From the
Expression of fetal
In humans and in a few other species, two ontogenetic switches
in globin gene expression occur within the
A
unique feature of hemoglobin switching in humans is the availability of
many deletion mutants associated with continued fetal
The HPFH syndromes resulting from large deletions of the human
In the present study we decided to
test the validity of the first hypothesis using as a model the HPFH-3
deletion, whose 3` breakpoint is located 30 kb downstream of the
The levels of human globin mRNAs in the erythroid tissues
of transgenic mice were estimated by comparison to the hybridization
signals obtained with various amounts of hemin-induced human K562 cell
RNA. The results are expressed as picograms of A
In this study we have tested the validity of the hypothesis
of imported enhancers
(5) derived from the 3` end of the human
The role of these sequences was further
investigated by testing the effects of the several fragments of this
region on the transcription of the fusion
We further investigated the
specificity of the HPFH-3 enhancer to activate other heterologous
globin and non-globin promoters. The enhancer was capable of activating
the human embryonic
In view of the functional detection of the
enhancer element, we pursued its detailed sequence analysis. The region
contained within the F fragment has been sequenced previously by
Henthorn et al. (10) . We further sequenced an
additional 431 bp downstream to encompass the 700-bp region that
exhibited the full enhancer activity by using as templates derivatives
of fragments A and G. The enhancer lies within a complex array of
multiple repeat elements including an inverted palindrome repeat and a
set of seven short tandem repeats consisting of a 41-bp sequence,
covering a region of 350 bp. It is noteworthy that a number of regions
within the enhancer exhibit homology with other known globin or
non-globin enhancers and display specific motifs for transcription
factors. Thus, the enhancer exhibits six regions of homology
(54-62%) with a conserved region of 24 bp, shared by both the
chicken 3`
The
generality of the enhancer mechanism on the generation of the HPFH
phenotype is further supported by two additional examples: ( a)
the Kenya HPFH deletion
(1) caused by an intergenic deletion
between the A
The data both from the present study and the other
studies mentioned above support the validity of the hypothesis that DNA
elements located within the 3` juxtaposed sequences of the HPFH
deletion mutations can override the developmental specificity of the
fetal
All transfections and assays were
performed at least in three separate experiments. Relative CAT activity
was determined by dividing the average percent conversion for each
fragment by the average percent conversion of the control
The
nucleotide sequence(s) reported in this paper has been submitted to the
GenBank/EMBL Data Bank with accession number(s) X81476.
We thank Dr. O. Smithies for the recombinant HPFH-3
bridging fragment, Drs. P. Henthorn and D. Mager for many fruitful
discussions and help, Drs. D. Bodine and A. Schechter for the CAT
plasmids, G. Houlaki for valuable help with the graphics and Bonnie
Lenk for excellent secretarial assistance.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-globin genes in individuals with the
deletion forms of hereditary persistence of fetal hemoglobin (HPFH) has
been attributed either to enhancement by 3` regulatory elements
juxtaposed to
-globin genes or to deletion of
-gene silencers
normally residing within the
-globin gene cluster. In the present
study, we tested the hypothesis of imported enhancers downstream of
-globin gene using the HPFH-3 deletion as a model. The abnormal
bridging fragment of 13.6 kilobases (kb) containing the A
-gene
with its flanking sequences and 6.2 kb of the juxtaposed region was
microinjected into fertilized mouse eggs. Twelve transgenic mice
positive for the fragment were generated. Samples from 11.5-day yolk
sacs, 16-day fetal liver, and adult blood were analyzed for A
-mRNA
using RNase protection assays. Three mice lacked A
expression in
the yolk sac indicating non-optimal integration site. Four expressed
A
-mRNA at the embryonic stage only, while two expressed
A
-mRNA in both embryonic and fetal liver erythroid cells. Since
the A
-gene with its normal flanking sequences and in the absence
of the locus control region is expressed only in embryonic cells of
transgenic mice, these data suggest that the juxtaposed sequences have
altered the developmental specificity of the fetal
-globin gene.
These sequences were further tested for the presence of an enhancer
element, by their ability to activate a fusion reporter gene consisting
of the CAT gene linked to the
-globin gene promoter, in erythroid
(K562) and non-erythroid (HeLa) cells. A 0.7-kb region located
immediately 3` to the breakpoint, enhanced chloramphenicol
acetyltransferase activity by 3-fold in erythroid cells. The enhancer
also activated the embryonic
-globin gene promoter by 2-fold but
not the adult
- or
-globin gene promoters. The enhancer
represents a region of previously known complex tandem repeats; in this
study we have completed the sequencing of the region encompassing the
0.7-kb enhancer element. Multiple areas of the enhancer region exhibit
homology to the core element of the simian virus 40 enhancer and to the
sequences of the human 3` A
- and the chicken 3`
-globin
enhancers. A consensus binding site for the erythroid specific GATA-1
transcription factor and seven consensus sites for the ubiquitous CP1
transcription factor are also included within the enhancer. These data
suggest that these sequences located immediately 3` to the breakpoint
of the HPFH-3 deletion, exhibit both the structure and the function of
an enhancer, and can modify the developmental specificity of the fetal
-globin genes, resulting in their continued expression during
adult life.
-globin gene cluster:
one from embryonic to fetal (
) early in development and
a second one from fetal to adult (
) during the
perinatal period. Experimental data so far suggest that these switches
reflect temporal and tissue-specific interactions between cis and
trans-acting regulatory elements of the
-globin gene cluster.
-gene
expression in the adult life. The deletions in these mutants occur
within the
-globin gene cluster and lead to clinical syndromes
characterized by increased production of fetal hemoglobin
(HbF)
(
)
in the adult life. The two syndromes,
i.e.
-thalassemia and hereditary persistence of
fetal hemoglobin (HPFH) exhibit discrete phenotypes regarding the
levels of HbF and its distribution in the peripheral blood
erythrocytes: HbF levels of 10-15% and heterocellular
distribution in
-thalassemia versus 20-30% HbF
levels and pancellular distribution in HPFH
(1, 2) .
-globin gene cluster provide a model to study the mechanisms of
fetal
-gene activation. So far, seven large deletions have been
described that are all associated with increased and continued
expression of fetal hemoglobin in adult life
(1, 2, 3, 4) . Three major hypotheses
have been proposed to explain this failure of normal switching; one
suggests that the increased
-gene expression reflects enhancement
by elements imported into the
-globin gene region
(5) , the
other considers that silencer sequences in the A
to
region
are removed by these deletions
(6) , while the third hypothesis
considers that the increased and continued expression of the fetal
-genes in adult life, reflects position effects on the fetal
-genes by the juxtaposed downstream chromatin structure
(7) . Recently, an enhancer element immediately 3` to the
deletion breakpoint of HPFH-1 deletion has been detected, which may
play a role in the continued expression of
-genes in HPFH-1 and
HPFH-2 mutations
(8) .
-gene, removing 48.5 kb of DNA
(9, 10) . Using
bioassays such as transgenic mice and transient assays in erythroid and
non-erythroid cells, we characterized an enhancer element 3` to
breakpoint of HPFH-3 that can modify the developmental specificity of
A
-gene, resulting in persistence of fetal
-globin gene
expression in adult life.
DNA Construct Microinjections
The abnormal
13.6-kb bridging fragment containing the normal fetal A-globin
gene, its flanking sequences, and 6.2 kb of juxtaposed sequences was
reconstituted as follows: the cloned
(10) 11.5-kb XbaI
fragment (kindly provided by Dr. O. Smithies) was ligated with the
2.0-kb HindIII- XbaI fragment containing the 5` end of
the A
-globin gene (Fig. 1). Prior to ligation, the
HindIII site had been destroyed and replaced by a
NotI site, to facilitate cleavage of the 13.6-kb reconstituted
insert as an intact NotI- SmaI fragment. The utilized
SmaI site resides 11 bp from the 3` XbaI site in the
pUC18 polylinker. This strategy also avoids the presence of plasmid
sequences in the construct that are deleterious in the transgenic mouse
bioassay.
Figure 1:
Stages of the reconstitution of the
abnormal 13.6-kb bridging fragment. This fragment contains the normal
fetal A-globin gene and its flanking sequences and about 6.2 kb of
juxtaposed sequences (shown as hatched bar). A,
normal arrangement of the region of the human
-globin gene locus
containing the fetal A
-gene and the
-pseudogene.
B, the 5` end breakpont of the HPFH-3 deletion occurs within
the Alu repeat (shown as a horizontal arrow) just 5` of the
-pseudogene and juxtaposes sequences (shown as a hatched
bar) normally residing 30 kb from the 3` end of the
-globin
gene. C, ligation of the cloned 11.5-kb XbaI fragment
with the 5` end of the A
-globin gene. D, reconstitution
of the abnormal 13.6-kb fragment used for microinjection into mouse
fertilized eggs N, NotI; X, XbaI;
H, HindIII. The normal HindIII site 5` of
the A
-globin gene has been destroyed and replaced by a
NotI site.
Transgenic Embryos and Fetuses
The 13.6-kb DNA
fragment following digestion with NotI- SmaI was
excised from the pUC18 vector and was purified following cesium
chloride gradient ultracentrifugation
(11) . Following dialysis,
the DNA was suspended in fresh TE buffer (10 mM Tris, 1 mM EDTA), extracted with phenol, phenol-chloroform, and chloroform,
and then subjected to ethanol precipitation. DNA concentration was
accurately measured using a Hoeffer TKO 100 DNA fluorometer. DNA
fragments of the reconstituted insert (3-6 ng/µl) were
microinjected into the pronuclei of (C57BL/6J CBA/J)F2 mouse
zygotes as described previously
(12, 13) . Transgenic
embryos and fetuses were identified by Southern blot analysis.
RNA Analysis from Embryonic, Fetal, and Adult
Tissues
Total cellular RNA was isolated from 11.5-day embryonic
yolk sacs, 16-day fetal livers, and adult blood of the various
transgenic lines, as described previously
(12, 13, 14) . The levels of A- mRNA were
measured with a
P-labeled SP6 polymerase-synthesized
antisense RNA probe (560 nt) specific for the 3` end of A
-globin
mRNA. The probe was hybridized and digested as described
(11, 13) resulting in a 165-nt A
-gene-specific protected
fragment.
-globin mRNA per
microgram of total mRNA. In these cells,
16 pg of
-mRNA/µg of total mRNA are detected
(11, 15) ,
while the G
/A
ratio is >10; (see Fig. 2, K562
line). Only the 165-nt fully protected fragment was considered for
the quantitation. Comparison of the A
-mRNA levels to the
endogenous mouse embryonic globin mRNA was performed with the same
human probe, since cross-hybridization with endogenous embryonic mRNA
results in a protected fragment of low molecular size.
Figure 2:
Expression of the HPFH-3 transgene in
embryonic blood, fetal liver, and adult blood measured by RNase
protection analysis. A, an RNA probe specific for the 3` end
of A-globin mRNA (560 nt) was hybridized and digested as described
(11, 13) resulting in a 165-nt protected fragment ( pf).
Lanes K562 1-6 contain 0, 0.03, 0.15, 0.75, 3, and 7.5
µg of total mRNA from hemin-induced K562 cells, respectively. The
smaller <85-nt protected fragment corresponds to cross-hybridization
with G
-specific mRNA. The remaining lanes show the analysis of 15
µg each (except in 1-7, 1.5 µg) of 11.5-day mouse
embryonic ( E) blood mRNA, 50 µg of 16-day fetal
( F) liver mRNA, and 20 µg of adult ( A) blood mRNA
from the six HPFH-3 transgenic lines 1-1,
1-4, 1-5, 1-7,
2-2, and 3-5. Cross-hybridization with
endogenous mouse embryonic globin mRNA results in low molecular size
fragments seen at the bottom of the gel for the E lanes.
Lane M contains
P-labeled size markers (pBR322
digested with HpaII), some of whose sizes are indicated on the
right. B, expression of the HPFH-3 transgene in 16-day fetal
liver. Lanes K562 1-4 contain 0, 0.03, 0.15, and 0.375
µg of mRNA, respectively. For comparison, 15 µg of embryonic
( E) blood mRNA from lines 1-4 and 1-5 and 100 µg of fetal ( F) liver mRNA from lines
1-4, 1-5, and 3-5,
respectively, were analyzed. Lanes 1, 2, 15,
42, 47, and 51 represent a total of 36, 66,
35, 32, 33 and 46 µg of total fetal liver mRNA from the
corresponding transient HPFH-3 transgenes 1, 2, 15, 42, 47, and 51,
respectively. Molecular size markers are as described in panel
A.
Ten
different contiguous (A, B, C, D, and F) or overlapping (F1, G, H, J,
and I) fragments encompassing the 6.2 kb region of the 3` juxtaposed
sequences were generated following digestion with appropriate
restriction enzymes, as shown in Fig. 3. The F and F1 fragments
differ in the sense that the latter contains purely sequences residing
immediately 3` to the breakpoint of the deletion, while F contains 66
bp of the Alu repeats residing immediately upstream of the breakpoint
(see Figs. 1 and 3). The isolated fragments were subcloned upon
modification of their ends in both orientations either into the
BamHI site (fragments A and B), SmaI site (fragments
C, D, F1, H, J, and I), or the AvaI- BamHI sites
(fragments F and G) of the polylinker region of the pUC18 and pUC19
plasmids. The resulting recombinant plasmids were then subjected to
SalI digestion, and the 2.1-kb SalI fragment derived
from plasmid -CAT-HPFH-3 Plasmid Constructions
-CAT (kindly provided by Dr. D. Bodine) was subcloned
into the SalI site in both orientations in all the plasmids
(Fig. 3). This fragment
(16) contains the human
G
-globin promoter gene (
384 to +36 relative to the cap
site) fused with the CAT gene. By employing the above strategy, the
fragments were subcloned into both orientations (5`
3` and
3`
5`) and in both positions (upstream and downstream) relative to
-CAT gene ( i.e. p
CATX1, p
CATX2, p
CATX3 and
p
CATX4) as shown in Fig. 3. For the analysis of the effects
of the enhancer element on the heterologous globin and non-globin
promoters, the individual promoter elements
(17) from
CAT,
CAT, and
CAT plasmids (kindly provided by Dr. A. Schechter)
and from the DHFR-CAT plasmid
(16) (kindly provided by Dr. D.
Bodine) were utilized. The F fragment, containing the enhancer element
was subcloned in both orientations, 3` to the promoter-CAT fusion
genes, i.e. into the BamHI (
CAT and
CAT),
HpaI (
CAT), and AvaI (DHFR-CAT) sites,
respectively.
Figure 3:
Experimental strategy followed for the
detection of enhancer-like elements within the 3` juxtaposed sequences
of the HPFH-3 deletion mutation. A, the 10 different
contiguous ( A-D and F) or overlapping ( F1,
G, H, J, and I) fragments
encompassing the 3` juxtaposed sequences (shown as a hatched
bar) that were used to detect enhancer activity. The numbers above each bar indicate the length of each fragment in kb.
B, the fragments (shown as black boxes) were
subcloned into the -CAT plasmid containing the
-promoter gene
(
384 to +36 from the cap site) fused with the CAT gene
(16). A, AvaI; H, HphI; B,
BamHI; Bg, BglII; Nc,
NcoI; K, KpnI; X,
XbaI.
Transfections and Assessment of CAT Activity
The
cells used in this study (K562 and HeLa) were grown in RPMI 1640 and
Dulbecco's modified Eagle's media (Life Technologies Inc.),
respectively, supplemented with 10% fetal calf serum. Following growth
in mid-log phase, the cells were plated in 10 ml of medium on
100-cmdishes. Twenty-four h after plating, the cells were
transfected with equimolar amounts ( i.e. about 10 µg) of
supercoiled DNA of the various constructs using DNA-calcium phosphate
precipitation, as described previously
(16, 18) . A
standard amount (3 µg) of plasmid pCH110
(19) was added to
each DNA-calcium phosphate preparation for normalization of the
transfection efficiency using the
-galactosidase assay
(20) . The total amount of DNA (20 µg/plate) was kept
constant by adding the appropriate amount of sheared salmon sperm DNA.
The protein concentration of the cell extract was quantitated using a
protein assay kit (Bio-Rad). Forty-eight h after transfection, the
cells were harvested, lysed, and the protein concentration of the
cleared lysate was determined as described
(21) , while CAT
activity was analyzed by the method of Gorman et al. (22) .
DNA Sequencing of the Enhancer Region
DNA
sequencing of both strands was performed using the dideoxy method of
Sanger et al. (23) . The DNA fragments (F, A, A3, and
G1) containing the enhancer sequences 3` of the breakpoint were
subcloned into the M13mp phage vector and were sequenced using the
Sequenase kit (United States Biochemical Co., Cleveland, OH), as
described previously
(24) .
Expression of the Human Fetal A
To investigate the pattern of expression of
the human fetal A-Globin HPFH-3 Gene
in Transgenic Mice
-globin gene fused with the 3` HPFH-3 juxtaposed
sequences during development, we analyzed the mRNA of 11.5-day
embryonic blood, 16-day fetal liver, and adult blood of 12 transgenic
mice. Six mice were independent founders, and six mice were used as
``transient'' transgenes sacrificed at day 16, followed by
analysis of their fetal liver mRNA, using an RNA protection assay. In
the six founder lines three mice failed to express the A
-gene in
11.5-day embryonic blood, probably due to integration into an inactive
chromatin region ( Fig. 2and ). The other three mice
expressed the A
-gene in levels ranging from 4 to 0.02% of the
endogenous mouse embryonic globin mRNA (). Of the last
three mice, two (lines 1-5 and 1-4) failed to express
further the A
-gene, while mouse 3-5 expressed the
A
-gene in 16-day liver, which is the organ of adult
erythropoiesis. An additional mouse (line 42) of the group of transient
HPFH-3 transgenic mice, expressed also the A
-gene in 16-day fetal
liver ( and Fig. 2). Thus, the human A
-gene in
the two mice, when fused with the 3` juxtaposed sequences of the HPFH-3
deletion, was expressed beyond the stage that is normally expressed,
i.e. the 11.5-day embryonic blood
(11, 13, 25, 26) . These data suggested
a putative regulatory role of these juxtaposed sequences on the
developmental specificity of the A
-gene.
Identification of an Enhancer 3` to Breakpoint of the
HPFH-3 Deletion
To further investigate the role of the 3`
juxtaposed sequences of the HPFH-3 deletion, 10 overlapping and
contiguous fragments spanning the entire 6.2 kb region were subcloned
in both orientations (5`3` and 3`
5`) and in both positions
(upstream and downstream) of the
CAT hybrid reporter gene
(Fig. 3) which has been shown to be enhancer-responsive in K562
cells
(16) . A series of 26 constructs were individually
transfected into uninduced K562 cells, hemin-induced K562 cells, and
HeLa cells, which were cultured for 48 h and were then analyzed for CAT
activity. I shows the results of the analysis for the
detection of the enhancer element. The enhancerless
CAT plasmid
was used as the reference basic plasmid, and its relative CAT activity
was considered as 1.0. The 11
CAT plasmid, carrying the
2.3-kb EcoRI fragment containing the 3` A
enhancer
(16) was used as a positive control. Of the 24 recombinant
plasmids tested, only plasmids with the F1, F, and G fragments (and to
a lesser extent the ones with the A fragment) demonstrated enhancer
activity by increasing almost 3-fold the average relative CAT activity
of the
CAT plasmid in induced K562 cells (I). This
activity was less pronounced in uninduced K562 cells. The increased
CAT expression by the F1, F, and G fragments was independent of
their orientation (5`
3` or 3`
5`) and position (upstream or
downstream) relative to
CAT, a feature consistent with other
transcriptional enhancers
(16, 27, 28, 29) . Analysis of the
effects of the F1, F, G, A, and H fragments, on the activation of the
CAT reporter gene, permitted the localization of the enhancer core
element immediately 3` to the breakpoint, extending for about 700 bp
downstream (Fig. 4). Furthermore, the comparable activity of the
F1 and F fragments ensures that the whole enhancer effect is actually
conferred by the 0.7 kb region and that there is no aberrant synergy
between the enhancer element and the residual Alu sequences contained
in fragment F. When the same recombinant
CAT plasmids were used to
transfect non-erythroid HeLa cells, both F and G fragments failed to
increase the level of CAT activity, suggesting that the enhancer
element is probably erythroid-specific (data not shown).
Figure 4:
Identification and completion of the
sequencing of the enhancer core element. A, analysis of the
effects of the F1, F, G, A, and
H fragments on the activation of the CAT gene localized
the enhancer element ( hatched bar) immediately 3` from the
breakpoint ( vertical arrow), extending downstream for about
700 bp. The region corresponding to the F fragment has been previously
sequenced by Henthorn et al. (10). Sequence analysis revealed
that the region immediately 3` to the breakpoint of the deletion
contains a complex array of repeated sequences (10). The 3` end half of
a perfect palindrome is indicated by the large open arrow,
starting just 3 bp before the breakpoint. A set of seven short tandem
repeats is shown as solid arrows. An interruption of 24 bases
of unrelated sequences (shown as hatched bars) occurs at the
fourth repeat. The CCAAT motif is shown as a dot. There are
two CCAAT motifs on the sixth repeat and a seventh CCAAT motif
( boxed) was found at the 3` end of the enhancer (see panel
B). At the 3` end of the enhancer region there is a consensus
(A/T)GATA(A/G) motif for the binding of the erythroid-specific
transcription factor GATA-1. B, sequencing of the novel 431 bp
region encompassing the 3` end of the enhancer element. The novel
sequences start from position 55 (corresponding to 466 nt from the
breakpoint of the HPFH-3 deletion) and end up at position 485. The
upstream 466 bp region starting from the 3` end of the breakpoint and
terminating close to the BamHI site ( GGATCC,
underlined by light broken line) has been sequenced previously
by Henthorn et al. (10); these sequences just upstream of the
BamHI site are highlighted. The motif for the SV40
core element ( GTGGTCTG) is boxed. The novel CCAAT motif is also boxed and the PstI site
( CTGCAG) is underlined with a heavy broken
line. The HphI site ( GTAACACTCACC) marking the
3` end of the F1 fragment is underlined with a solid
line.
Activation of Heterologous Promoters by the HPFH-3
Enhancer Element
To test the ability of the 3` enhancer element
to increase transcription from other globin or non-globin promoters,
fragment F (exhibiting the highest activity) was subcloned 3` to the
CAT gene in both orientations (5`3` and 3`
5`) into vectors
containing several hybrid genes such as
-globin-CAT,
-globin-CAT,
-globin-CAT, and DHFR-CAT
(16, 17) . These plasmids along with the control
CAT plasmid were used to transfect induced or uninduced K562 cells
by DNA-calcium phosphate coprecipitation. After 48 h following
transfection, the cells were assayed for CAT activity. Fragment F
significantly increased the expression of
-CAT plasmid carrying
the embryonic
-globin gene promoter by 2.3-fold (data not shown).
On the contrary, no effect was documented on the transcriptional
activity of the adult
- and
-globin genes, while there was a
repression by 3.8-fold of the DHFR-CAT activity (data not shown). These
data suggest that the effect of the 3` enhancer of the HPFH-3 deletion
is restricted on fetal and embryonic globin gene promoters, while it
has a repressing effect on non-globin promoters such as the
housekeeping DHFR gene promoter.
Nucleotide Sequence Analysis of the 3` Enhancer of HPFH-3
Deletion
Based on the information of the CAT assays described
above, the region of the enhancer element was localized immediately 3`
to the breakpoint of the HPFH-3 deletion extending for about 700 bp
downstream. The region encompassing most of the F fragment had been
previously sequenced by Henthorn et al. (10) extending
466 bp from the 3` breakpoint. Since primarily fragments F, F1, and G
and to a lesser extent the A fragment were activating the CAT
fusion gene (I and Fig. 4), they provided evidence
that the region of the enhancer extends further downstream for an
additional 200 bp (Fig. 4 A). We therefore performed DNA
sequencing starting from the 3` breakpoint of the deletion and extended
it to an additional 431 bp downstream from the end of the 3` normal
sequence previously characterized
(10) as shown in
Fig. 4B. The sequence analysis of the 3` enhancer
(Fig. 4, A and B) revealed that this region
contains a complex array of repeated sequences
(10) . These
include ( a) the 3` half end of a perfect palindrome (80 bp
each) starting just 3 bp before the breakpoint, ( b) a set of
seven short tandem repeats (shown as black solid arrows in
Fig. 4A) consisting of a 41-bp sequence, repeated
totally or partially with one interruption of 24 bp of unrelated
sequences (shown as hatched bars in Fig. 4 A) at
the fourth repeat
(10) . At the 3` end of the first, second,
third, and seventh repeat there is a CCAAT motif (shown as a dot in Fig. 4 A) for the binding of the ubiquitous CP1
transcription factor. Two CCAAT motifs exist on the sixth repeat. An
additional CCAAT motif was found downstream near the 3` end of the
newly sequenced DNA region (Fig. 4, A and B).
Within each of these repeats there are five regions which exhibit
54-62% homology with a conserved region of 24 bp, shared
extensively (17 of 21 bp) by both the chicken 3`
-enhancer,
residing about 410 bp 3` to the chicken
-gene poly(A)
addition site
(30, 31) and by the human 3`
A
-enhancer residing about 480 bp 3` to the A
-gene poly(A)
site
(16) . This conserved element between these two enhancers
is not found elsewhere in the human
-globin gene cluster
(16) , although the short repeats of 41 bp where the conserved
24-bp sequence resides are repeated elsewhere in the human genome
(10) . A sixth region of homology with these enhancers is found
upstream of the breakpoint within the 5` portion of the 160-bp
palindrome but in an inverted orientation
(10) . At least 27
regions were found within the sequenced area of 897 bp which show
homology (62-87%) with the SV40 enhancer core sequence GTGG(A/T)
(A/T) (A/T)G
(22) . Finally, at position +348 from the
breakpoint, the enhancer region contains the consensus motif AGATAA for
the binding of the erythroid-specific transcription factor GATA-1,
which participates in the regulation of the majority of
erythroid-specific genes and erythroid-specific enhancers
(32, 33) .
-globin cluster, as a result of large deletions, to the vicinity
of the fetal
-globin genes, leading to their continued expression
in the adult life. We have used as a model the HPFH-3 deletion which
removes 48.5 kb of DNA of the
-cluster including the
- and
-globin genes and results in high levels of expression of the
-genes. We have reasoned that the 3` juxtaposed sequences 3` to
the breakpoint might contain regulatory elements that can modify the
developmental expression of the fetal
-globin genes. To address
this question we reconstituted the abnormal 13.6-kb bridging fragment
as it is formed in the heterozygote individual with the HPFH-3 deletion
(9, 10) containing the normal fetal A
-globin gene,
its flanking sequences, and 6.2 kb of juxtaposed sequences derived from
a region normally located 30 kb downstream of the
-globin gene.
The bridging fragment was then microinjected into the fertilized mouse
eggs, and the developmental expression of the
-globin gene was
assayed in the generated transgenic mice. Previous studies from several
laboratories
(11, 34) have established that the human
-globin genes, when introduced as individual fragments containing
the minimum of flanking sequences, display exclusively an embryonic
pattern of expression, i.e. being expressed only in the
11.5-day yolk sac cells but not in definitive erythroid cells of later
stages such as the fetal liver cells or the adult blood erythrocytes.
Therefore, this system provides the opportunity to test the effect of
additional sequences on the developmental program of the fetal
-genes. When these 3` juxtaposed sequences were added as part of
the bridging fragment in tandem with the individual A
-globin
fragment, the transgene was expressed in two mice beyond the embryonic
stage, its activity being detectable in the 16-day fetal liver, the
organ of adult erythropoiesis. These qualitative data are in marked
contrast to previous transgenic mice studies using the A
-gene
fragment in isolation, where expression was restricted to yolk sac
cells
(11, 34) and suggest that the presence of the 3`
juxtaposed sequences 3` to the A
-gene was capable in altering
partially the developmental specificity of the fetal gene. The human
fetal genes when introduced individually into transgenic mouse exhibit
exclusive embryonic pattern of expression; however, their level of
expression represents only a fraction of that of the endogenous mouse
genes
(11, 34) . On the contrary, when the individual
-globin genes are linked with sequences containing the locus
control region (LCR), a position-independent, copy number-dependent,
and high level of erythroid-specific expression is achieved
(25, 35, 36, 37) . However, the temporal
specificity of the
-genes is lost in the presence of the LCR
sequences, resulting in their expression in all stages of development:
yolk sac, fetal liver, and adult erythroid cells
(25, 35) . Our construct did not contain LCR sequences
and therefore permitted us to assay directly solely the effects of the
3` juxtaposed sequences on the developmental expression of
-genes.
Our assay was quite sensitive in revealing the effect of these
sequences on the developmental and/or transcriptional specificity of
the 3` juxtaposed sequences.
CAT reporter gene in
erythroid (K562 cells) and non-erythroid HeLa cells. Fragments
delineating a region of about 700 bp immediately 3` to the breakpoint,
activated the
CAT gene nearly 3-fold, regardless of their
orientation or position in respect to the
CAT genes, primarily in
induced K562 cells but not in HeLa cells. Thus, these data revealed an
enhancer element located 3` to the breakpoint of the HPFH-3 deletion
exhibiting an erythroid specificity. The levels of activation of
CAT gene by the HPFH-3 enhancer were comparable (although slightly
lower) to those of HPFH-1 enhancer detected previously using the same
CAT plasmids in K562 cells
(8) . However, in the latter
case, there was considerable variation of the fold activation depending
on the position and orientation of the enhancer. Furthermore, the
HPFH-1 enhancer also exhibited a minimal activity in HeLa cells, in
contrast to the HPFH-3 enhancer.
-globin promoter but had no effect on the
adult
- or
-globin promoters in K562 cells. These data imply
specificity of the enhancer on fetal/embryonic promoters. We have
previously shown
(17) that the
CAT fusion gene is inactive
in K562 cells, in contrast to the
CAT gene. Therefore, it is
conceivable that the lack of activation of
-promoter by the HPFH-3
enhancer could alternatively reflect a variable enhancer strength,
since a considerable activation of the
CAT gene was achieved in
K562 cells by the powerful and non-stage-specific enhancer element
contained within the 5` hypersensitive site 2 (5` HS2) of the LCR
(38) . It is interesting that the HPFH-3 enhancer down-regulated
the DHFR-CAT gene by nearly 4-fold. A similar effect has been observed
with the 3` A
-enhancer
(16) exerting strong repression on
the same DHFR-CAT gene but in 293 cells, which constitutively express
adenovirus E1A protein, a known repressor on some viral and cellular
enhancers
(39, 40, 41) . It is not clear why
this repression occurred in K562 cells in our case, since these cells
do not express the E1A protein and also why the
and
promoters when linked to the same enhancer failed to be down-regulated
in the same cell line.
-enhancer
(30, 31) and the human 3`
A
-enhancer
(16) . Twenty-seven regions within the enhancer
exhibit homology (62-87%) with the SV40 enhancer
(22) core sequence GTGG(A/T) (A/T) (A/T)G. Similar homologies to
the chicken 3`
-globin gene enhancer, the human 3` A
-gene
enhancer, and to the SV40 core enhancer element have also been detected
within the region of the recently described HPFH-1 enhancer, located 3`
of the breakpoint of the HPFH-1 deletion
(8) . Furthermore,
seven consensus CCAAT motifs for the ubiquitous CP1 transcription
factor are found within the enhancer while at the end of the set of
repeats, the enhancer exhibits the consensus motif AGATAA for the
erythroid-specific transcription factor GATA-1 which binds specifically
to erythroid-specific promoters and enhancers
(32, 33) .
A GATA-1 motif has been found also within the HPFH-1 enhancer
(8) . Therefore, the HPFH-3 enhancer element shares features
with other globin and non-globin enhancers, and probably its function
may be mediated by the additional binding of erythroid and
non-erythroid factors. The structural and functional properties of the
juxtaposed 700-bp region located immediately 3` to the breakpoint of
the HPFH-3 deletion documented in this study are consistent with an
enhancer element that has the properties of modifying the developmental
specificity of the fetal
-genes and thus maintaining a high level
of expression in the adult erythroid cells. The effects of the enhancer
on the continued transcription of the
-promoters may be mediated
either via the enhancer sequences per se and/or by additional
parameters that are associated with the nature of the chromatin status
of the juxtaposed sequences that promotes and maintains the active
configuration of the
-genes
(7, 42, 43) .
Thus, the presence of this juxtaposed enhancer element can explain the
resulting HPFH phenotype in the HPFH-3 deletion. A similar enhancer 3`
to the breakpoint of HPFH-1 juxtaposed to the vicinity of the
-genes has been proposed to contribute to the HPFH phenotype for
both the HPFH-1 and HPFH-2 deletions
(8) . It is conceivable
that the HPFH-3 enhancer might be operating also for the generation of
the phenotype of HPFH-4 deletion, since their 3` deletion breakpoints
are differing only by about 2.0 kb
(3, 10) .
- and the
-genes, results in a fusion
A
-gene and in a continued high level expression of both
G
- and A
-genes with a pancellular distribution of fetal
hemoglobin in erythrocytes
(1) . Without excluding the
possibility that the deletion of putative negative elements between the
A
- and
-genes have contributed to the HPFH phenotype, the
high levels of expression of both G
- and A
-genes cis to
the deletion can be readily explained by the juxtaposition of the 3`
enhancer
(11, 34, 44) to the proximity of
the A
(2 kb) and G
(6 kb) promoters; ( b) we
have recently
(45) identified an additional enhancer element 3`
to the breakpoint of the HPFH-6 deletion, originally described as Thai
(A
)° thalassemia
(4) . This enhancer, located
53 kb from the 3` end of the
-globin gene, seems to be responsible
for the generation of the HPFH phenotype, since its loss by the Chinese
(A
)° thalassemia, a perfectly comparable deletion to
HPFH-6, results in a (
)° thalassemia phenotype,
associated with much lower output of fetal hemoglobin and
heterocellular distribution in erythrocytes
(46) . The presence
of this enhancer also near the 3` breakpoints of the recently described
Yunnanese (A
)° thalassemia
(47) can explain
the efficient production of fetal hemoglobin and thus the mildness of
the phenotype of a homozygote for the Yunnanese deletion ( i.e. Hb 10.7 g/dl, no splenomegaly) compared to the severe phenotype of
the Chinese (A
)
thalassemia deletion
(46) .
-genes and are responsible for the resulting discrete
phenotype. These data do not rule out the role of putative negative
elements
(48) between the A
- and
-genes that are
removed by these HPFH deletions and which may independently contribute
to the HPFH phenotype. Further studies will be needed to clarify and
characterize the exact role of these enhancer elements in their natural
positions within the cluster.
Table:
Expression of HPFH-3 transgenes in embryonic
blood, fetal liver, and adult blood
Table:
Expression of transient HPFH-3 transgenes in
fetal liver
Table:
Relative CAT
activity in uninduced () and hemin-induced K562 (+) cells,
transfected with the various plasmids containing the individual
fragments of the juxtaposed region
CAT
plasmid. CAT values were normalized based on protein quantitation,
Lac-Z assays, and confirmation of the amount of DNA used. Equimolar
amounts of DNA from each plasmid were used for the transfection
experiments. As a positive control for the CAT assay system, the pRSV
CAT plasmid (16, 22) was used, which increased consistently CAT
activity 35-40-fold in both K562 and HeLa cells. The relative CAT
activity of the rest of the 12 recombinant plasmids (not shown)
containing fragments H, J, I, B, C, and D, was similar to that of the
control reference
CAT plasmid, i.e., 1.0.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.