The
- 175T
-
C Mutation Increases Promoter Strength in Erythroid Cells:
Correlation With Evolutionary Conservation of Binding Sites for
Two Trans-Acting Factors
By Deborah L. Gumucio, Wendy K. Lockwood, Janice L. Weber, Ann M. Saulino, Kathleen Delgrosso, Saul Surrey,
Elias Schwartz, Morris Goodman, and Francis S. Collins
A point mutation at position -175 has been detected in 'y
as well as 'y globin genes in individuals with hereditary
persistence of fetal hemoglobin (HPFH). To prove that this
single point mutation results in increased promoter
strength, we transfected erythroid and nonerythroid cell
lines with constructs containingnormal and mutant promoters linked to the bacterial chloramphenicol acetyl transferase (CAT) gene. Differences in transfection efficiency
were controlled by cotransfection of pRSVgpt. In K562
erythroleukemia cells, the - 175 HPFH promoter directed
three- to fourfold more CAT activity than its wild type
counterpart. However, in HeLa cells the two promoters
were similar in strength. The -195 to -165 region of the
y-globin promoter contains binding sites for two proteins:
a ubiquitously distributed octamer binding protein. OBP,
and the erythroid-specific protein, GF-I W e find that while
the GF-1 binding site is highly conserved among related
primate y-globin genes, the octamer binding site is not.
The evolutionary conservation of GF-1 as well as its
erythroid-specific distribution suggest that this protein is
important in y-globin gene expression. A role for OBP in
the regulation of y-globin, if any, must have arisen recently
in primate evolution.
0 1990 by The American Society of Hematology.
E
occurs in the eighth base of a conserved octamer sequence,
ATGCAAAT, which has been shown to be an important
transcriptional control element in a variety of other genes."-I3
We and others have shown that the y-globin octamer
sequence is recognized by a ubiquitously distributed octamer
binding protein (OBP).'4-'s Furthermore, the HPFH mutation at -175 dramatically reduces the binding of this
p r ~ t e i n , ' ~ . ' suggesting
~-'~
that, in this context, the octamer
element may represent a repressor binding site. A second,
erythroid-specific binding factor, named by various investigators GF- 1,I7 NF-El ,16,19.20 GATAAG binding protein,2'
Eryf-1,22 or EF-ya,I4 recognizes sequences adjacent to the
octamer binding ~ i t e . ' ~ *Interactions
'~-'~
between these two
proteins may be important in the regulation of y gene
expression.
To provide functional proof that a y-globin promoter
bearing the -175 HPFH mutation is capable of upregulating a linked reporter gene, we determined the relative
strength of normal and - 175 mutant y-globin promoters in a
transient expression assay in both erythroid and nonerythroid cells. To further assess the possible roles of OBP and
GF-1 in mediating this over-expression, we tested whether
the binding sites for these proteins are conserved in related
primate y-globin genes.
LUCIDATION of the molecular mechanisms that control developmentally programmed switches in gene
expression is a major goal of molecular biology. The human
fetal to adult hemoglobin switch provides a well-characterized model system in which to study these mechanisms. This
switch is characterized by reduced expression of the paired
y-globin genes (Gy and Ay) and increased expression of the
adult (6 and 0) globin genes. In some individuals, the switch
is incomplete and expression of the y genes persists in adult
life. This benign condition, known as hereditary persistence
of fetal hemoglobin (HPFH), can result from deletions
within the globin cluster. However, in a subset of individuals
no deletions are apparent and the only defect seems to be a
point mutation in the promoter region of the overexpressed y
gene.
HPFH point mutations have been detected at positions
-202, -198, -196, -175, -161, -158,and -117,relative
to the start site for y gene transcription.'.'' The T
C
substitution a t position - 175 is of particular interest since it
-
From the Departments of Internal Medicine and Human Genetics, University of Michigan. and the Howard Hughes Medical
Institute, Ann Arbor, MI; Division of Hematology, Department of
Pediatrics and Human Genetics, Children's Hospital of Philadelphia, PA;and the Department of Anatomy and Cell Biology, Wayne
State University School of Medicine. Detroit, MI.
Submitted July 28, 1989; accepted September 29, 1989.
Supported by Grants No. 1-1100 from the March of Dimes,
HL28157 and HL33940 from the National Institutes of Health
(NiH).and by a comprehensive Sickle Cell Center Grant (HL38632)
from the NIH. D.L.G. acknowledges support from NIH National
Research Service Award I F32 HLO7654-01. F.S.C. is an Associate
Investigator of the Howard Hughes Medical Institute.
Address reprint requests to Deborah L. Gumucio, PhD, Howard
Hughes Medical Institute. University of Michigan, I I50 WMedical
Center Dr, 4570 MSRB II, Ann Arbor. MI 48109-0650.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C.section I734 solely to
indicate this fact.
0 1990 by The American Society of Hematology.
0006-4971/90/7503-0022$3.00/0
756
.
MATERIALS AND METHODS
Cell lines. Transfections were performed using K562 human
erythroleukemia cellsz3or human cervical carcinoma cells (HeLa).24
Both lines were maintained in Dulbecco modified Eagle medium
(Hazelton Laboratories, Lenexa, KS) supplemented with 10%fetal
calf serum. In one experiment, 25 Mmol/L hemin was added to K562
cells for 4 days before electroporation in an effort to further induce
y-globin expre~sion.'~The chloramphenicol acetyl transferase
(CAT)/GPT ratio in this experiment was similar to values obtained
in the absence of hemin. Because hemin is highly toxic to cells, the
remainder of experiments were performed without added hemin.
DNA. The promoter region (-299 to +35) of a wild type y'
gene (yCAT) as well as a promoter containing the -175 T
C
mutation (-/-17SCAT) were cloned next to the CAT gene.26The
pRSVgpt plasmidz7was used as a control for transfection efficiency.
Plasmid DNAs were purified by alkaline lysis and cesium banding.
-
Blood, Vol 75, No 3 (February 1). 1990: pp 756-761
- 175T
-t
757
C INCREASES PROMOTER STRENGTH
Transfections. K562 or HeLa cells (1 x lo7 cells/transfection)
were washed in phosphate buffered saline (PBS) and resuspended in
400 pL of ice cold PBS (K562) or Hepes buffered saline (HeLa).
DNA (20 pg pRSVgpt and 40 pg of y test plasmid, ?CAT, or
y-175CAT) was added in a volume of 100 pL. Cells and DNA were
subjected to an electrical pulse of 200 V, 700 microfarads (K562) or
200 V, 1,300 pF (HeLa) using a BTX Transfector 300 electroporator (Biotechnologies and Experimental Research, Inc, San Diego,
CA). For K562 transfections, sheared salmon sperm DNA was
added to a final concentration of 250 pg/mL. Immediately after
electroporation, cells were returned to culture medium. After 48
hours, cells were harvested by centrifugation, washed 2 times with
PBS, and resuspended in 100 to 150 p L of 0.25 mol/L Tris, pH 7.8.
Extracts were prepared by three cycles of freezing and thawing.
Assays for CAT26and GPTZswere performed according to published
procedures. After thin-layer chromatography and autoradiography,
converted substrate was quantitated by scintillationcounting.
For calculations of promoter strength, all CAT results were
normalized by GPT activities. Student's r test was used to determine
whether differences between means were significant.
Gel retardation. Nuclear extracts were prepared as previously
described.14Gel retardation was performed with syntheticoligonucleotide probes that were labeled with Klenow polymerase (New
England Biolabs, Beverly, MA) to a specific activity of IO5cpm/ng.
Binding reactions and electrophoresis were performed as described.14
RESULTS
For each transfection, both CAT and GPT activities were
assayed using four different volumes of extract. The percent
conversion of radioactive substrate was plotted for each
volume, and linear regression was performed. Final calculations include only those experiments in which excellent
linearity (9 > .92) of CAT and GPT activity could
be demonstrated. The slopes (m) of these lines were used
A. GPT
to determine the relative strength of each promoter as
"CAT/"GPT. Final values for relative promoter strength
(RPS) of the - 175 plasmid were calculated as: (CAT-,,,/
GPT-,75)/(CATw,/GPTw,).
Figure 1 summarizes the results of transfections done in
K562 cells. Data are grouped and presented as the average
slope of C A T or GPT activity for each plasmid on a given
day. Three different plasmid preparations were used throughout the course of the experiments. Daily variations were
noted in transfection efficiency as evidenced by random
scatter in the G P T slopes (Fig 1A). However, despite this
variation, C A T activity driven from the - 175 promoter was
always greater than that driven from the wild type promoter
(Fig 1B). When data were corrected for transfection efficiency (Fig lC), the - 175 promoter was found to be 3.7
0.5-fold (mean f SEM, n = 5 ) stronger than its wild type
counterpart (P5 .005).
Analysis of Fig 1 shows that no promoter interference
occurred between the cotransfected pRSVgpt and either of
the test CAT constructs. First, the level of G P T activity did
not directly correlate with cotransfection of either C A T
construct (Fig 1A). Second, an experiment done in the
presence of hemin, where both C A T and GPT activities were
several fold higher (Fig 1, day 1, stars), showed similar G P T
activities with both cotransfected test plasmids (GPT slope
for yCAT cotransfection = 2.99; G P T slope for y-175CAT
cotransfection = 2.83); in the same experiment, CAT activity was 3.55-fold greater for the -175yCAT construct
( C A T slope f o r ? C A T = 7.01; C A T slope f o r
y-175CAT = 24.9). Similarly, analysis of raw data from
transfections done in HeLa cells showed no evidence of
promoter interference.
C. CAT/GPT
6. CAT
b
-175
WT
-175
WT
-175
WT
Fig 1. Transient expression in K562 cells. (A) GPT activities. Each point represents the average slope of GPT activity resulting from
cotransfection of pRSVgpt with the wild type (WT) plasmid, YCAT, or the - 175yCAT plasmidon a given day. Slopes were calculated from
assays using four different volumes of extract as described in the text. Results for W T and -1 75 plasmids on the same day are connected
with a straight line. (*I, Day 1 (hmhadded, see Materials and Methods): WT n = 1; - 175 n = 1. (01, Day2: W T n = 1; - 175 n = 1. (0).
Day 3: W T n = 2; - 175 n = 2. N),Day 4 WT n = 4; - 175 n = 2. (0).Day 6: WT n = 2; - 175 n = 2. (B) CAT activities. Data are
presented as daily averages of CAT slopes for each plasmid as in A. Symbols represent days as described in A. (C) CAT/GPT ratios. Points
represent daily average CAT slopes as plotted in B divided by daily average GPT slopes as plotted in A. Symbols represent days as
described in A.
758
GUMUCIO ET AL
Figure 2 compares the RPS of the - 175 promoter in K562
and HeLa cells. The results indicate that the up-regulation
caused by the -175 HPFH promoter is detectable in
erythroid cells only. In HeLa cells, the relative strengths of
the two promoters were not significantly different (- 175
RPS = 1.07 + 0.09,n = 3). Data from individual transfections showed that the CAT/GPT ratio of the wild type
promoter was similar in K562 cells (CAT,,/GPT,, = 5.09 *
1.36,n = 10) and HeLa cells (CAT,,/GPT,, = 7.97 f 2.93,
n = 4). However, the - 175 promoter was stronger in K562
cells (CAT~,,,/GPT_,,, = 19.41 + 4.32, n = 8) and
unchanged in HeLa (8.35 f 3.04,n = 4).
The similar ratios of CAT/GPT observed for the y
promoter in K562 and HeLa cells did not result from lowered
CAT and GPT activity in HeLa cells. Clearly, the y
promoter used in these experiments lacks elements that
control cell specificity of activity. Others have also observed
significant activity of the y promoter in HeLa and other
nonerythroid cells.'7s'8~29330
Several methods were used to determine whether the
augmentation in CAT activity mediated by the -175 promoter was the result of increased transcription originating
from the correct messenger RNA (mRNA) initiation site.
Total RNA was isolated from K562 cells 24 hours after
transfection and attempts were made to analyze CAT
transcripts by S1 -nuclease mapping, RNase protection, or
6.0
a
5.0
..T
.G
...
.G
Orang
Gibbon
Spider
Galago
Fig 3. Sequences of the -175 region from related primate
y-globin promoters. The positions of the octamer sequence,
ATGCAAAT, and two TATCT motifs that interact with a single
GF-1 m o l e c ~ l e 'are
~ indicated. Dots indicate identity with the
human sequence. Sequences are from orangutan (Pongo
pygmaeut?'), spider monkey (Ateles geot7roy~).gibbon (Hylh a t e s la?), and galago (G8lago C r a s s i c a u d ~ t i ~ ~ ) .
polymerase chain reaction. No CAT transcripts could be
detected by any of these techniques, suggesting that CAT
mRNA is highly labile in these cells. After submission of this
manuscript, Nicolis et a13' reported detection of yCAT
constructs using polyA selected RNA. In those studies, a
three- to fourfold increase in the levels of correctly initiated
yCAT RNA was demonstrated in cells transfected with the
- 175 HPFH promoter.
Previous studies have shown that two proteins bind the
y-globin promoter in the region between - 185 and - 170: a
ubiquitously distributed octamer binding protein, OBP, and
an erythroid-specific protein, GF- 1 .I4-', If these two proteins
are important for the regulation of y-globin expression, it
might be expected that their binding sites would be conserved
in closely related primate species. To examine this question,
we performed gel retardation assays using oligonucleotide
probes corresponding to the - 175 region of human, orangutan, spider monkey, gibbon, and galago. While the upstream portion of the bipartate GF-1 binding site is completely conserved, two changes in the downstream site occur
in the galago sequence (Fig 3). One of these changes is
identical to the -175 T
C HPFH mutation. Changes
occur in the consensus octamer binding site in all sequences
except the human promoter. Figure 4 shows that all primate
promoters retain the capability of binding GF- 1. In contrast,
OBP binds only to the human y-globin promoter.
-
4.0
-175
RPS
3.0
-
2.0
1.0
K562
HeLa
Fig 2. Relative strength of the -175 HPFH promoter in K562
and HeLa cells. -175 RPS is defined as: (CAT-,,,/GPT_,,,)/
(CAT,/GPT,).
For K562 cells, 10 transfections of yCAT and eight
transfections of 7-175 CAT were performed on 5 separate days.
For HeLa cells, four transfections of each plasmid were done on 3
separate days. Points represent daily averages of -175 RPS in
each cell type.
DISCUSSION
The T
C mutation at - 175 was first observed in the Gy
gene of an American black family6 and a family from
Northern Sardinia.' Recently, a third individual with this
mutation was reported; this time the mutation was detected
in the promoter of the Aygene." In each case, the high level
of HbF (17% to 38%) was derived from predominant
expression of the y gene bearing the promoter mutation.
These observations provide good circumstantial evidence
that the -175 site marks a sequence that is important for
y-globin gene expression.
The data presented here demonstrate that a promoter with
the - 175 HPFH mutation is 3.7-fold stronger than the wild
type promoter in K562 erythroleukemia cells. Because differences in the relative strengths of the wild type and -175
HPFH promoters were small, great care was taken to control
experimental variables. All test CAT constructs were cotransfected with the pRSVgpt plasmid. Results were then ex-
- 175T
-
759
C INCREASES PROMOTER STRENGTH
Fig 4. 0 . 1 retardation assay testing the conservation of
OBP and GF-1 binding sites.
Double-stranded oligonucleotide probos for the
176 region of the human f-globin gene
and four related primetes bequences shown in Fig 3)were
Inbeled and incubated with 6 #g
of K682 nuclear extract in the
presence of 600 ng of poly (dldC1. Binding reactions and electrophoresia were performed as
previously described." The positions of protein-DNA complexes formed with OEP and
GF-1 are indicated.
i
pressed as the ratio of CAT/GPT activity. providing an
internal normaliration that corrects for sample to sample
variations in transfection efficiency. In addition. GPT and
CAT assays were performed using four diNerent volumes of
extract. Regression lines were calculated to assure that all
four points fell in the linear portion of the saturable curves.
Final values for relative promoter strengths were then
calculated. not from single points. but from the slopes of
these regression lines. This approach allows the accurate
detection of small differences in promoter strength. despite
the daily variations in transfection eficicncy that are inherent to the clectroporation procedure.
The T C change at - 175 is the only difference between
the two promoters tested. Thus, this report provides direct
proof that this single base change causes upregulation of a
linked gene. However. the three- to fourfold increment in
relative promoter strength that we observe in these transient
expression assays is far less than the more than 100-fold
upregulation of the mutated gene in vivo.'.".'" It is possible
that the fetal-like expression pattern of K562 cells (which
express t and y, but not 6 ) does not provide an optimum
background against which to discern an increase in y-globin
expression. The existing level of y expression in these cells
may dampen the observed erect of the upmutation. which is
most apparent in the adult in vivo. It is also possible that the
relatively small promoter fragment used in these studies
(-299 to 35) lacks other determinants that may interact
with the - 175 site to result in full augmentation of expression. Other investigators observed similarly small effects of
the - I17 HPFH mutation (three- to fourfold upregulation)
even after transfection of a 3.3-kilobase (kb) Hindlll fragment containing entire "y-globin gene.M In another study.
I .4-fold upregulation of a y' gene carrying the - 1 I7 HPFH
mutation was seen in a noncrythroid cell after transfection of
cosmid clones containing most of the 6 globin locus.w
However. none of these constructs contain the recently
described dominant control region located upstream from the
y-globin gene. which directs high-level, tissue-specific exprcssion of homologous and hetcrologous prom~tcrs.".'~The
presence of this locus may influence promoter strength in cis
-
1
1
HUMAN
ORANG
GALAGO
GIBBON
SPIDER
in a manner similar to that recently proposed for developmental regulation of globin switching in chicken erythroid ccIIs.Iv
It would be of interest to test the activity of normal and
HPFH y-globin promoters in the context of a minilocus
containing this control region.
The - I75 HPFH mutation occurs in the eighth base of a
conserved octamer sequence. We and others have shown that
an ORP with the same gel mobility and ubiquitous distribution as NF-A I I ' binds to the y-globin octamer sequence.""*
In addition. an erythroid-specific protein, GF-I binds to an
area that overlaps the ORP site.'' I ' In gel shift experiments.
the presence of the - 175 mutation reduces the binding of
ORP by 12-fold,'' but does not affect the binding of GF- 1 .''.IHowever, one recent study demonstrates that this mutation
does produce subtle alterations in the footprint of GF-I on
the DNA.'- Therefore. either one or both of these proteins
could conceivably be involved in upregulation of the - 175
mutant promoter. The results presented here show that the
increase in promoter strength caused by the - 175 mutation
is erythroid-specific. While this manuscript was being prepared and reviewed. three reports appeared in the literature
that corroborate this finding.".'"."
The erythroid-specific nature of the - 175 upregulation
suggests that despite the dramatic elfect of this mutation on
O R P binding. the mechanism of overexpression probably
docs not involve relief from active repression by a ubiquitous
ORP. The same conclusion has been reached in two very
recent studies that have used site-directed mutagenesis to
further analyze the contribution of both ORP and GF-I to
y-promoter strength."." In these studies. it was concluded
that while an intact GF-I site is necessary to observe
upregulation in the presence of the - 175 HPFH mutation.
other mutations that destroy octamer binding do not result in
increased promoter strength.
The experiments presented here demonstrate that the
octamer binding site in the human y promoter is actually a
very recent acquisition. The prosimian galago y-globin gene.
which like that of the rabbit and mouse is expressed as an
embryonic locus and switched o r at the beginning of fetal
life." lacks the octamer site. Moreover. the site is absent even
760
GUMUCIO
in some closely related simian primate genes, which like the
human gene have been recruited to a fetal expression pattern.
Other simian primate y genes (gorilla and rhesus monkey33)
d o possess a functional OBP site (data not shown). Therefore, the acquisition of a n octamer binding site does not
correspond with the phenomenon of fetal recruitment of
y-globin expression. The role, if any, of this sequence and its
cognate protein in the control of y-globin gene regulation has
yet to be determined.
The GF-1 binding site, in contrast, is present and functional in all y-globin promoters tested. We recently found
that the rabbit y gene also retains a GF-1 site in the
homologous position (data not shown). T h e erythroid-
ET AL
specific distribution of GF-1 suggests that it may be a n
important determinant of promoter strength in the presence
of the - 175 HPFH mutation. Strong evolutionary conservation of its binding site also points toward a significant role for
GF-1 in y-globin gene expression. The recent cloning of this
interesting protein should facilitate further investigation of
its regulatory fun~tion.4O.~’
ACKNOWLEDGMENT
The authors thank Drs David Bodine and Timothy Ley for many
helpful discussions. We are also grateful to Heidi Heilstedt and Kris
Blanchard for help with the CAT assays, and Bernice Sandri for
secretarial help.
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