From the
Transcriptional regulation of the human
The human
Both
genetic and molecular data (Higgs et al., 1990) have indicated
that the appropriate erythroid lineage- and developmental
stage-specific expression of the human
The
molecular mechanisms of how the
A number of nuclear
factors bind in vitro to several different sequence motifs of
the
The functional
contributions of these nuclear factor binding motifs to the enhancement
of human embryonic
The plasmids
containing different site-directed mutants are named according to the
following examples. The plasmids containing the wild type HS-40 are
pHS-40-
For transient expression in dimethyl sulfoxide
(Me
The relatively low levels of globin
promoter activities from enhancerless plasmids were measured by the
sensitive human growth hormone (GH) assay (Selden et al.,
1986). The level of HS-40-directed
Data from either GH assay or RNA primer
extension analysis were calibrated against the chloramphenicol
acetyltransferase activities expressed from co-transfected pSV2CAT
plasmids (Gorman et al., 1982), although the chloramphenicol
acetyltransferase activities usually were constant within the range of
±10%. The quantities of the endogenous
The HS-40 enhancer consists of a total of four GATA-1 motifs,
two NF-E2/AP1 motifs, and one GT motif, as defined by nuclear factor
binding studies in vitro (Jarman et al., 1991;
Strauss et al., 1992) (Fig. 1 A). Except for
GATA-1(a), which exhibited no genomic footprint and was apparently
non-functional in previous Bal31 deletion analysis (Zhang et
al., 1993), we have introduced 2- or 3-bp substitutions into each
of the above mentioned motifs (Fig. 1 B). The enhancer
effects of these HS-40 mutants on the
As exemplified by the primer extension analysis in
Fig. 2 and summarized in Fig. 3the enhancer function of HS-40 on
As summarized in Fig. 3, all mutations
introduced into HS-40 greatly decreased its enhancer effect on the
For the first time, site-directed mutagenesis has been used
to systematically analyze the functional roles of different nuclear
factor-DNA complexes of HS-40 in the transcriptional activation of
human
Our data indicate that at least
GATA-1(c) is involved in the final stage of transcriptional activation
of the
The
NF-E2/AP1 binding domain of HS-40 consists of two motifs separated by 6
bp, 5`-ATGACTCAGTG-3` and 5`-TGCTGAGTCAT-3`, both of which can bind
NF-E2 as well as AP1 (Jarman et al., 1991; Strauss et
al., 1992; Andrews et al., 1993a). As we have discovered,
contrasting with 5`HS-2, the NF-E2/AP1 motifs of the
The
opposite effects of the 3- and 1-bp mutations of the 3`NF-E2/AP1 motif
thus strongly suggest that binding of NF-E2 factor to the 3`NF-E2/AP1
motif, and possibly the 5`NF-E2/AP1 motif as well, represses the
HS-40-mediated
The apparent negative regulatory effect of NF-E2
binding on the HS-40 enhancer-directed expression of
-like globin
genes, embryonic
2 and adult
, during erythroid development
is mediated by a distal enhancer, HS-40. Previous protein-DNA binding
studies have shown that HS-40 consists of multiple nuclear factor
binding motifs that are occupied in vivo in an erythroid
lineage- and developmental stage-specific manner. We have
systematically analyzed the functional roles of these factor binding
motifs of HS-40 by site-directed mutagenesis and transient expression
assay in erythroid cell cultures. Three of these HS-40 enhancer motifs,
5`NF-E2/AP1, GT II, and GATA-1(c), positively regulate the
2-globin promoter activity in embryonic/fetal erythroid K562 cells
and the adult
-globin promoter activity in adult erythroid MEL
cells. On the other hand, the 3`NF-E2/AP1 motif is able to exert both
positive and negative regulatory effects on the
2-globin promoter
activity in K562 cells, and this dual function appears to be modulated
through differential binding of the ubiquitous AP1 factors and the
erythroid-enriched NF-E2 factor. Mutation in the GATA-1(d) motif, which
exhibits an adult erythroid-specific genomic footprint, decreases the
HS-40 enhancer function in dimethyl sulfoxide-induced MEL cells but not
in K562 cells. These studies have defined the regulatory roles of the
different HS-40 motifs. The remarkable correlation between genomic
footprinting data and the mutagenesis results also suggests that the
erythroid lineage- and developmental stage-specific regulation of human
-like globin promoters is indeed modulated by stable binding of
specific nuclear factors in vivo.
-like globin gene family is clustered on
chromosome 16. The transcriptionally active members of the gene cluster
are arranged in the order of their expression during erythroid
development: 5`-
2 (embryonic)-
2 (fetal/adult)-
1
(fetal/adult)-
1 (fetal/adult, minor)-3` (Higgs et al.,
1989). Of these members,
2 is transcribed predominantly in yolk
sac, the embryonic erythroid tissue. As development proceeds, the
expression of
2 is shut off, and the two
-globin genes,
2 and
1, are turned on in fetal liver. Finally, in adult
humans, the two
-globin genes are expressed at high levels in
nucleated erythroblasts originated from appropriate stem cells in the
bone marrow. In the adult and fetal erythroblasts, however, low levels
of
2 and
1 messages could still be detected (Albitar et
al., 1989; Chui et al., 1989). The expression of these
-like globin genes, similar to the
-like globin family
members, is regulated predominantly at the level of transcription
(Collins and Weissman, 1984; Karlsson and Nienhuis, 1985).
-like globin gene family
is, like the
-like globin family (Orkin, 1990; Stamatoyannopoulos,
1991), modulated by the so-called locus control region
(LCR).
(
)
The
-LCR is located at
approximately 40 kilobase pairs upstream of the
2-globin gene, and
its functional domain has been mapped, by transgenic mice and DNA
transfection studies, to a 300-bp sequence containing an
erythroid-specific DNase I-hypersensitive site (HS-40). Similar to the
regulation of the
-like globin genes by
-LCR, the
-LCR
or HS-40 confers high levels of erythroid-specific expression of linked
human
2- or
-globin genes in transgenic mice or stably
integrated cell lines (Jarman et al., 1991; Sharpe et
al., 1992; Vyas et al., 1992; Sharpe et al.,
1993; Gourdon et al., 1994). It also behaves as a classical
enhancer for the promoter activity of
2- or
-globin genes in
transiently transfected erythroid cell culture (Pondel et al.,
1992; Ren et al., 1993; Zhang et al., 1993).
-LCR controls
2- or
-globin gene transcription are not well understood. By analogy to
what have been hypothesized for the
-LCR function (Grosveld et
al., 1987; Tuan et al., 1989; Evans et al.,
1990; Forrester et al., 1990; Engel, 1993), the
-LCR
could play a dominant role in the formation of an active chromatin
structure of the entire
-like globin locus domain. It could also,
like other eukaryotic gene enhancers, facilitate the assembly of active
transcriptional initiation complexes at the
2- or
-globin
upstream promoters. All of these regulatory processes are likely to be
modulated by diffusible nuclear factors, as suggested by previous
chromosome transfer and cell fusion experiments (Deisseroth and
Hendrick, 1979; Baron and Maniatis, 1986).
-LCR (Jarman et al., 1991; Strauss et al.,
1992). Subsequent genomic footprinting work has demonstrated the
formation in vivo of specific nuclear factor-DNA complexes at
a subset of these sequence motifs in erythroid cells (Strauss et
al., 1992; Zhang et al., 1993). Interestingly, a single
set of nuclear factor binding motifs is found in the
-LCR as well
as in the
-LCR (Fig. 1 A). These motifs include GATA-1,
which binds a factor specifically expressed in erythroid, megakaryotic,
and mast cell lineages (Martin et al., 1990; Romeo et
al., 1990); the NF-E2/AP1 motif that can bind either NF-E2, which
has a similar cell type specificity of expression as GATA-1, or the
widely expressed AP1 factor (Andrews et al., 1993a, 1993b;
Chan et al., 1993; Chang et al., 1993; Ney et
al., 1993; Caterina et al., 1994; Igarashi et
al., 1994); and the GT motif, which binds Sp1, USF, or TEF (Jarman
et al., 1991; Ellis et al., 1993).
2-globin promoter activity by HS-40 have been
partially analyzed before (Zhang et al., 1993). The results
suggested that one of the four GATA-1 motifs, GATA-1(c) (Fig. 1),
and both NF-E2/AP1 motifs played positive regulatory roles (Zhang
et al., 1993). However, the mutant HS-40 sequences used in the
above study were generated mainly by Bal31 deletion, which could remove
more than one functional motif during successive deletion steps. This
would have prohibited the definitive assignment of the
positive/negative role of a particular motif. Furthermore, the previous
analysis was carried out only with transfected K562 cells, a human
erythroid cell line of embryonic/fetal origins (Lozzio and Lozzio,
1975). Thus, it did not allow us to assess the functional significance
of the developmental stage-specific difference of nuclear factor
binding patterns within HS-40, as seen in previous genomic footprinting
analysis of HS-40 in adult versus embryonic/fetal erythroid
cells (Strauss et al., 1992; Zhang et al., 1993).
Figure 1:A, nuclear factor binding
motifs of HS-40. The motifs that bind nuclear factors in vitro are indicated by the blank boxes on the left map. Those motifs that bind nuclear factors in vivo in K562 cells ( K) and human adult erythroblasts
( E) are indicated by the filled or hatched boxes. Genomic footprints are similar for motifs with
filled boxes, while those of the hatched motifs are different between the two types of erythroid
cells. The three GATA-1 motifs shown are GATA-1(b), GATA-1(c), and
GATA-1(d), respectively, as arranged on HS-40 from left to
right. The two NF-E2/AP1 motifs are 5`NF-E2/AP1 and
3`NF-E2/AP1, also arranged from left to
right. The single GT motif is termed GT II throughout the
text. The maps are constructed from the data of references (Jarman
et al., 1991; Strauss et al., 1992; Zhang et
al., 1993). B, mutants of HS-40 motifs. The mutations of
the nuclear factor binding motifs of HS-40 were generated by
site-directed mutagenesis. For each motif and its flanking DNA, the
wild type sequence is shown in full, with the central binding site of
nuclear factor(s) boxed. The mutated base or bases are
indicated by downward arrows. Note that two different
mutants of the 3`NF-E2/AP1 motif, I and II, have been generated and
analyzed (see text for details).
To gain further insights into the functions of the multiple nuclear
factor-DNA complexes of HS-40, we have generated various HS-40 mutants
by site-directed mutagenesis. The activity of either the embryonic
2- or the adult
-globin gene promoter cis-linked to these
HS-40 mutants was then assayed after transient expression in K562 cells
and in MEL, a mouse adult erythroid cell line (Friend et al.,
1971). As shown below, the present study has clearly defined the
positive/negative roles of different HS-40 DNA motifs, including a dual
role of the 3` NF-E2/AP1 motif. The functionality of these motifs also
correlates remarkably well with their erythroid lineage- and
developmental stage-specific occupancies by nuclear factors in
vivo.
Plasmid Constructs and Site-directed
Mutagenesis
We first constructed pBlue-HS-40 by blunt end
ligation of a 373-bp EcoRI- BamHI fragment containing
the HS-40 enhancer (Higgs et al., 1990) into the
HindIII and EcoRI sites of pBluescript II
KS(-). Site-directed mutagenesis was conducted using a
commercially available kit, Mutatorsite-directed
mutagenesis kit (Stratagene). Different oligonucleotides were designed
and synthesized so that they were homologous to specific target regions
in the wild type enhancer but with one or more base mismatches in the
core binding sites for different nuclear factors. Many of them also
contained specific restriction enzyme sites in order to facilitate the
identification of the mutants. HS-40 mutations were first introduced
into the plasmid pBlue-HS-40. The wild type and different HS-40
mutant-containing fragments were then isolated from agarose gels after
XbaI digestion of pBlue-HS-40 or the above pBlue-HS-40-based
plasmids and cloned into the XbaI site of p-
597GH (Zhang
et al., 1993), which contains 597 bp of the
2-globin
promoter region (-559 to +38) linked to the human growth
hormone reporter gene in p0GH (Nichols Institute). Each of the enhancer
fragments was also cloned, by blunt end ligation, into the
HindIII site of pB-
590GH, which contains a 590-bp
HindIII- DdeI fragment of the human
-globin
promoter (-574 to +21) located immediately upstream of the
human growth hormone gene of p0GH. Only plasmids containing HS-40 in
the genomic orientation relative to the globin promoters were used in
the transfection assay. All mutant plasmids were identified by
restriction enzyme cutting and dideoxynucleotide sequencing.
Corresponding to the lower strand of HS-40, the mutant oligonucleotides
used for site-directed mutagenesis of the HS-40 enhancer are:
GATA-1(b), 5`-GTTGGCCCAGaatTCTGCTCCCTCAAGT-3`; 5`NF-E2/AP1,
5`-CAGAAGCACTGAtcgATGGTTGGCCCAG-3`; 3`(NF-E2/AP1)-I,
5`-CCCCCACAGGATcga-TCAGCAGTCCTGT-3`; 3`(NF-E2/AP1)-II,
5`-CCCCCACAGGATGACTCAGaAGTCCTGT-3`; GT II,
5`-CCCACCTCCACgtgCACAGGATGACTC-3`; GATA-1(c),
5`-GCACCAGAGGTTGaatTCAGCAGTACCA-3`; GATA-1(d),
5`-CTTCCCTCTCAGgTtAACAGGAGGGGGA-3`.
597GH and pHS-40-B-
590GH; the plasmids containing a
mutation in the 5`NF-E2/AP1 motif of HS-40 are pHS-40
(5`NF-E2/AP1)-
597GH and pB-HS-40 (5`NF-E2/AP1)-
590GH, etc.
Transient Transfection Analysis
The maintenance of
the erythroid K562 and MEL cells in culture is as described (Zhang
et al., 1993; Reddy and Shen, 1993). DNA transfection was
accomplished by electroporation (Potter et al., 1984).
Procedures for transfection of K562 cells are similar to those
previously described (Zhang et al., 1993) except that the
cells were harvested at the densities of 5-8 10
cells/ml. It was observed that the lower densities of cells at
harvesting prior to the electroporation gave more reproducible data of
transient expression.
SO)-induced MEL, the cells growing in Dulbecco's
modified Eagle's medium containing 10% fetal bovine serum, 50
units/ml penicillin, and 50 µg/ml streptomycin were harvested at
the densities of 1-5
10
cells/ml. A total of
10
cells was collected by centrifugation and resuspended in
0.4 ml of medium containing 10 µg of the test plasmid(s), 1 µg
of pSV2CAT (Gorman et al., 1982), and 50 µg of sheared
salmon sperm DNA. After electroporation, the cells were grown in media
with 2.5% fetal bovine serum and 2% Me
SO for 5 days, with
1:1 dilution on the 3rd day.
2-globin promoter activities in
K562 cells and HS-40-directed
-globin promoter activities in
Me
SO-induced MEL cells were measured by the GH assay and/or
RNA primer extension (Sambrook et al., 1989), which gave
comparable results.
2 mRNAs were also
used to standardize the expression levels of transfected plasmids. The
average expression level for each plasmid was derived from three or
more independent DNA transfection experiments. The details of the above
GH assay, chloramphenicol acetyltransferase assay, and RNA primer
extension analysis are as described previously (Sambrook et
al., 1989; Zhang et al., 1993).
2-globin promoter activity
in K562 cells and on the
-globin promoter activity in
Me
SO-induced MEL cells were then measured by transient
expression assay. Under our experimental conditions, the wild type
HS-40 enhances the activity of cis-linked embryonic
2-globin
promoter in K562 cells by 160 ± 15-fold and that of cis-linked
adult
-globin promoter in Me
SO-induced MEL cells by 13
± 2-fold.
2-globin promoter activity in K562 cells was not affected by
mutations either in the GATA-1(b) motif (Fig. 2 A, lane 1), which is consistent with our previous result (Zhang
et al., 1993), or in the GATA-1(d) motif
(Fig. 2 A, lane 6). However, the HS-40
enhancer function dropped by 40% when the GATA-1(c) motif was mutated
(Fig. 2 A, lane 5).
Figure 3:
Summary of functional roles of
individual nuclear factor binding motifs of HS-40 enhancer. The motifs
in the 355-bp enhancer are labeled as described in Fig. 1 A except for GATA-1(d), which is half-filled to indicate its
occupancy by nuclear factors, presumably GATA-1, in adult erythroid
cells but not in embryonic/fetal erythroid cells. The small x in the lines below the top diagram indicates the individual motifs
changed by site-directed mutagenesis. The enhancer effects of wild type
and mutant HS-40 sequences on the 2-globin promoter activity in
K562 cells and on the
-globin promoter in Me
SO-induced
MEL cells were analyzed by transient expression assay. The relative
enhancer strengths, with the wild type ( WT) HS-40 being 100%,
were calculated from RNA primer extension and GH assay data as
described in the text and are listed. The standard deviations, as
derived from two to four independent transfection experiments, are
listed in the parentheses.
Figure 2:
A, example of primer extension assay of
RNAs from cells transfected with plasmids containing 2-GH hybrid
gene cis-linked with wild type HS-40 ( lane 7) or with
HS-40 enhancer mutated at GATA-1(b) ( lane 1);
5`NF-E2/AP1 ( lane 2); 3`NF-E2/AP1, the 1-bp mutation
( lane 3); GT II ( lane 4); GATA-1(c)
( lane 5); GATA-1(d) ( lane 6);
lane 8 is from enhancerless plasmid p-
597GH.
B, primer extension assay of RNAs from K562 cells transfected
with plasmids: lane 1, wild type p-HS-40-
597GH;
lane 2, p-HS-40(3`NF-E2/AP1-II)-
597GH; lane 3, p-
597GH; lane 4,
p-HS-40(3`NF-E2/AP1-I)-
597GH. The endogenous
2-globin mRNAs
gave primer extension products 93 nucleotides ( nt) long, while
the major primer extension product from
2-GH hybrid RNAs of
transfected plasmids is 64 nucleotides long.
Mutation of
either 5`NF-E2/AP1 or GT II motif essentially abolished the enhancer
function of HS-40 in K562 cells (Fig. 2 A, lanes 2 and 4). Surprisingly, mutant HS-40 with the
1-bp mutation in the 3`NF-E2/AP1 motif, 3`(NF-E2/AP1)-II
(Fig. 1 B), exhibited a 2-3-fold higher level of
enhancer activity than the wild type (Fig. 2 A, lane 3 and Fig. 2 B, lane 2).
This is in contrast to the previous observation of similar base
mutation in a dimeric NF-E2/AP1 motif, which greatly repressed the
enhancer function of 5`HS2 or -LCR (see ``Discussion'').
On the other hand, the 3-bp mutation of 3`NF-E2/AP1 motif,
3`(NF-E2/AP1)-I (Fig. 1 B), greatly decreased the HS-40
enhancer function by 75% (Fig. 2 B, lane 4).
-globin promoter activity in Me
SO-induced MEL cells.
Interestingly, the increase of HS-40 enhancer function in K562 cells
due to the 3`NF-E2/AP1-II mutation was not observed in MEL cells. Also,
mutation of the GATA-1(d) motif, which is genomic footprinted only in
the adult erythroid environment (Strauss et al., 1992; Zhang
et al., 1993), decreased the HS-40 enhancer function in MEL
cells by 60%.
-like globin upstream promoters by the
-LCR in
erythroid cells at different developmental stages. Since only transient
expression assay has been used, our data most likely reflect the
structure-function relationship during the final stage of
transcriptional activation when the globin promoters are presumably
physically associated with the HS-40 enhancer. Our primary targets for
site-directed mutagenesis of HS-40 are those sequence motifs that bind
specific nuclear factors in vitro and/or in vivo (Fig. 1). Despite the fact that genomic footprints most
likely reflect only relatively stable binding of proteins to DNA helix
and that nuclear factor(s) functioning at an earlier stage of human
-like globin gene regulation may remain bound to its cognate DNA
sequence(s), our data have demonstrated a remarkable correlation
between the erythroid-specific occupancy of specific nuclear factor
binding motifs at the HS-40 enhancer and their regulatory roles in the
transcriptional activation of the human
-like globin promoters in
transiently transfected erythroid cell cultures.
Positive Regulatory and Neutral Roles of Different GATA-1
Motifs
Several previous studies have analyzed the function of
the single GATA-1 motif in the 5`HS-2 enhancer of -LCR. Mutation
in the GATA-1 motif of a synthetic core 5`HS-2 fragment showed no
effect on the level of expression of a linked human globin gene in
multicopy transgenic mice (Ellis et al., 1993). The same
construct exhibited a 40% reduction of
-globin gene expression in
stably integrated MEL cells (Ellis et al., 1993). Mutation of
the same GATA-1 motif in a native 5`HS-2 environment, however, did not
affect the expression of a cis-linked
-globin promoter when stably
integrated in K562 cells as a single copy (Miller et al.,
1993). Finally, GATA-1 mutation showed no effect on the 5`HS-2 enhancer
function on
-globin promoter in transiently transfected K562 cells
(Gong and Dean, 1993). It was suggested by these workers that GATA-1
motif may function by conferring position-independent expression of
linked genes, instead of acting as a classical enhancer element.
However, interpretation of the above data was complicated by 5`HS-2
being only a part of the
-LCR.
-like globin promoters by HS-40 in embryonic/fetal as well
as in adult erythroid cells, although the effect of GATA-1(c) mutation
on HS-40 enhancer-directed activation of the
2-globin promoter in
K562 cells is not as great as the GT II or NF-E2/AP1 motifs
(Fig. 3; see below). Interestingly, mutation of GATA-1(d) motif,
which is bound with nuclear factor(s) in the adult erythroid
environment but not in K562 cells (Strauss et al., 1992; Zhang
et al., 1993), indeed has no effect on HS-40 enhancer function
in K562 cells. It is involved, however, in the transcriptional
activation of the human
-globin promoter by HS-40 in transiently
transfected MEL cells (Fig. 3). Since the above three GATA-1
mutations would prohibit binding of the GATA-1 factor (Ko and Engel,
1993), our data indicate that nuclear factor-DNA complexes formed at
the GATA-1(c) and GATA-1(d) motifs play active roles in the erythroid
cell-specific and developmental stage-specific enhancer function of the
HS-40 or
-LCR. On the other hand, mutation in the GATA-1(b) motif,
which is occupied by nuclear factors in both K562 cells and adult
erythroblasts (Zhang et al., 1993), showed no effect in
transfected K562 (Figs. 2 and 3). We suspect that in erythroid cells of
embryonic/fetal lineage such as K562, this GATA-1 motif is involved at
an earlier regulatory step of HS-40 function such as the assembly of an
active chromatin structure.
The GT II Motif Is Essential for HS-40 Enhancer
Function
The function of the GT motif of 5`HS-2 of the -LCR
has been analyzed previously. Its mutation has only a minimal effect on
the enhancer function of either a synthetic 5`HS-2 fragment in stably
integrated cells (Ellis et al., 1993) or a native 5`HS-2 in
transgenic mice (Caterina et al., 1991). The drastic decrease
in the enhancer effect of HS-40 carrying a mutation in its GT motif, GT
II (Fig. 3), strongly suggests that the nuclear factor-DNA
complex(es) at the GT motif is an essential and integral part of the
multiple protein-DNA complexes formed in HS-40 during its final step of
activating the
-like globin promoters in erythroid cells of
different developmental stages. Sp1 is the major protein in K562
nuclear extract that binds the wild type HS-40 GT motif. However, it
cannot bind to the mutant GT motif.
(
)
The close
proximity of GT II to the 3`NF-E2/AP1 motif also implies that nuclear
factor-DNA complexes formed at these two motifs may directly interact
with each other.
Positive and Negative Regulatory Roles of the NF-E2/AP1
Motifs
The function of the tandemly arranged, dimeric NF-E2/AP1
motifs of 5`HS-2 of the -LCR has been extensively studied
previously by the analysis of mutated 5`HS-2 or
-LCR in
transiently transfected cells, in stably integrated cell lines, or in
transgenic mice. These results have demonstrated that intact dimeric
NF-E2/AP1 motifs are required for 5`HS-2 to function as a potent
erythroid-specific enhancer. However, the dimer motifs do not play any
role in
-LCR or 5`HS-2-mediated, integration site-independent
expression of different human
-like globin promoters (Moi and Kan,
1990; Ney et al., 1990a, 1990b; Caterina et al.,
1991; Talbot and Grosveld, 1991; Miller et al., 1993).
-LCR, or
HS-40, can function as either a positive or negative regulatory
element. Introduction of a 3-bp mutation into the central core of
either the 5` or 3` NF-E2/AP1 motif (Fig. 1 B)
significantly lowered the HS-40 enhancer function to 5-25% of the
wild type level (Fig. 3). However, when a GC base pair at one
boundary of the 3`NF-E2/AP1 binding site is changed to TA
(3`(NF-E2/AP1)-II, Fig. 1 B), the activity of the HS-40
enhancer, when placed in the genomic orientation relative to the
2-globin promoter in the plasmid, is increased by a factor of
2-3-fold in K562 cells. This is interesting since extensive
studies have shown that the same 1-bp mutation abolishes the binding of
NF-E2, but not AP1, to the sequence
(5`-GCTGAGTCAT-3`/3`-CGACTCAGTA-5`), which is identical in the
5`NF-E2/AP1 motif of the 5`HS-2 enhancer, 3`NF-E2/AP1 motif of HS-40,
and the NF-E2/AP1 site in the porphobilinogen deaminase promoter
(Mignotte et al., 1989; Moi and Kan, 1990; Ney et
al., 1990b; Talbot and Grosveld, 1991; Andrews et al.,
1993a; Miller et al., 1993). On the other hand, the 3-bp
mutation would greatly affect the binding of both NF-E2
(3) and
AP1 (Smeal et al., 1989; Abate et al., 1991).
2-globin promoter activity, while the binding of
other AP1 factor(s) to the same motif(s) positively regulates the HS-40
function on
2 promoter expression in K562 cells. Given that K562
is an erythroid cell line trapped during the embryonic to fetal
transition (Lozzio and Lozzio, 1975) and the relative concentration of
NF-E2 to AP1 in erythroid cells appears to increase as development
proceeds (Andrews et al., 1993a), it is likely that
transcriptional activation and silencing of the human
2-globin
gene during embryonic to fetal erythroid development is partly
modulated by the competitive binding of positive regulatory AP1 and
negative regulatory NF-E2 factors to the
-LCR. Of course, we
cannot rule out the possible involvement of other nuclear factors
capable of binding to the NF-E2/AP1 motif(s) in the positive and
negative regulation of the HS-40 enhancer effect (Chan et al.,
1993; Chang et al., 1993; Caterina et al., 1994;
Igarashi et al., 1994; Kataoka et al., 1994; Kerppola
and Curran, 1994).
2-globin
promoter activity is reminiscent of the negative regulation of several
non-erythroid eukaryotic gene systems mediated through binding of the
c-Fos-c-Jun complex at AP1 binding sites (Nicholson et al.,
1990; Lopez et al., 1993; Hsu et al., 1994). It is
also known that specific nuclear factor binding DNA motif(s) can
function as either a positive or negative regulatory element, depending
on the cellular environment (Miner and Yamamoto, 1991). Furthermore, an
enhancer could sometimes function as a silencer (Jiang et al.,
1993).
SO, dimethyl sulfoxide; GH, growth hormone.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.