From the Laboratory of Chemical Biology, NIDDK, National Institutes
of Health, Bethesda, Maryland 20892 and Departments of
Computer Science and Engineering and § Biochemistry and
Molecular Biology, Center for Gene Regulation, The Pennsylvania State
University, University Park, Pennsylvania 16802
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
We have previously reported, on the
basis of transfection experiments, the existence of a silencer element
in the 5'-flanking region of the human embryonic () globin gene,
located at
270 base pairs 5' to the cap site, which provides negative
regulation for this gene. Experiments in transgenic mice suggest the
physiological importance of this
-globin silencer, but also suggest
that down-regulation of
-globin gene expression may involve other
negative elements flanking the
-globin gene. We have now extended
the analysis of
-globin gene regulation to include the flanking
region spanning up to 6 kilobase pairs 5' to the locus control region
using reporter gene constructs with deletion mutations and transient
transfection assays. We have identified and characterized other strong
negative regulatory regions, as well as several positive regions that
affect transcription activation. The negative regulatory regions at
3 kilobase pairs (
NRA-I and
NRA-II), flanked by a positive control element, has a strong effect on the
-globin promoter both in erythroid K562 and nonerythroid HeLa cells and contains several binding
sites for transcription factor GATA-1, as evidenced from DNA-protein
binding assays. The GATA-1 sites within
NRA-II are directly needed
for negative control. Both
NRA-I and
NRA-II are active on a
heterologous promoter and hence appear to act as transcription
silencers. Another negative control region located at
1.7 kilobase
pairs (
NRB) does not exhibit general silencer activity as
NRB
does not affect transcription activity when used in conjunction with an
-globin minimal promoter. The negative effect of
NRB is
erythroid specific, but not stage-specific as it can repress
transcription activity in both K562 erythroid cells as well as in
primary cultures of adult erythroid cells. Phylogenetic DNA sequence
comparisons with other primate and other mammalian species show unusual
degree of flanking sequence homology for the
-globin gene, including
in several of the regions identified in these functional and
DNA-protein binding analyses, providing alternate evidence for their
potential importance. We suggest that the down-regulation of
-globin
gene expression as development progresses involves complex, cooperative
interactions of these negative regulatory elements,
NRA-I/
NRA-II,
NRB, the
-globin silencer and probably other negative and
positive elements in the 5'-flanking region of the
-globin gene.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The expression of the individual genes of the human -globin
cluster is regulated in both a developmental and a
tissue-dependent manner. The developmental "switches"
in expression follow the sequential arrangement of the globin genes,
beginning at the 5' region of the gene cluster and including the five
active
, G
, A
,
, and
-globin genes (1). The effort to
understand the mechanism of hemoglobin switching has focused on
localizing the cis-acting DNA sequence elements which are involved in
regulating globin gene expression, and identifying and characterizing
the transcription factors or proteins that bind to those DNA motifs or
related proteins (2, 3). Each globin gene and its immediate flanking
region appear to contain sufficient information for developmentally correct expression as suggested by transgenic mouse experiments (4-7).
Phylogenetic footprinting has been used to identify evolutionarily conserved regions and other potential protein binding sites in the
globin gene cluster (8-10). Located at the distal 5' region of the
-globin cluster immediately upstream of the embryonic
-globin
gene are the DNase I hypersensitive sites (HS 1 to HS 5)1 of the locus control
region (LCR) (6-13 kb 5') that are important in controlling
transcription and replication of the
-globin cluster. The proposed
role of the LCR in developmental regulation is controversial. Studies
in transgenic mouse show that linkage of the LCR to individual globin
gene results in much higher expression in vivo, and an apparent alteration in the developmental specificity of the
- and
-globin genes, depending on proximity and arrangement of the
transgene (11-13). In contrast, developmental specificity of expression of human
-globin gene appears to be more autonomous and
does not require a particular arrangement with respect to the fetal
- or adult
-globin genes. DNA constructs lacking the LCR show
developmental switching of globin genes in transgenic mice showing the
LCR is expendable for developmental regulation, at least in this
assay.
We have previously identified an -globin gene silencer (
GS),
using reporter gene transfection assays, in vitro
transcription and DNA-protein binding assays, located in the region
between
300 bp and
250 bp 5' to the
-globin gene cap site
(14-16). The potential biological significance of the silencing
activity of
GS was supported by in vivo studies using
transgenic mice (7, 17, 18). Additional studies have revealed other
cis-acting regulatory elements further 5' to the
-globin gene (9,
20, 21), including a positive regulatory element, located at
700 bp,
and a negative regulatory element located at about
400 bp. In
general, the 5' region of the
-globin gene provides much of the
activity for developmental regulation of the
-globin gene expression
as evidenced from transgenic mouse studies (7). However, the expression
of limited levels of the human
-gene (5-10% of the mouse
y or
) with constructs in which the silencer has been mutated
(18)2 suggests that other
important negative regulatory elements may exist around the
-globin
gene.
In the present study, we have investigated the functional role of the
-globin gene 5'-flanking region up to
6 kb, which includes HS 1, and have identified several functionally important cis-elements that
markedly affect expression driven by the
-globin promoter.
Construction of serially deleted mutants enabled us to systematically
study the positive and negative cis-acting elements involved in
-globin control. We observed multiple regulatory sequences in this
region and focused on several strong negative elements located in the
regions around
1.7 and
3.0 kb. In all cases, the negative elements
are flanked by positive regulatory regions. These elements contain
several DNA-protein binding motifs, including the erythroid specific
transcription factor GATA-1. DNA sequences in the regulatory region
located at
1.7 kb are conserved in all mammals examined, whereas the
DNA sequences located at
3.0 kb are present only in the prosimian
primate orangutan, galago, and human. Our data suggest that in addition
to the
GS and the stage-specific positive element located more
proximal to the
-promoter, expression of the
-globin gene
including specifically its down-regulation during development involves
multiple positive and negative elements.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Plasmid Constructions--
An -globin promoter/reporter gene
construct was made by linking human
-globin gene containing 5'
sequences from the promoter +46 to
6073 bp 5' of the cap site, to a
luciferase reporter gene (LUC)-coding plasmid pGL-Basic
(Promega), generating a parent construct p
6073 that includes DNase I
HS 1 at about
5 kb. A series of 5'-deletion mutants were made by
linearizing p
6073 with SacI and SpeI followed
by exonuclease III digestion, at 1-min intervals. The ends of the
deleted mutants were filled in with the Klenow fragment of DNA
polymerase I and self-ligated. A second set of 5' series of deletions
was made from p
3028 to generate smaller deletion mutants. The 5'
ends of the deletion mutants were determined by dideoxy sequencing.
Cell Culture--
The human erythroleukemia K562 and HeLa cells
were grown in RPMI 1640 or AMEM medium (Biofluid, Rockville, MD),
respectively, supplemented with 10% fetal bovine serum,
L-glutamine and penicillin/streptomycin. Primary human
adult erythroid cells (hAEC), were grown in a two-phase liquid culture
system as described previously (20). Briefly, mononuclear cells from
the peripheral blood of normal donors, isolated on a Ficoll-Hypaque
gradient, were grown in -minimal essential medium with 10% fetal
calf serum and 10% conditioned medium collected from 5637 human
bladder carcinoma cells (phase I). After 7 days the cells were washed
and recultured in liquid medium supplemented with 1 unit/ml recombinant
erythropoietin (phase II).
Transient Transfection Assays-- Both K562 and HeLa cells were transfected by electroporation with Gene Pulser (Bio-Rad) at 250 V (220 V for HeLa) and 960 µF with a plasmid DNA amount ranging from 10 to 40 µg. Transfections with hAEC were carried out after 10-11 days of incubation by combining phase II cultured cells from different donors. Transfected cells were collected and lysed after 48 h of incubation, and 20 µl of the cell lysate were used to determine luciferase activity analyzed with a Monolight 2010 luminometer (Analytical Luminescence Laboratory, San Diego, CA), in which the substrate D-luciferin was automatically injected. The results are expressed as the average of at least three experiments with the activity of luciferase normalized to the amount of protein used in each experiment. A construct containing the LUC reporter gene under control of the SV40 promoter was used separately as the positive control to establish a value for promoter activity of 1.0.
In Vitro DNA Foot Printing--
DNA probes were made by labeling
sense primers with [-32P]dATP followed by polymerase
chain reaction amplification to generate DNA fragments. The probes
range from
3198 to
2898 bp 5' for
NRA-I/
NRA-II and from
1838 to
1588 bp 5' for
NRB. The labeled probes were purified by
SpinBind (FMC, Rockland, ME). The mixtures of probe (20,000 cpm) and
nuclear extract (50-100 µg) were incubated for 30 min on ice
followed by the addition of DNase I (0.25-0.5 unit) and incubation for
4 min at room temperature. Equal volumes of stop solutions containing
400 µg/ml proteinase K were added and samples incubated for 30 min at
37 °C, and 2 min at 70 °C. After phenol/chloroform extraction and
ethanol precipitation the DNA samples were dissolved in loading buffer
and analyzed on 6% polyacrylamide sequencing gels.
Electrophoretic Mobility Shift Assays--
Gel shift studies
were carried out by annealing a pair of oligonucleotides, labeled with
[-32P]dATP followed by SpinBind (FMC, Rockland, ME)
gel purification. The reactions were carried out on ice for 30 min in a
15-µl total volume and loaded onto a 4% polyacrylamide gel. In
competition experiments, an unlabeled probe or the same fragment with
mutation with 12.5-100-fold molar excess was included in the reactions as indicated. Oligonucleotide sequences for gel shift are as follows with the mutated bases underlined:
NRA II-1G: 5'-CCCAG AGCTG TATCT
TAATTGT;
NRA II-
1G: 5' CCCAG AGCTG GCGCC
TAATTGT.
DNA Sequence Analysis--
Pairwise alignments of the DNA
sequences from the -globin gene clusters of human, galago, rabbit,
and mouse were computed using the program SIM (21) and displayed as
percent identity plots (22). In a percent identity plot, all the
gap-free aligning segments in the region of interest are automatically
plotted as a series of horizontal lines (each between the coordinates
of the human sequence present in a gap-free alignment) placed along the
y axis according to the percent identity in each aligning segment. Notable features in the human sequence are also placed along
the x axis. The simultaneous alignment of these four DNA sequences were obtained from the Globin Gene Server
(www.globin.cse.psu.edu) (23). The region encompassing
NRA in
human and the homologous regions from orangutan (EMBL accession no.
X05035) and galago (GenBankTM accession no. U60902) were
aligned simultaneously using the program YAMA2 (24). In the displays of
the multiple alignments, boxes are drawn around blocks of at least six
columns where each column has an identical nucleotide in at least 75%
of the positions; this is equivalent to requiring invariant columns for
alignments of three sequences.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The Presence of Negative Element(s) in the 5'-Flanking Sequences of
Human -Globin Gene--
The human embryonic epsilon globin (
)
5'-flanking sequence was linked to the luciferase reporter gene and
tested by transient transfection in K562 cells, a human erythroleukemia
cell line that expresses embryonic and fetal globin genes. As shown in
Fig. 1A, the transcription
activity of
-promoter in transfected cells measured as luciferase
reporter gene activity varies greatly with different lengths of
5'-flanking sequences. A high level of activity 2.5-fold greater than
the SV40 promoter was observed for the minimal
-promoter construct
p
177, as expected given the active transcription activity of the
endogenous
-globin gene in K562 cells. The
GS in the region of
300 to
250 bp (14) and other negative elements located at
419 bp
(25) contribute to the lowered reporter gene activity of p
883 when
compared with that of the minimal
-promoter construct (p
177).
Extending the 5' region to encompass HS 1, we find that the
transcription activity of p
6073 is 10-fold lower than that of
p
883 suggests the existence of one or more strong negative
element(s) in the region from
800 to
6000 bp.
|
Transcriptional Activity Profile of the -Globin Gene
Promoter--
We have studied the transcriptional activity profile of
this region of the
-globin gene-flanking sequences in detail by
constructing a series of deletion mutants extending up to 6 kb 5' of
the human
-globin gene linked to luciferase reporter gene. The
transcriptional activities of these reporter gene constructs were
tested in transient transfection assays in embryonic/fetal erythroid
K562 and nonerythroid HeLa cells (Fig. 1A). In K562 cells,
transcription activity of the
-globin gene minimal promoter was
comparable with that of SV40, in contrast to HeLa cells in which the
-globin minimal promoter activity is only 10% of that SV40.
Analysis of the deletion mutants in these cells revealed several
regulatory regions flanking the
-globin gene 5' extending from
883
bp to HS 1. A striking feature of the behavior of the reporter gene
constructs is that positive regulatory regions are generally flanked by
negative regulatory regions, i.e. certain constructs appear
as "spikes" in the graph. The two most striking combinations of
this type are a pair of positive (
PRA) and negative regions
(
NRA-I/
NRA-II) located between
2.8 and
3.1 kb that are active
in both K562 cells and HeLa cells and a pair of positive (
PRB) and
negative (
NRB) regions located around
1.7 kb that function only in
K562 cells. Another, less potent regulatory pair includes the positive regulatory region between
1995 bp and
1747 bp flanked on the 5'
side by a negative regulatory that functions in both K562 and HeLa
cells. The positive region between
1084 and
1135 bp and an overall
negative region between
1135 and
1460 bp are active only in K562
cells. Additional positive regulatory regions (Fig. 1A) are
localized between
2385 and
2772 bp and between
3199 and
3329 bp
that increase transcription activity by about 3-fold in K562 cells, and
between
3329 and
3986 bp that increases transcription activity in
HeLa cells. Other negative regulatory regions that reduce transcription
activity are localized between
883 and
1084 bp,
2000 and
2385
bp, and
3986 and
4442 bp, and are active in both K562 cells and
HeLa cells. Extending the 5' region from
4442 to
6073 bp further
decreases reporter gene activity in K562 cells.
Conserved DNA Sequences in the 5'-Flanking Region of Mammalian
-Globin Genes--
A summary of the results of the deletion series
are shown in Fig. 1B (top panel), aligned with
graphs of the sequence matches observed in pairwise comparisons of the
human sequence with that of other mammals. In these percent identity
plots, the percent identity (from 50 to 100%) for each gap-free
aligning segment is plotted using the coordinates of the human
sequence, and notable features such as exons and interspersed repeats
are placed along the horizontal axis (22). Fig. 1B shows the
percent identity plots for alignments of the human sequence with that
from the prosimian primate galago, from rabbit, and from mouse as three panels, including the region from HS 1 of the LCR through the
-globin-coding sequence. In general, almost all of the galago sequence aligns with a high similarity to the human sequence. Extensive
matches are also seen for comparisons of the human sequence with rabbit
and mouse, although a roughly 1.6-kb segment between HS 1 and the
-globin gene does not match (corresponding to about
4-2.4 kb in
the human). Matching sequences extending this far 5' to the gene are
not characteristic of all mammalian globin genes. For instance, the
5'-flanking region of the human
-globin gene matches with that of
galago to about
3000 bp, and with mouse to about
770 (23). The
regions delineated in the results of the deletion series as
NRA-I/
NRA-II and
NRB show significant regions of matching in
those comparisons. Thus the simultaneous alignment of these sequences
is helpful in analyzing this region in more detail, as described below.
However, regions comparable to human
NRA-I/
NRA-II and
PRA are
found only in orangutan and galago, and only this pairwise alignment is
informative, in contrast to greater cross-species matching more
proximal to the
-globin gene itself.
Characterization of NRB--
The tissue-specificity of
NRB
was further examined by comparison of the two constructs, p
1747 and
p
1707, in human adult erythroid primary cells (hAEC) as well as in
the K562 and HeLa cell lines (data not shown). The decrease in
transcription activity of p
1747 compared with p
1707 is
erythroid-specific as observed in both K562 and hAEC cells but not in
HeLa cells, suggesting the erythroid-specific property of
NRB.
Protein binding to the
NRB was studied by in vitro DNase
I footprinting with nuclear extracts from both K562 and HeLa cells. Two
strongly protected regions were detected only with K562 nuclear
extracts (Fig. 2). These footprints are
located around
1752 to
1735 bp and
1718 to
1710 bp and overlap
with regions that are conserved in the 5' region of corresponding
embryonic globin genes in mouse, rabbit, and galago (Fig. 2,
bottom).
NRB alone, however, does not act as a true
silencer. Interestingly, no significant negative activity is observed
when
NRB is linked directly to the
minimal promoter and tested
in either K562 or HeLa cells, when linked to a heterologous promoter
transcription activity is again reduced (Fig.
3). This suggests that
NRB alone may
exhibit negative regulation depending on the promoter, but does not act
as a true silencer.
|
|
Characterization of NRA-I and
NRA-II--
The region between
3127 and
2902 bp which is active in both K562 cells and HeLa cells,
has a much stronger negative effect in the erythroid cells (Fig.
1A), perhaps related to GATA-1 binding (Fig.
4). This region contains two negative
control regions,
NRA-I (
3127 to
3071 bp) and
NRA-II (
3028
to
2902 bp), each associated with a decrease in reporter gene
activity. In K562 cells, the region separating these two motifs (
3071
and
3028 bp) exhibits a modest positive effect (Fig. 1A).
The combined effect of
NRA-I and
NRA-II in the 225-bp region
reduces transcription activity 20-fold when added back to construct
p
2902 to create p
3127. The negative effects of
NRA-I and
NRA-II were also observed in HeLa cells with about a 13-fold
increase in transcription activity comparing p
2902 with p
3127.
The activity of p
3127 is 3-4-fold lower than the
-globin minimal
promoter construct, p
177.
|
|
Multiple Protein-binding Sites Identified in NRA-I and
NRA-II--
To attempt to identify the sequence motif responsible
for the negative effect of
NRA-I and
NRA-II, we carried out DNase I footprint analysis and correlated the results with aligned DNA sequences from this region. Since the sequence corresponding to
NRA
is not present in mouse or rabbit, we reasoned that it would be
informative to look at additional primate species. The only other
primate species for which sequence data extends this far is the
orangutan, and a simultaneous alignment of human, orangutan, and galago
sequences is shown in Fig. 6B.
Fig. 6A shows the DNase I footprinting assay of region
NRA. The probe was generated by a polymerase chain reaction with
32P-labeled primer, and the nuclear extract from K562 cells
was used in the reactions. Several regions are footprinted by DNase I
digestion designated as FP1-FP5. These include a conserved
progesterone receptor binding motif (FP1) and a GATA-1 binding motif
(FP2). A major footprinted region (FP3) appears within the region
3071 and
3028 bp which exhibits a small positive effect on
transcription activity when comparing the constructs p
3028 with
p
3071 in K562 cells. This footprinted region (FP3) is included
within a block of sequence that is invariant among human, orangutan,
and galago. Two minor footprinted regions (denoted FP4 and FP5) are at
potential GATA-1 binding motifs in
NRA-II at about
2976 and
2949
bp, respectively. An inverted AGATAG sequence appears in the region corresponding to FP4 in the galago
-globin 5'-flanking region and
the region corresponding to FP5 is only partially conserved in this
comparison. Although two of the GATA1 binding sites have mismatches in
galago that would be expected to decrease binding affinity, these
binding sites are identical between orangutan and human.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
It has been noted for some time that the -globin gene and its
flanking regions are more conserved among mammals than are the
- or
-globin genes (26, 27). Additional DNA sequences and development of
new sequence alignment software have continued to show homology
throughout much of the 5'-flanking region, extending to HS 1 of the
LCR. This homology is highly suggestive of extensive regulatory
sequences. Previous studies have revealed multiple, conserved
regulatory elements in the 800 bp proximal to the cap site of the human
-globin gene. Conserved CCAAT and CACC motifs are needed for
function of the proximal promoter (28), a highly conserved GATA motif
at
160 bp is needed for response to the HS 2 enhancer (29), and the
-globin silencer (
GS) (14) between
300 and
250 bp contains
conserved binding sites for GATA1 and YY1 (8, 15, 16). Additional
regulatory elements are observed further 5', such as the negative
element located at
419 (25, 30). Multiple positive regulatory
elements have also been identified within the first 800 bp 5' to the
-globin gene, and at least two of them function in a synergistic
manner (25, 31). Each of these additional cis-acting regulatory
sequences between
800 and
300 bp correspond to evolutionarily
conserved sequences (8, 9, 23, 32). The assumption that the sequence
conservation results from selection for a common regulatory function
was verified by observing a similar pattern of positive and negative
regulatory elements 5' to the rabbit
-globin gene (9).
Data in this report from the transient transfection assay of a series
of deletion mutants show that multiple negative and positive cis-acting
regulatory elements are found even more distally to the -globin
gene, extending to HS 1 of the LCR. As illustrated in Fig.
1B, DNA sequences corresponding to many but not all of these
regulatory elements are conserved in other mammals. Two prominent pairs
of negative and positive regulatory elements in the
6000- to
800-bp
region, A and B, were studied in more detail. The highest level of
reporter gene activity was observed for p
2902, in contrast to the
low level of activity observed for p
2807, p
3028, and p
3127.
These activities of these constructs localized a strong positive
regulatory region (
PRA) between
2807 and
2902 and a negative
regulatory region (
NRA) consisting of two subregions between
3127
and
3071 (
NRA-I) and between
3028 and
2902 (
NRA-II). Both
NRA-I and
NRA-II also function when combined with a heterologous (SV40) promoter, with
NRA-II, exhibiting a stronger negative regulatory effect (Fig. 5).
Our work shows the importance of the erythroid transcription factor,
GATA-1, in these distal sites. GATA-1 has been found to be a repressor
of the -globin gene in vivo (33) and appears to be
involved in negative regulation of the erythropoietin gene (34). We
have found it to be involved in the activity of
GS (15).
Site-directed mutagenesis of each of the two potential GATA-1 binding
sites located in
NRA-II decrease its negative effect, and mutation
of both sites restored most the SV40 promoter activity (Fig. 5). These
results demonstrate that the negative regulation of
NRA-II is
directly related to the two GATA-1 binding sites. The fact that
NRA-II is active in both K562 and HeLa cells suggests that GATA-1
(expressed in K562 cells) and possibly other GATA factors (expressed in
HeLa cells) can suppress transcription of the
-globin gene. Whether
this would be necessary in nonerythroid cells in which the globin
chromatin is in a closed conformation is not clear. Mutation of GATA-1
site located in
NRA-I does not change the negative effect (data not
shown).
Unlike the other cis-regulatory elements in the 5'-flanking region of
the -globin gene, the DNA sequences of the human
NRA and
PRA
regions are not conserved in non-primate mammals, and are found only in
the primates human, orangutan, and galago (Fig. 6B). Since
mutations in this region have a strong phenotype in transfected cells,
it appears that the function of this region is limited to primates. A
complex array of positive and negative cis-regulatory elements are
revealed by the deletion/transfection analysis. Likewise, the in
vitro footprinting shows multiple binding sites. One of the long
strings of invariant nucleotides in the human-orangutan-galago
alignment (11 bp long) corresponds to FP3 (Fig. 6A), which
is in a region implicated in positive regulation (between
3071 and
3028). In other cases the correspondence between the footprints and
the invariant strings of nucleotides is not as strong. For instance,
two of the three GATA binding sites in
NRA contain mismatches
between human and galago, suggesting that some of the function observed
for
NRA may be specific to higher primates. Regulation of the
-and
-globin genes is distinctive in higher primates, with
considerably more expression of the
-globin gene compared with that
of the
-globin gene in primitive erythroid cells but abundant
expression of the
-globin gene in fetal definitive erythroid cells.
In most other mammals (including galago), the
-globin gene ortholog
is expressed at an equal or higher level than the
-globin gene
ortholog in primitive cells, and neither are expressed in definitive
cells (fetal or adult). Thus some but not all of the regulatory
elements in the
NRA/
PRA may be distinctive to higher primates.
Consistent with this hypothesis, we find that the GATA-1 binding sites
are identical between orangutan and human. However, the orangutan
sequence is very similar to human overall, and investigation of the
sequence of more distantly related simian species would provide a clear
test of the hypothesized function in higher primates. The GATA-1
binding site at
208, implicated in silencing of the
-globin gene
(17), is also found in the human sequence but not in prosimian mammals
or representatives or other mammalian orders, again consistent with a
function only in higher primates.
The second prominent pair of positive and negative regulatory elements
is NRB/
PRB. The negative regulation exhibited by
NRB is seen
only in erythroid cells (data not shown). The strong negative effect of
NRB on the
-globin gene promoter occurs only when it is in its
natural position (Figs. 1 and 3), but it does not act alone on the
proximal promoter (to
177) of the
-globin gene or a heterologous
promoter such as SV40. This suggests that the negative effect of
NRB
may require interaction with downstream sequences in the 5'-flanking
region or other negative elements. A similar cooperative mechanism has
also been proposed for the several positive elements located with
800
of the
-globin gene, which do not function in isolation (20).
DNA-protein binding assays reveal two footprinted regions in
NRB
with K562 cell nuclear extracts, which are absent with HeLa cell
nuclear extract (Fig. 2). Both protected regions correspond to blocks
of sequences, or phylogenetic footprints, conserved in human, galago,
rabbit and mouse. Thus in the case of
NRB, three independent lines
of investigation, i.e. functional analyses of deletion
constructs, in vitro DNA-protein binding data, and analyses
of DNA sequence conservation, generate congruent results, all showing
that this is an important regulatory region in many and possibly all
orders of mammals.
It is interesting to note that this type of deletion analysis points to
the existence of positive and negative elements as frequently close to
each other, essentially in a tandem arrangement along the -globin
gene 5'-flanking sequences. In addition to
NRA/
PRA and
NRB/
PRB, we have also localized pairs of positive and negative
elements generating smaller effects from
2385 to
1747 bp and from
1460 to
1084 bp (Fig. 1A). Several of these regulatory
regions contain conserved sequences previously identified as
phylogenetic footprints (8). The positive region from
1707 to
1511
bp with erythroid specificity identified in this study has been shown
to contain a conserved YY1 binding site and can bind YY1 very strongly
(8), as well as GATA-1. YY1 is a ubiquitous transcription factor with
dual action (35). The negative regions from
1460 to
1135 bp (active
in K562 cells) and
1084 to
883 bp (active in both K562 and HeLa
cells) identified in this study have binding motifs for YY1 and GATA-1.
The positive region from
1153 to
1084 bp (active in K562 cells)
contains a potential GATA-1 binding site (8). The previously
characterized
GS element from
300 to
250 bp also contains
binding sites for both YY1 and GATA1. The manner in which YY1 and GATA1
function in both positive and negative regulation of the
-globin
gene is an important matter for further study. The detection of GATA-1
binding proteins, such as FOG (36), may point to complex protein
assembly mechanisms mediating these effects.
We suggest that the down-regulation of -globin gene expression as
development progresses involves cooperative interactions of the
negative regulatory elements located around
4.5,
3,
1.7, and
0.3 kb (
GS), plus specific motifs located in the other general negative regions identified in the 5'-flanking region examined in this
study (Fig. 1A). In particular, the reporter activity of
construct p
6073, which contains about 6 kb of 5'-flanking sequences,
is only 3% of that for the proximal
-globin promoter, p
177 (Fig.
1A). This suggests that, even though along 6 kb of 5'-flanking sequences there are several positive as well as negative control elements, the net effect is negative on the
-globin gene promoter, despite the fact that this construct contains HS 1. This
could be the reason that when the
-globin silencer around
275 is
deleted or mutated, the expression in adult transgenic mice of the
human
-globin transgene linked to an LCR is only 5-10% as compared
with the level of the endogenous mouse
y or
gene
(18).2 Additional aspects of the silencing process may be
apparent when the
-globin gene is linked with the LCR and other
genes within the
-globin gene cluster. Other experiments in
transgenic mice suggest that control of
-globin gene expression may
not be strictly autonomous and that in addition to the LCR, other
regulatory elements flanking the 5' region of the
-globin gene may
affect expression of the genes located more 3' in the cluster. Studies
using human YAC constructs containing the
-globin gene cluster with
the LCR showed that deletion of the
-globin silencer region also
affected
-globin gene expression as well (19). Our new results
identifying even more cis-acting regulatory elements in the 5' flank of
the
-globin gene illustrate the complexity of the mechanisms of
-globin gene silencing, and they are a further step in improving
understanding of the joint regulation of the entire
-globin gene
cluster.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank C. Barrow for technical assistance
and S. Shapiro for providing the plasmid which has been used to
generate p6073.
![]() |
FOOTNOTES |
---|
* This work was supported in part by research grants from the National Institutes of Health, PHS R01 LM05110 (to W. M.), PHS R01 LM05773, and PHS R01 DK27635 (to R. H.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence should be addressed: Laboratory of Chemical Biology, NIDDK, NIH, Bldg. 10, Rm. 9N-307, 10 Center Dr., MSC 1822, Bethesda, MD 20892-1822. Tel.: 301-496-5408; Fax: 301-402-0101.
1 The abbreviations used are: HS, hypersensitivity site; LCR, locus control region; GS, gene silencer; hAEC, human adult erythroid cell; NR, negative regulatory region; PR, positive regulatory region; bp, base pair(s); kb, kilobase pair(s).
2 B. Peters, unpublished data.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|