which express ap, r and e, and the cells of the definitive
lineage present at later stages, which express bH and bA.
(aA and aD are expressed in both lineages). r-globin mRNA
accumulation correlates with the presence of primitive lineage erythroid cells in chicken embryogenesis. Conversely,
bA mRNA correlates with the appearance of definitive lineage cells. These data are consistent with the clonal model
of hemoglobin switching (see Introduction). Direct analysis of the globin proteins (Fucci et al., 1983, 1987; Mahoney
et al. 1977; Schalekamp and Van Goor, 1984), and similar
analysis of the mRNA species (Lois and Martinson, 1989)
present in purified populations of primitive and definitive
cells support this view. Furthermore, immunofluorescence
studies using antibodies specific for embryonic and adult
hemoglobins demonstrate lineage-specific restriction at the
level of the individual cell (Beaupain, 1985; Shimizu,
1976). Such studies also show that this restriction of globin
expression occurs even at the earliest stages of primitive
and definitive erythroid differentiation in the yolk sac
(Beaupain, 1985). This would suggest that the program for
selecting which globin genes are to be expressed is activated at early times in development, perhaps at the stem
cell level. Indeed, experiments using chicken/quail chimeras
indicate that primitive and definitive erythroid cells may
originate from two distinct, separate, precursor cell populations (Dieterlen-Lievre, 1975; for review, see Dieterlen-
Lievre, 1988). The basis for this selection program is not
known, but the mechanism responsible for it is maintained
throughout the lifetime of the cell and is thus amenable to
study by the methods used in this paper.
Sequences in the r 5 flanking region needed for lineage
and tissue specific activity
How is r-globin expression confined to the primitive lineage? To address this question, we examined the properties
of the 456 bp region immediately upstream of the r gene.
Plasmids containing this region coupled to the cat gene
were introduced into both primary chick embryo fibroblasts
and primary erythroid cells. Expression of cat was observed
only in the erythroid cells; furthermore, expression in erythroid cells was limited to the primitive lineage when sufficient chick serum was present to give optimal levels of
expression. The 456 bp r promoter region thus contains sufficient information to specify both tissue- and lineagespecific expression. We therefore set out to determine the
elements in the region that are responsible for this behavior.
As a preliminary step, we studied the effect of sequential 5′ deletions of the promoter. As shown in Fig. 2,
removal of the region between –246 bp and –181 bp in the
promoter results in a 70% reduction in cat expression in
primitive lineage cells; deletion of an additional 83 bp
reduces expression nearly to background. Neither the fulllength construction nor any of those with truncated promoters is active in definitive cells, arguing against a definitive cell-specific repressor.
Role of GATA and Sp1 sites in transcriptional activity
Our deletion studies identified promoter regions containing
binding sites for trans-acting factors. Next, we showed with
mobility shift experiments that actual binding occurs in
these regions. These experiments demonstrate that Sp1,
GATA-1, and BGP1 factors all bind to their defined cognate sites in the r-upstream region. No other factors were
r-globin switching
1159
Fig. 7. (A) Smears of blood samples used for the
RNA expression course were stained by the MayGrünwald Giemsa method and then scored (out of
several random fields of ~300 cells) for the presence
of primitive and definitive erythroid cells. (B)
Accumulation of r-, bA-,aA-globin RNA and GATA1 RNA per cell (5 3 106 cells/primer extension
reaction) as a function of chick development. Each
RNA was measured using a specific oligonucleotide
probe in a primer extension assay. The products of the
extension assay were quantitatively analyzed on a
Molecular Dynamics PhosphorImager. The highest
value for each RNA was arbitrarily set to 1. (C)
Estimations of the amounts of Sp1, GATA-1 and
PAL factors as a function of development measured
as described in Materials and methods. Values for
each factor were normalized to the highest value
measured for that particular factor. Sp1 was measured
using the dsRGP2.0 probe, GATA-1 with the
dsRGP3.0 probe and PAL with the ap PAL probe
(see Materials and methods). The amounts of factors
were measured with extract from 1.4 3 106 erythroid
cells per lane. Inset shows linear response of assay for
Sp1 in crude nuclear extract.
identified under the conditions used in these experiments.
In one case, both Sp1 and BGP1 were shown to bind to the
same site (BGP1 proximal/Sp1distal). A similar dual BGP1/Sp1
binding site has been observed in the promoter of the a
subunit of the chicken acetylcholine receptor gene (Piette
et al., 1989).
Interestingly, either Sp1 site can interact with Sp1 to form
a double band in gel shift experiments. In addition, members of the doublet vary in mobility depending on whether
primitive or definitive cell extracts are used. The doublet
bands of the primitive extract have a slightly but reproducibly higher mobility than those seen in the definitive
extracts. Furthermore, mixing experiments show that the
definitive extract pattern dominates the primitive extract
pattern (data not shown). These observations suggest that
the Sp1 complexes formed with definitive extracts are in
some way different from those in the primitive extracts,
possibly because of a covalent modification (such as phosphorylation or glycosylation) or an association with an as
yet unidentified co-factor (such as those reported by Dynlacht et al. (1991) to be associated with and necessary for
the activity of TFIID), and will be the subject of further
investigations.
Cluster mutations in the GATA-1, Sp1proximal or TFIID
sites in the r-upstream element individually result in a pro-
nounced loss of transcriptional activity, indicating that binding of any one of these factors to its site is crucial to rglobin gene transcription. A similar requirement for a functional GATA site and a functional CACCC site (which can
bind Sp1 as well as CON; Jackson et al., 1989) has been
reported for the erythroid-specific promoter of the human
porphobilinogen deaminase gene (Frampton et al., 1990).
Lineage-specific variation in the abundance of Sp1 and
GATA-1
Genes regulated by ubiquitous transcription factors may
nonetheless be affected by changes in the abundance of
those factors. We find that the abundance of several duplex
DNA-binding factors differs significantly in primitive and
definitive lineage cell nuclei. In particular, the amounts of
Sp1 and GATA-1 per nucleus decrease by a factor of about
10 as definitive red cells replace primitive cells in the circulation. Similar decreases in the amounts of GATA-1 in
chicken red cells have been reported elsewhere (Nicolas et
al., 1991; Perkins et al., 1989). Since the nuclear volumes
are similar in the two lineages, their intranuclear factor concentrations must also differ 10-fold. This difference is not
due to uniform losses of all binding activities during nuclear
1160
M. E. Minie and others
Fig. 8. (A) Schematic of proposed mechanism for the restriction of r-globin gene activity to the primitive erythroid lineage by
transcription factor abundance. Note that the association of Sp1 and GATA-1 depicted in the figure is not meant to imply an experimental
observation of such an interaction, but instead represents simply one possible manner of interaction between the two factors. (B) Plot of
fractional occupancy of r-globin Sp1 and GATA sites as a function of relative factor concentration, calculated from the equations in the
appendix.
extraction, as the amount of PAL factor detected remains
constant between days 4 and 12.
GATA-1 mRNA also undergoes a decrease in cellular
abundance as development proceeds (Fig. 7B). The lower
abundance of GATA-1 protein in primitive lineage cells
presumably reflects these lower levels of mRNA. It is
equally possible to argue that the lower levels of GATA-1
protein result in lower levels of mRNA, since the GATA1 promoter contains active GATA-1 binding sites (Hannon
et al., 1991). This autoregulation may be an important component of the switching mechanism.
We note that Whitelaw et al. (1990) report an increase
in GATA-1 mRNA at later stages of erythroid development
in the mouse. This may reflect a real difference between
chicken and mouse erythropoiesis.
Earlier reports from this laboratory (Jackson et al., 1989)
examined the change in the abundance of PAL protein
during development; the relative amounts of protein present
at different stages were calculated by normalizing to the
abundance of Sp1. By this measure PAL abundance
appeared to increase 10-fold between days 5 and 11 of
embryogenesis. Our present results show that this change
in the PAL/Sp1 ratio is attributable to a decrease in Sp1
concentration; in the 4-12 day period, PAL concentration
in fact remains constant as noted above. Previous measurements of other factors, such as GATA-1 and BGP1,
were also normalized to Sp1. These factors are now seen
also to undergo a decrease in abundance per cell between
days 4 and 12 of development. Nonetheless, there is clearly
a major increase in PAL concentration that occurs subsequent to day 12, the developmental stages which were the
focus of the earlier work.
Other ubiquitous transcription factors have been shown
to vary in their abundance in a tissue-specific manner
(Mitchell et al., 1991; Saffer et al., 1991; Snape et al., 1990,
1991), and these differences have been implicated in devel-
opmental regulation. For example, it has been proposed that
the protamine-2 gene in mouse spermatocytes is regulated
in part by changes in the concentration of Sp1 during spermatogenesis (Saffer et al. 1991; Bunick et al., 1990). It has
also been suggested that high cellular concentrations of Sp1
play a role in early hematopoiesis in mouse embryos (Saffer
et al., 1991). Similarly, it has been shown that AP-2 varies
in abundance in a tissue-specific manner in both Xenopus
and mouse development, and that high abundance of that
factor in certain cells may drive the expression of several
genes, including the keratin gene in Xenopus epidermal
tissue (Mitchell et al., 1991; Snape et al. 1990, 1991). Thus,
there is significant evidence that seemingly ubiquitous transcription factors vary in abundance in a developmentally
specific manner, and that this variation may play a role in
the stage-specific regulation of gene activity.
Our experiments do not rule out the possibility that other
sites proximal or distal to the r promoter also contribute to
expression of that gene. There may be other elements within
the promoter region that we examined that serve to modulate expression in a lineage-specific manner. Our results
show, however, that the Sp1 and GATA-1 binding sites are
critical to the lineage-specific expression revealed by our
transient expression studies. Furthermore, we show that the
reduction in abundance of Sp1 and GATA-1 that accompanies progression from the primitive to the definitive lineage would be sufficient to have a major effect on occupancy of these sites in vivo. Thus, sufficient information
for stage-specific expression would be contained in principle in the interactions with Sp1 and GATA-1 sites of the
upstream region that we have studied.
A model mechanism for the restriction of r-globin gene
activity to the primitive lineage
While the cellular basis of the hemoglobin switch in chick-
r-globin switching
ens appears to be explained by the existence of two separate erythroid lineages, the molecular mechanisms underlying the expression of specific globin genes in each cell
type remains unclear. We propose that the differences in
the abundances of Sp1 and GATA-1 between primitive and
definitive cells play a role in the cell type restriction of
globin gene expression in chicken and, in particular, may
explain the stage-specific response of the r promoter
described in this paper (Fig. 8A).
We have shown that both the Sp1proximal and GATA-1
sites of the r-globin gene promoter are necessary for the
activation of transcription. It seems reasonable to speculate
that these sites would be occupied in the factor-rich primitive cell. As shown in Fig. 8B (and in the Appendix), it is
possible to develop a quantitative description of how such
a system might work. Under suitable conditions of binding
constants and factor abundance, a 10-fold decrease in Sp1
and GATA-1 concentrations could alter the fraction of promoter sites occupied by both factors by nearly two orders
of magnitude. We suggest that the level of expression of r
is determined by the level of occupancy of the Sp1 and
GATA-1 sites by these factors; the calculated differences
would be quite sufficient to explain our results, and to
account for stage-specific regulation of r expression.
Implicit in this model is the assumption that the observed
effect of the AGATAAG and GGGGTGGGG cis-acting
elements in raising levels of r expression is due to the binding of GATA-1 and Sp1. The gel shift experiments show
that these are the greatly predominant binding components
in vitro, and transactivation studies in fibroblasts have
shown that expression from promoters carrying GATA-1
binding sites are stimulated by GATA-1 expression (Evans
and Felsenfeld, 1991). Nonetheless, we cannot rule out the
possibility that the effective trans-acting species in vivo are
in fact minor components unrelated to GATA-1 and Sp1,
or that only suitably modified versions of these factors are
capable of transactivation, or that the rate-limiting step in
control of transcription is the binding of undetected cofactors. We believe, however, that the model we propose gives
a simple explanation of the observations.
Further experiments could be designed to assess the
effect of altered site occupancy by manipulating the levels
of GATA-1 and Sp1 within erythroid cells, or by altering
the binding site affinities for these factors. Interestingly,
nature has already hinted at the result of such experiments.
The human Ag- and Gg-globin genes, which also exhibit
cellular restriction (Peschle et al., 1984, 1985), both exhibit
abnormal expression in patients with non-deletional hereditary persistence of fetal hemoglobin (HPFH) diseases. In
some of these patients, naturally occurring promoter mutations in Sp1 and GATA-1 binding sites have increased the
affinities of these sites for their cognate factors (Mantovani
et al., 1988; Ronchi et al., 1989; Sykes and Kaufman, 1990).
Transient transfection experiments have shown that these
mutants result in higher levels of promoter activity, and this
higher activity may result in the observed loss in cellular
restriction (Martin et al., 1989; Nicolis et al., 1989; Ronchi
et al., 1989). Such mutations suggest that simple alterations
in the affinities of either Sp1 or GATA sites for their cognate factors can result in the loss of lineage specificity.
It is instructive to compare r-globin gene expression with
1161
the definitive lineage-specific expression of the bA-globin
gene. The bA-globin gene promoter has Sp1 sites, and the
b/e enhancer downstream of the gene has a pair of GATA1 sites. In the absence of other mechanisms, and assuming
that our model is correct, the higher abundance of Sp1 and
GATA-1 in primitive than in definitive lineage cells might
be expected to result in a higher level of expression of bAglobin in primitive lineage cells, particularly given that the
Sp1 and GATA-1 sites in the neighborhood of bA have
twice the affinity of those near r (data not shown and
Letovsky and Dynan, 1989). The bA gene is presumably
prevented from being expressed in primitive lineage cells
by its absolute requirement for another factor, NF-E4, that
is found only in definitive lineage cells (Choi and Engel,
1988; Gallarda et al., 1989) and may cross-bind the Sp1
sites in the bA promoter (Evans et al., 1990). Stage-specific
expression of the bA gene thus depends upon a stagespecific regulatory factor. In contrast, expression of the r
promoter, as well as the ap globin gene (J. Knezetic and
G. Felsenfeld, unpublished), may be controlled by erythroid
lineage-specific differences in the concentration of ubiquitous factors. There is no reason a priori why individual
members of the globin families should make use of identical regulatory mechanisms. As information becomes available about the other a- and b-globins, it will be interesting
to see how many depend for stage specificity on the action
of special factors, and how many of these genes make use
of concentration-dependent mechanisms like those that we
have proposed here for the r-globin gene.
Appendix
A. Effects of concentration on binding of multiple transacting factors
We wish to estimate the effects of concentration on the
binding of Sp1 and GATA-1 to their sites in the r promoter. The data presented in this paper suggest that both
sites must be occupied for effective promoter function, and
that there is an approximately 10-fold difference in intranuclear concentration of each factor between 4-day and 12day embryonic cells. We therefore must calculate the fraction of sites occupied simultaneously by both factors as a
function of concentration. We first analyze the equations
for equilibrium binding to demonstrate the overall binding
behavior, and then use our binding data to estimate the
occupancy of promoter sites in vivo as a function of developmental stage.
B. Calculation of binding behavior
We assume that the promoter DNA can be doubly complexed to Sp1 and GATA-1 (DSG), singly complexed to
either one (DS or DG) or protein-free (D). If the binding
of S is independent of G,
K1 = (DS)/(D)(S) = (DSG)/(DG)(S) ;
K2 = (DG)/(D)(G) = (DSG)/(DS)(G) .
It is also essential to take into account the binding to nonspecific sites, with concentration N, and binding constant
KN, assumed for simplicity of illustration to be the same
for both factors. The non-specific binding constant
1162
M. E. Minie and others
KN = (NS)/(N)(S) = (NG)/(N)(G) .
Because non-specific DNA-binding sites are in vast excess,
the initial concentration N0 is unaffected by binding of S
and G, and this DNA acts as a buffer for the free concentrations of these factors. The activity of the promoter
depends on the concentration (DSG) of doubly occupied
sites. If the ratio of open plus singly occupied sites to
doubly occupied sites is f,
f=
KNN0
K1K2S0G0
[K1S0 + K2G0 + KNN0],
where S0 and G0 are the initial (total) concentrations of S
and G. For the sake of illustration and simplicity, we
assume that the occupancy by S and G of their DNA sites
is about the same, i.e. K1S0 = K2G0. This simplification
does not affect the general conclusions; in the next section,
more accurate, individual estimates are made of K1, K2, S0
and G0. The equation for f can be recast to give the fraction Q of all sites that are doubly occupied, as a function
of the total concentration of factor S0 (= G0):
Q = r2/(1 + 2r + r2),
where r = K 1S0/KNN0 = LS 0 and L is a constant. The equation allows us to calculate this fraction as a function of the
change in concentration of factors. The result is shown in
Fig. 8B, where both S0 and G0 are allowed to vary coordinately over a 20-fold range. For an approximately 10-fold
decrease in concentration (between 4 and 12 days), the
effective occupancy can change from 25% to 0.8%.
C. Occupancy of r promoter sites within the nucleus
The quantitative gel mobility shift data presented here, in
conjunction with the above analysis, make it possible to
estimate the fractional occupancy of the Sp1 and GATA
sites within the nucleus. We have previously measured the
specific affinity constant of chicken Sp1 (about 2.5 3 109,
cited in Clark et al., 1990); it is similar to that measured
for human Sp1 (Letovsky and Dynan, 1989). We have also
estimated the affinity constant of GATA-1 for a strong
binding site as ~109 (G. Felsenfeld and C. Trainor, unpublished data). Typically, non-specific binding constants are
about 10 4-fold smaller than specific constants. We can use
this information to estimate the number of regulatory factor
molecules present in nuclei: in 5-day cells, 15000 copies of
Sp1 and 4500 copies of GATA-1; in 9-day cells, 1300 of
Sp1 and 440 of GATA-1. To calculate fractional occupancy
of sites in the nucleus (see calculation above), we assume
that about 10% of the DNA in chromatin is accessible for
non-specific binding within the nucleus. We then calculate
that Q, the fraction of r promoter sites occupied simultaneously by Sp1 and GATA-1 is 0.079 in 5-day cells, and
0.0013 in 9-day cells, a 60-fold decrease, consistent with
the general calculation presented above. The point of the
general calculation above is to show that this behavior is
to be expected over a considerable range of input parameters, and is not highly sensitive to the actual values of binding constants, factor abundances or nuclear DNA concentrations.
We thank Drs A. Wolffe, J. Grasso, E. Bresnick, J. Hesse, D.
Clark, C. Trainor and all of our other collegues at the Laboratory
of Molecular Biology for their insightful discussions and critical
reviews of this work. Special thanks go to Drs J. Hesse and M.
Lieber for their advice on transfection protocols and timely
encouragement. Dr. Knezetic’s sharing of plasmids and unpublished observations are also gratefully acknowledged, as is the gift
of anti-Sp1 sera from Drs S. J. Clark and R. Tjian.
References
Beaupain, D. (1985). Line-restricted hemoglobin synthesis in chick
embryonic erythrocytes. Cell Diff. 16, 101-107.
Beaupain, D., Martin, C. and Dieterien-Lievre, F. (1979). Are
developmental hemoglobin changes related to the origin of stem cells and
site of erythropoiesis? Blood 53, 212-225.
Blackman, M. A., Tigges, M. A., Minie, M. E. and Koshland, M. E.
(1986). A model system for peptide hormone action in differentiation:
interleukin-2 induces a B lymphoma to transcribe the J chain gene. Cell
47, 609-617.
Bruns, G. A. P. and Ingram, V. M., (1973). The erythroid cells and
haemoglobins of the chick embryo. Philos. Trans. R. Soc. Lond. Ser. B.
Biol. Soc. 266, 225-305.
Bunick, D., Johnson, P. A., Johnson, T. R. and Hecht, N. B. (1990).
Transcription of the testis-specific mouse protamine 2 gene in a
homologous in vitro transcription system.Proc. Natl. Acad. Sci. U.S.A.
87, 891-895.
Buratowski, S., Hahn, S., Sharp, P. A. and Guarente, L. (1988). Function
of a yeast TATA element-binding protein in a mammalian transcription
system. Nature 334, 37-42.
Chirgwin, J. M., Przybyla, A. E., MacDonald, K. J. and Rutter, W. J.
(1979). Isolation of biologically active RNA from sources enriched in
ribonuclease. Biochemistry 18, 5294-5299.
Choi, O.-R. B. and Engel, J. D. (1988). Developmental regulation of bglobin gene switching. Cell 55, 17-26
Clark, S. P., Lewis, C. D. and Felsenfeld, G. (1990). Properties of BGP1, a
poly(dG)-binding protein from chicken erythrocytes. Nuc. Acids Res. 18,
5119-5126.
Coll, J. and Ingram, V. M. (1978). Stimulation of heme accumulation and
erythroid colony formation in cultures of chick bone marrow cells by
chicken plasma. J. Cell Biol. 76, 184-190.
Dieterlen-Lievre, F. (1975). On the origin of haemopoietic stem cells in the
avian embryo: an experimental approach. J. Embryol. Exp. Morph. 33,
607-619.
Dieterlen-Lievre, F. (1988). Birds. In Vertebrate Blood Cells, (ed. A. F.
Rowley and N. A. Ratcliffe) pp. 257-336. Cambridge University Press.
Dodgson, J. B. and Engel, J. D. (1983). The nucleotide sequence of the
adult chicken a-globin genes. J. Biol Chem. 258, 4623-4629.
Dodgson, J. B., Stadt, S. J., Choi, O-R., Dolan, M., Fischer, H. D. and
Engel, J. D. (1983). The nucleotide sequence of the embryonic chicken
b-type globin genes. J. Biol Chem. 258, 12685-12692.
Dolan, M., Sugarman, B. J., Dodgson, J. B. and Engel, J. D.
(1981).Chromosomal arrangement of the chicken b-type globin genes.
Cell 24, 669-677.
Dynlacht, B. D., Hoey, T. and Tjian, R. (1991). Isolation of coactivators
associated with the TATA-binding protein that mediate transcriptional
activation. Cell 66, 563-576.
Emerson, B., Nickol, J. and Fong, T. C. (1989). Erythroid-specific
activation and derepression of the chick b-globin promoter in vitro. Cell
57, 1189-1200.
Evans, T., and Felsenfeld, G. (1991). trans-Activation of a globin
promoter in nonerythroid cells. Mol. Cell Biol. 11, 843-853.
Evans, T., Felsenfeld, G. and Reitman, M. (1990). Control of globin gene
transcription. Ann. Rev. Cell Biol. 6, 95-124.
Evans, T., Reitman, M. and Felsenfeld, G. (1988). An erythrocytespecific DNA-binding factor recognizes a regulatory sequence common
to all chicken globin genes. Proc. Natl. Acad. Sci. U.S.A. 85, 5976-5980.
Frampton, J., Walker, M., Plumb, M. and Harrison, P. R. (1990).
Synergy between the NF-E1 erythroid-specific transcription factor and
the CACCC factor in the erythroid-specific promoter of the human
porphobilinogen deaminase gene. Mol. Cell Biol. 10, 3838-3842.
Fucci, L., Cirotto, C., Tomei, L. and Geraci, G. (1983). Synthesis of
r-globin switching
globin chains in the erythopoietic sites of the early chick embryo. J.
Embryol. Exp. Morph. 77, 153-165.
Fucci, L., Vitale, E., Cirotto, C. and Geraci, G. (1987). Evidences that
hemoglobin switch in the chick embryo depends on erythroid cell line
substitution. Cell Differentiation. 20, 55-63.
Gallarda, J. L., Foley, K. P., Zhuoying, Y. and Engel, J. D. (1989). The bglobin stage selector element factor is erythroid-specific
promoter/enhancer binding protein NF-E4. Genes Dev. 3, 1845-1859.
Groudine, M. and Weintraub, H. (1982). Propagation of globin DNase Ihypersensitive sites in absence of factors required for induction: a
possible mechanism for determination. Cell 30, 131-139.
Hamburger, V. and Hamilton, H. L. (1951). A series of normal stages in
the development of the chick embryo. J. Morph. 88, 49-92.
Hannon, R., Evans, T., Felsenfeld, G. and Gould, H. (1991). Structure
and promoter activity of the gene for the erythroid transcription factor
GATA-1.Proc. Natl. Acad. Sci. U.S.A. 88, 3004-3008.
Hesse, J. E., Nickol, J. M., Lieber, M. R. and Felsenfeld, G. (1986).
Regulated gene expression in transfected primary chicken erythrocytes.
Proc. Natl. Acad. Sci. U.S.A. 83, 4312-4316.
Ingram, V. M. (1972). Embryonic red blood cell formation. Nature 235,
338-339.
Jackson, D. P., Evans, T., Nickol, J. M. and Felsenfeld, G. (1989).
Developmental modulation of protein binding to b-globin gene
regulatory sites within chicken erythrocyte nuclei. Genes Dev. 3, 18601873.
Kadonaga, J. (1990). JK Lab Techniques. 3rd Edition.
Kemper, B., Jackson, P. D. and Felsenfeld, G. F. (1987). Protein-binding
sites within the 5′ DNAase I-hypersensitive region of the chicken aD globin gene. Mol. Cell Biol. 7, 2059-2069.
Knezetic, J. A. and Felsenfeld, G. (1989). Identification and
characterization of a chicken a-globin enhancer. Mol. Cell Biol. 9, 893901.
Landes, G. M., Villeponteau, B., Pribyl, T. M. and Martinson, H. G.
(1982). Hemoglobin switching in chickens: is the switch initiated post
transcriptionally? J. Biol. Chem. 257, 11008-11014.
Lewis, C. D., Clark, S. P., Felsenfeld, G. and Gould, H. (1988). An
erythrocyte-specific protein that binds to the poly (dG) region of the
chicken b-globin gene promoter. Genes Dev. 2, 863-873.
Letovsky, J. and Dynan, W. S. (1989). Measurement of the binding of
transcription factor Sp1 to a single GC box recognition sequence. Nuc.
Acids Res. 17, 2639-2653.
Li, R., Knight, J. D., Jackson, S. P., Tjian, R. and Botchan, M. (1991).
Direct interaction between Sp1 and the BPV enhancer E2 protein
mediates synergistic activation of transcription. Cell 65, 493-505.
Lieber, M. R., Hesse, J. E., Nickol, J. M. and Felsenfeld, G. (1987). The
mechanism of osmotic transfection of avian embryonic erythrocytes:
analysis of a system for studying developmental gene expression. J. Cell
Biol. 105, 1055-1065.
Lois, R. and Martinson, H. G. (1989). Chicken globin gene transcription is
cell lineage specific during the time of the switch. Biochemistry 28, 22812287.
Lucas, A. M. and Jamroz, C. (1961). Atlas of avian hemotology.
Agricultural Monograph 25, USDA.
Mahoney, K. A., Hyer, B. J. and Chan, L. (1977). Separation of primitive
and definitive erythroid cells of the chick embryo. Dev. Biol. 56, 412-416.
Maniatis, T., Fritsch, E. F. and Sambrook, J. (1983). Molecular Cloning:
a Laboratory Manual. New York: Cold Spring Harbor Laboratory.
Mantovani, R., Malgaretti, N., Nicolis, S., Rochi, A., Giglioni, B. and
Ottolenghi, S. (1988). The effects of HPFH mutations in the human gglobin promoter on binding of ubiquitous and erythroid specific nuclear
factors. Nuc. Acids Res. 16, 7783-7797.
Martin, D. I., Tsai, S-F. and Orkin, S. H. (1989). Increased g-globin
expression in a non-deletion HPFH mediated by an erythroid-specific
DNA-binding factor. Nature 338, 435-438.
Minie, M. E. (1986). The regulation of J chain gene transcription: Role of
chromatin structural changes and protein-DNA interactions. Ph.D.
Thesis, U. C. Berkeley.
Mitchel, P. J., Timmons, P. M., Herbert, J. M., Rigby, P. W. J. and
Tjian, R. (1991). Transcription factor AP-2 is expressed in neural crest
cell lineages during mouse embryogenesis. Genes Dev. 5, 105-119.
Nickol, J. M. and Felsenfeld, G. (1988). Bidirectional control of the
chicken b- and e-globin genes by a shared enhancer. Proc. Natl. Acad.
Sci. U.S.A. 85, 2548-2552.
Nicolas, R. H., Partington, G., Major, G. N., Smith, B., Carne, A. F.,
1163
Huskisson, N. and Goodwin, G. (1991). Induction of differentiation of
avian erythroblastosis virus-transformed erythroblasts by the protein
kinase inhibitor H7: analysis of the transcription factor EF1. Cell Growth
Differ. 2, 129-135.
Nicolis, S., Antonella, R., Malgaretti, N., Mantovani, R., Gilglioni, B.
and Ottolenghi, S. (1989). Increased erythroid-specific expression of a
mutated HPFH g-globin promoter requires the erythroid factor NFE-1.
Nuc. Acids Res. 17, 5509-5516.
Nikinmaa, M. (1990). Vertebrate Red Blood Cells: Adaptations of Function
to Respiratory Requirements. New York: Springer-Verlag.
Pain, B., Woods, C. M., Saez, J., Flickinger, T., Raines, M., Peyrol, S.,
Moscovici, C., Mocovici, M. G., Kung, H.-J., Jurdic, P., Lazarides, E.
and Samarut, J. (1991). EGF-R as a hemopoietic growth factor receptor:
the c-erbB product is present in chicken erythrocytic progenitors and
controls their self-renewal. Cell 65, 37-46.
Perkins, N. D., Nicolas, R. H., Plumb, M. A. and Goodwin, G. H. (1989).
The purification of an erythroid protein which binds to enhancer and
promoter elements of haemoglobin genes. Nuc. Acids Res. 17, 1299-1314.
Peschle, C., Migliaccio, A. R., Migliaccio, G., Pentrini, M., Calandrini,
M., Russo, G., Mastroberardino, G., Presta, M., Gianni, A. M., Comi,
P., Giglioni, B. and Ottolenghi, S. (1984). Embryonic>fetal Hb switch
in humans: studies on erythroid bursts generated by embryonic
progenitors from yolk sac and liver. Proc. Natl. Acad. Sci. U.S.A. 81,
2416-2420.
Peschle, C., Mavillio, F., Care, A., Migliaccio, G., Migliaccio, A. R.,
Salvo, G., Samoggia, P., Petti, S., Guerriero, R., Marinucci, M.,
Lazzaro, D., Russo, G. and Mastroberardino, G. (1985). Haemoglobin
switching in human embryos: asychrony of z>a and e>g-globin switches
in primitive and definitive erythropoietic lineage. Nature 313, 235-238.
Piette, J., Klarsfeld, A. and Changeux, J.-P. (1989). Interaction of nuclear
factors with the upstream region of the a-subunit gene of chicken muscle
acetylcholine receptor: variations with muscle differentiation and
denervation. EMBO J. 8, 687-694.
Reitman, M. and Felsenfeld, G. (1988). Mutational analysis of the chicken
b-globin enhancer reveals two positive-acting domains. Proc. Natl. Acad.
Sci. U.S.A. 85, 6267-6271.
Reitman, M. and Felsenfeld, G. (1990). Developmental regulation of
topoisomerase II sites and DNase I-hypersensitive sites in the chicken bglobin locus. Mol. Cell. Biol. 10, 2774-2786.
Reitman, M., Lee, E., Westphal, H. and Felsenfeld, G. (1990). Siteindependent expression of the chicken bA-globin gene in transgenic mice.
Nature 348, 749-752.
Ronchi, A., Nicolis, S., Santoro, C. and Ottolenghi, S. (1989). Increased
Sp1 binding mediates erythroid-specific overexpression of a mutated
(HPFH) g-globulin promoter. Nuc. Acids Res. 17, 10231-10241.
Saffer, J. D., Jackson, S. P. and Annarella, M. B. (1991). Developmental
expression of Sp1 in the mouse. Mol. Cell Biol. 11, 2189-2199.
Samarut, J. (1978). Isolation of an erthropoietic stimulating factor from the
serum of anemic chicks. Exp. Cell Res. 115, 123-126.
Samarut, J. (1979). Erythroietin-like factor production by young chick
blastoderms. Dev Biol. 70, 278-282.
Samarut, J. and Nigon, V. (1976). In vitro development of chicken
erythropoietin-sensitive cells. Exp. Cell Res. 100, 245-248.
Schalekamp, M. and van Goor, D. (1984). Haemoglobin and globin
synthesis in the isolated primitive and definitive erythroid cells of chicken
embryos. Evidence for a non-clonal mechanism at the haemoglobin
switch. J. Embryol. Exp. Morph.84, 125-148.
Shimizu, K. (1976). Identification of hemoglobin types contained in single
chicken erythrocytes by fluorescent antibody technique. Devl Biol. 48,
317-326.
Singh, H., Sen, R., Baltimore, D. and Sharp, P. A. (1986). A nuclear factor
that binds a conserved sequence motif in transcriptional control elements
of immunoglobulin genes. Nature 319, 154-158.
Snape, A. M., Jonas, E. A. and Sargent, T. D. (1990). KTF-1, a
transcriptional activator of Xenopus embryonic keratin expression.
Development 109, 157-165.
Snape, A. M., Winning, R. S. and Sargent, T. D. (1991). Transcription
factor AP-2 is tissue-specific in Xenopus and is closely related or identical
to keratin transcription factor 1 (KTF-1). Development 113, 283-293.
Stalder, W. E., Larson, A., Engle, J. D., Dolan, M., Groudine, M. and
Weintraub, H. (1980). Tissue-specific DNA cleavage in the globin
chomatin domain introduced by DNase I. Cell 20, 451-460.
Sykes, K. and Kaufman, R. (1990). A naturally occurring gamma globin
mutation enhances Sp1 binding activity. Mol. Cell Biol. 10, 95-102.
1164
M. E. Minie and others
Townes, T. M., Lingrel, J. B., Chen, H. Y., Brinster, R. L. and Palmiter,
R. D. (1985). Erythroid-specific expression of human b-globin genes in
transgenic mice. EMBO J. 4, 1715-1723.
Whitelaw, E., Tsai, S-F., Hogben, P. and Orkin, S. (1990). Regulated
expression of globin chains and the erythroid transcription factor GATA1 during erythropoiesis in the developing mouse. Mol. Cell Biol. 10,
6596-6606.
Yamamoto, M., Ko, L. J., Leonard, M. W., Bueg, H., Orkin, S. H. and
Engle, D. J. (1990). Activity and tissue-specific expression of the
transcription factor NF-E1 multigene family. Genes Dev. 4, 1650-1662.
(Accepted 11 May 1992)