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(Received for publication, October 11, 1995; and in revised form, December 14, 1995) From the
Using DNase I
footprinting and electrophoretic mobility shift assays, we show that
the region immediately upstream of the 4.1-kb start site is occupied
mainly by the ubiquitous factor Sp1. In contrast, the region adjacent
to the 3.9-kb start site is bound by multiple proteins which include
the tissue-restricted factor AP2, a mammary gland-specific form of
CTF/NF1, Sp1, as well as a candidate negative regulatory factor that
represses transcription from the 3.9-kb start site. These data
experimentally support our conclusion that the 3.9-kb start site has
been introduced into the mammalian
The synthesis of lactose is catalyzed by the
protein heterodimer, lactose synthetase (EC 2.4.1.22), which is
assembled from We have shown that the murine (4) and
bovine (5) The 4.1-kb start site is predominantly used in
all somatic cells and tissues examined. An exception is found in the
mid- to late pregnant and lactating mammary gland, where the 3.9-kb
start site is preferentially utilized(6) . This switch to the
predominant use of the 3.9-kb start site is coincident with the
cellular requirement for increased levels of In this study, we
have focused on verifying those predictions of our model pertaining to
the transcriptional regulation of the
We have previously shown that the cellular requirement for
To determine whether these, or other, sequence
elements do in fact bind nuclear factors, and if this binding is
tissue-specific, a single end-labeled DNA fragment containing the
Figure 1:
DNase I footprinting
analyses of the region adjacent to the 3.9-kb transcriptional start
site. A, the DNA fragment containing
Figure 2:
The location of the DNase I protected
regions and the nuclear factor binding motifs in the 5`-flanking region
of the
FP-1, a rather weak
footprint seen with all three extracts, is located between +36 to
+60. It contains a GC-rich element (5`-GGGCGCG-3`) which is
similar to a sequence motif (5`-GGGCGGC-3`) found just upstream
(+24 to +30) of FP-1. Although this upstream region was not
protected, a hypersensitive site indicative of a protein-DNA
interaction, was seen at position +29 (Fig. 1B).
Footprints FP-2 and FP-4 were also obtained with all three extracts but
were more clearly observed on the coding strand (Fig. 1B) compared to the noncoding strand (Fig. 1A), where the interactions were primarily
characterized by the presence of hypersensitive sites (indicated by the arrows). FP-2 (-34 to +2) contains an inverted GT
box (5`-CCCACCC-3`) and FP-4 (-119 to -87) an inverted GA
box (5`-CCCTCCC-3`). In contrast to footprints FP-1, FP-2, and FP-4,
footprint FP-3 was most prominent with the LMG extract and footprint
FP-5 was clearly LMG-specific (Fig. 1A, lane
4). FP-3 (-70 to -42) is a complex region that
contains multiple overlapping protein binding motifs: a CTF/NF1
half-site (5`-TGGC-3`), a GC-rich element (5`-GGGCGGC-3`) identical to
that found at +24 to +30, an AP2 site (5`-GCCTGCGGG-3`), and
an Sp1 site (5`-GGGCGGG-3`). The only motif noted within FP-5
(-162 to -140) is a perfect AP2 site (5`-GCCGCAGGC-3`).
Because FP-5 extended to the extreme 5`-end of the DNA probe, an
overlapping fragment (-295 to +55) was used to more
precisely map its 5`-boundary (Fig. 1C). This analysis
revealed an additional LMG-specific, DNase I-protected region (FP-6,
-185 to -165) that also contains an AP2 site
(5`-TCCCGCGGC-3`). The above data obtained from the DNase I
footprinting analysis corroborates our previous studies (6) and
shows that the region adjacent to the 3.9-kb start site is recognized
by mammary gland-specific as well as ubiquitous factors. In order to
characterize the nuclear proteins interacting with the protected sites,
double-stranded oligonucleotides corresponding to the footprinted
regions were analyzed by EMSA using nuclear extracts from mouse
L-cells, brain tissue, and LMG.
A
protein-DNA complex (Fig. 3A, indicated by the solid arrow) of similar mobility was seen with all three
extracts (lanes 2-4), with the brain extract giving the
most intense band. It should be noted that, even though footprint FP-1
was rather weak, the protein-DNA complex as visualized by EMSA was
quite strong. This is due to the fact that EMSA is a more sensitive
DNA-protein binding assay than the DNase I protection
assay(13) . The formation of the complex was extract-dependent,
as it was not seen in the control reaction performed in the absence of
the nuclear extract (Fig. 3A, lane 1). The
specificity of binding was demonstrated by competition assays in which
unlabeled oligo 1 was preincubated with L-cell nuclear extract followed
by the addition of labeled oligo 1. As seen in Fig. 3B,
preincubation with a 100-fold molar excess of unlabeled oligo 1 greatly
diminished complex formation (lane 2) and preincubation with a
500-fold molar excess abolished complex formation (lane 3).
Figure 3:
Characterization by EMSA of the putative
negative regulatory factor that binds to the FP-1 site. A,
labeled oligo 1 (+20 to +59), spanning the FP-1 site, was
incubated without (-, lane 1) or with nuclear extract
from L-cells (L, lane 2), brain (Br, lane 3), and lactating mammary gland (LMG, lane
4) and subjected to electrophoresis on a 5% nondenaturing,
polyacrylamide gel. The solid arrow indicates the position of
the specific protein-DNA complex and the open arrow that of
the free probe. B, competition experiments in which none
(-, lane 1) or a 100- or 500-fold molar excess of
unlabeled oligo 1 (lanes 2 and 3) or oligo Sp1,
containing the consensus Sp1 recognition sequence (lanes 4 and 5), was incubated with the L-cell extract prior to the
addition of labeled oligo 1. C, L-cell nuclear extract and
labeled oligo 1 were incubated without (-, lane 1), or
with irrelevant serum (IS, lane 2) or anti-Sp1
antibodies (Sp1, lane 3).
Since the GC-rich elements (GGGCGGC and GGGCGCG) contained within
oligo 1 are similar to the Sp1 recognition sequence (GGGCGGG), an
oligonucleotide containing the consensus Sp1 site (oligo Sp1, Table 1) was also tested in competition assays with labeled oligo
1 as the probe. Oligo Sp1 was not an effective competitor, even at a
500-fold molar excess (Fig. 3B, lanes 4 and 5), indicating that the protein recognizing oligo 1 was not
Sp1 or an Sp1 family member. This conclusion was verified by showing
that polyclonal antibodies against human Sp1, which cross-react with
the mouse protein, neither inhibited nor caused a supershift (retard
the mobility) of the specific protein-DNA complex (Fig. 3C, lane 3). The anti-Sp1 antibodies
were shown to supershift authentic Sp1 in a control experiment (data
not shown, also see Fig. 4). Analogous experiments performed
using oligo 1 and nuclear extracts from brain and LMG gave results
similar to those described for L-cells (data not shown).
Figure 4:
Sp1 or Sp1-related nuclear factor(s)
binds to the FP-2 site. A, labeled oligo 2 (-47 to
-10) spanning the FP-2 site was incubated without (-, lane 1) or with nuclear extract from L-cells (L, lane 2), brain (Br, lane 3), and lactating
mammary gland (LMG, lane 4) or Sp1 protein (Sp1, lane 5), and analyzed by EMSA. The position of
the two protein-DNA complexes (I and II) is shown by
the solid arrows (see footnote 3). The open arrow indicates the position of the free probe. B, identical to A except labeled oligo Sp1 was used. C, unlabeled
oligo 2 (lanes 2 and 3) or oligo Sp1 (lanes 4 and 5) at the indicated molar excess was used as
competitor for the formation of specific protein-DNA complexes between
labeled oligo 2 and L-cell nuclear extract. D, L-cell nuclear
extract and labeled oligo 2 were incubated without (-, lane
1) or with irrelevant serum (IS, lane 2) or
anti-Sp1 antibodies (Sp1, lane
3).
We had
previously identified a sequence motif between -15 and -6
with a weak similarity to a negative element described by Kageyama and
Pastan(16) . However, EMSA using an oligonucleotide containing
this sequence motif failed to demonstrate any protein binding (data not
shown). Therefore, the protein binding to oligo 1, which we term GC
binding factor (GCBF), is the candidate for the negative
regulatory factor. Both GC-rich elements in oligo 1 appear to be
important for high affinity binding, as two separate oligonucleotides
containing either of the GC-rich elements showed very weak binding
(data not shown). GCBF is predicted to have a broad tissue distribution
as the 3.9-kb transcript is down-regulated in most somatic tissues.
Consistent with this prediction, a preliminary survey has established
that this factor is also present in liver, lung, and kidney (data not
shown).
To compare the binding of nuclear
factors in each nuclear extract to the consensus Sp1 site, EMSAs were
conducted using oligo Sp1. As seen in Fig. 4B, an
identical pattern of bands with mobilities similar to those observed
with oligo 2 was obtained, except that all the bands were
proportionally more intense. These results suggest that the same
factor(s) that binds the GT box (oligo 2) somewhat weakly, binds the GC
box (oligo Sp1) strongly. To confirm this, competition experiments
using oligo 2 and oligo Sp1 were performed with the L-cell extract (Fig. 4C). A 50-fold molar excess of unlabeled oligo 2
had little effect on binding of labeled oligo 2 (compare lane 2 to the reaction lacking the competitor oligonucleotide in lane
1). A 250-fold molar excess of unlabeled oligo 2 (lane 3)
resulted in partial competition with a proportionate weakening of both
bands. In contrast, a 50- or a 250-fold molar excess of unlabeled oligo
Sp1 (lanes 4 and 5), abolished the formation of both
complexes. The formation of the two protein-DNA complexes with oligo 2
was also inhibited by anti-Sp1 antibodies (Fig. 4D, lane 3). These data demonstrate that complex I and II,
obtained upon incubation of the L-cell nuclear extract with oligo 2,
are specific and result from the binding of Sp1 or Sp1-like proteins,
which have a greater affinity for the GC box (oligo Sp1) than the GT
box (oligo 2). Analogous experiments established that Sp1 or a
related family member also binds the FP-4 site which contains an
inverted GA box (CCCTCCC). Similar results were obtained when these
experiments were repeated using brain and LMG nuclear extracts (data
not shown).
Figure 5:
Identification of mammary gland-enriched
and ubiquitous transcription factors interacting at the FP-3 site. A, labeled oligo 3 (-82 to -37) spanning the FP-3
site was incubated without (-, lane 1) or with nuclear
extract from L-cells (L, lane 2), brain (Br, lane 3) and lactating mammary gland (LMG, lane
4) and analyzed by EMSA. The four protein-DNA complexes (I-IV) are indicated by the solid arrows. The open arrow indicates the position of the free probe. B, a 250-fold molar excess of oligo 3 (lane 2), oligo
Sp1 (lane 3), oligo AP2 containing the consensus AP2 site (lane 4), or oligo C/N containing the consensus CTF/NF1 site (lane 5), was used as competitor for the binding of nuclear
factors present in the LMG extract to labeled oligo 3. C, a
binding reaction containing labeled oligo 3 and LMG extract was
performed in the presence of irrelevant serum (IS, lane
1), anti-Sp1 antibodies (Sp1, lane 2), anti-AP2
antibodies (AP2, lane 3), or anti-CTF/NF1 antibodies (C/N, lane 4). Reactions shown in lanes 1 and 4, and lanes 2 and 3 were from two
separate experiments. D, labeled oligo C/N (lanes
1-3) or oligo 3 (lane 4) was incubated with the
nuclear extract from the indicated tissue.
To demonstrate if the formation of complexes I-IV was
specific and corresponded to the four putative protein binding motifs
identified in this region (Sp1, AP2, GC-rich element, and CTF/NF1
half-site; see Fig. 2), competition assays, using labeled oligo
3 and unlabeled oligo Sp1, oligo AP2, and oligo C/N containing the
respective consensus binding site as competitors, were performed. The
results of one such assay, using the LMG extract, showed that addition
of a 250-fold molar excess of unlabeled oligo 3 inhibited complexes
I-III; complex IV was partially diminished (Fig. 5B, lane 2), suggesting that the
formation of all four complexes is specific. Complex I formation was
abolished in the presence of unlabeled oligo Sp1 (Fig. 5B, lane 3) and anti-Sp1 antibodies (Fig. 5C, lane 2), confirming that Sp1 or an
Sp1-like protein binds to the perfect Sp1 motif (GGGCGGG) at the FP-3
site. Similar results were obtained with the nuclear extract from
L-cells (data not shown). It was surprising that Sp1 binding to this
site was weak since both L-cells and the LMG contain relatively high
levels of Sp1 (see Fig. 4B). This may be due to
competition between multiple factors binding to overlapping sequence
elements at the FP-3 site. Complex II formation was abolished in the
presence of a 250-fold molar excess of unlabeled oligo AP2 (Fig. 5B, lane 4) and anti-AP2 antibodies (Fig. 5C, lane 3), confirming that nuclear
factor AP2 binds to the AP2 motif (GCCTGCGGG) at the FP-3 site. A
number of observations led to the conclusion that complex III, which
was seen with all three nuclear extracts, may result from the binding
of GCBF or a GCBF-like factor to the GC-rich sequence (GGGCGGC): (i)
The GC-rich motif at the FP-3 site is identical to the GCBF binding
site (+24 to +30) upstream of FP-1. (ii) The mobility of
complex III is similar to that of the complex seen with oligo 1. (iii)
Complex III formation is highest in brain and GCBF levels are also
highest in this tissue. (iv) The formation of complex III is not
inhibited by an excess of unlabeled oligo Sp1 (Fig. 5B, lane 3), nor by anti-Sp1 antibodies (Fig. 5C, lane 2), as noted for GCBF. Complex IV formation, which is
unique to the LMG, was abolished in the presence of a 250-fold molar
excess of unlabeled oligo C/N (Fig. 5B, lane
5) and greatly diminished by anti-CTF/NF1 antibodies (Fig. 5C, lane 4), indicating that CTF/NF1 or
a CTF/NF1-like factor binds to the CTF/NF1 half-site (TGGC). This
nuclear factor has a greater affinity for the palindromic consensus
CTF/NF1 site than the half-site, as oligo C/N competed more effectively
for the formation of complex IV than oligo 3 (compare complex IV in lanes 5 and 2, Fig. 5B). It is
noteworthy that inhibition of complex IV (either by oligo C/N or
anti-CTF/NF1 antibodies) enhanced the formation of complex II,
suggesting that there is competition between binding of CTF/NF1 and AP2
to their respective site. However, CTF/NF1 appears to preferentially
bind at the FP-3 site, as complex IV is the major band seen with the
LMG nuclear extract. Analogous experiments using L-cell nuclear
extract showed that complex II formation was not competed by unlabeled
oligo AP2 but it was competed by unlabeled oligo C/N (data not shown).
These results are consistent with the fact that AP2 is a
tissue-restricted transcription factor that is present in LMG but not
in L-cells or brain (23) (see Fig. 6A).
Therefore, in the LMG, complex II formation is due to AP2, whereas in
L-cells it is due to CTF/NF1. These results suggest that two different
forms of CTF/NF1, with varying mobilities, exist in the LMG and
L-cells. To test this directly, labeled oligo C/N was incubated with
each nuclear extract (Fig. 5D). An intense,
heterogeneous band with mobility comparable to complex II was observed
with all three extracts (lanes 1-3), consistent with the
widespread distribution of CTF/NF1(24) . A higher mobility
band, similar to complex IV, was seen only with the LMG extract (lane 3), suggesting the presence of a mammary gland-specific
form of CTF/NF1 which we term, MG-C/N. Although CTF/NF1 is abundant in
all three tissues, its binding to oligo 3 is reduced in L-cells and
absent in brain. This may be attributed to the fact that the ubiquitous
form of CTF/NF1 has a greater affinity for the full palindromic binding
motif (TGG(C/A)(N
Figure 6:
Identification of AP2 as the nuclear
factor that binds to the FP-5 site. A, oligo 5 (-162 to
-127), spanning the FP-5 site, was labeled and incubated without
(-, lane 1) or with nuclear extract from L-cells (L, lane 2), brain (Br, lane 3),
and lactating mammary gland (LMG, lane 4) and
analyzed by EMSA. The solid arrow designates the position of
the protein-DNA complex obtained with the LMG extract. The open
arrow indicates the position of the free probe. B, a 50-
or a 250-fold molar excess of either oligo 5 (lanes 2 and 3) or oligo AP2 (lanes 4 and 5) was used as
a competitor for the formation of the specific protein-DNA complex
between labeled oligo 5 and LMG nuclear extract. C, a binding
reaction with labeled oligo 5 and LMG nuclear extract was performed
without (-, lane 1) or with irrelevant serum (IS, lane 2) or anti-AP2 antibodies (AP2, lane 3). The position of the supershifted band in lane 3 is shown.
In summary, the results from the
EMSA are in agreement with the DNase I footprinting analysis and
confirm that the major interaction at the FP-3 site is mammary
gland-specific and is the result of binding a mammary gland-specific
form of CTF/NF1 (MG-C/N) to the CTF/NF1 half-site.
Analogous experiments using an oligonucleotide spanning the
FP-6 site gave similar results, although the intensity of the complex
was reduced (data not shown). Since the regions represented by FP-5 and
FP-6 were equally well protected in the DNase I protection assay (Fig. 1C), these data suggest cooperative binding to
the two AP2 sites. For example, binding of AP2 at the FP-5 site may
stabilize binding at the FP-6 site. More importantly, these results
show that the mammary gland-specific interactions at the FP-5 and the
FP-6 sites are due to the binding of AP2 or an AP2-like protein that is
absent in L-cells and brain.
Six
protected regions (FP-7 to FP-12), demarcated by hypersensitive sites,
were seen when the DNA fragment (-474 to +55) was analyzed
by the DNase I footprinting assay (Fig. 7; FP-7 is better
visualized in the bottom half of Fig. 1C). FP-7, FP-8,
and FP-9 were observed with nuclear extracts from all three tissues and
each footprint contains an inverted GC or GT box (Fig. 2).
FP-10, FP-11, and FP-12 were seen with the L-cell and LMG extracts but
not with the brain extract. FP-11 was more pronounced with the LMG
extract compared to the L-cell extract on the noncoding strand,
however, on the coding strand both extracts showed equivalent
protection (data not shown). FP-10 and FP-11 contain an imperfect,
inverted GC box and an inverted GA box, respectively (Fig. 2).
The protection of the FP-12 region was qualitatively different between
the L-cell and LMG extracts; the L-cell extract showed better
protection at the top (3`)-half of FP-12, whereas the LMG extract
protected the bottom (5`)-half better. The reason for this became
apparent when an inspection of this protected sequence revealed
overlapping binding sites for AP2 (absent in L-cells) and Sp1 (present
in L-cells and LMG).
Figure 7:
DNase I footprinting analysis of the
region immediately upstream of the 4.1-kb transcriptional start site. A
DNA fragment containing the
Oligonucleotides corresponding to FP-7 to FP-12
were then analyzed by EMSA and protection at each site was shown to be
the result of binding by Sp1 or a related family member (data not
shown). As expected, the oligonucleotide corresponding to the FP-12
site also showed weak binding by AP2 with the LMG extract. These data
confirm that Sp1, or a family member, interacts at multiple sites in
the immediate vicinity of the 4.1-kb start site and that the region
upstream of this start site may well function as a housekeeping
promoter. Consistent with this conclusion is the correlation between
the levels of Sp1 binding activity and the 4.1-kb mRNA in the three
different tissues tested. Brain which has
Figure 8:
DNase I footprinting analysis of the
region between -474 to -805. A DNA fragment containing the
We have recently shown that a 798-bp genomic fragment
spanning the male germ cell start site is sufficient to target
expression of the reporter gene, With respect to
In the present study we have used DNase I protection and EMSAs to
determine if these cis-acting elements identified by ``paper
analysis'' do in fact bind the corresponding trans-acting factors.
The results are summarized in Fig. 9and reveal a modular
arrangement of binding sites. The cluster of sites adjacent to the
3.9-kb start site bind the mammary gland-enriched factors, MG-C/N and
AP2, the ubiquitous factor Sp1 and a putative negative regulatory
factor, GCBF. The cluster of sites located just upstream of the 4.1-kb
start site bind Sp1 or related family members. These data agree
remarkably well with the model we previously proposed, although several
modifications were noted. The sequence motif (-15 to -6)
similar to the negative element described by Kageyama and Pastan (16) and the sequence motif (-9 to +1) similar to
the binding site for MAF, a factor shown to be involved in the mammary
gland-specific expression of mouse mammary tumor virus (MMTV) (32) , did not show protein binding.
Figure 9:
Schematic showing the sites bound by
trans-acting factors as determined by DNase I footprinting and EMSAs.
The positions of the binding sites for various nuclear factors present
in the lactating mammary gland (LMG), L-cells and brain tissue
in the
While the ubiquitous
form of CTF/NF1 binds to the palindromic CTF/NF1 site at -495 to
-507, this factor is unlikely to be functionally involved in the
regulation of the 4.1-kb transcript, since the promoter deletion
analysis in L-cells (6) shows that the
Although GCBF or a
GCBF-like protein appears to bind to the GC-rich motif at the FP-3 site
( Fig. 2and Fig. 5), this binding does not seem to have a
negative effect, as a reduction in CAT activity was not observed when
the FP-3 region was included in one of the
Our data show that both the ubiquitous form of
CTF/NF1 and MG-C/N bind with higher affinity to the palindromic
sequence than to the half-site, but the half-site in FP-3 is notable in
that it binds MG-C/N with higher affinity than the ubiquitous form.
This may result from cooperative interaction with AP2, which also binds
at the FP-3 site. It has been proposed that CTF/NF1 binding may be
stabilized by interactions with factors bound to adjacent
sites(25) . The FP-13 site, containing the palindromic
CTF/NF1 sequence, binds both forms and shows equivalent protection
using nuclear extract from L-cells, brain, or LMG. While this might
suggest that this site is involved in 4.1-kb mRNA expression, we think
it unlikely as
AP2 also appears to be involved in 3.9-kb mRNA expression
as it is found only in the LMG and not in L-cells or brain. The close
proximity of the three AP2 sites to the CTF/NF1 half-site just upstream
of the 3.9-kb start site suggests that these factors may function
cooperatively, as proposed for MMTV, to increase transcription from the
3.9-kb start site. The three additional AP2 sites and the palindromic
CTF/NF1 site, located upstream of the 4.1-kb start site, may function
in an enhancer-like capacity. A redundancy of cis-acting elements
involved in tissue-specific expression has been noted in other genes.
For example, multiple binding sites for factors (CTF/NF1 and mammary
gland factor (MGF)) critical for mammary gland-specific expression of
the whey acidic protein gene are found in the promoter proximal and
distal regions, and it has been suggested that interaction at both
sites is necessary for high level expression(52) .
As discussed, the
3.9-kb
With the recruitment of
In summary, the results presented in this study support the
conclusion that the presence of the 3.9-kb start site in the mammalian
The nucleotide sequence(s) reported in this paper has been submitted
to the GenBank(TM)/EMBL Data Bank with accession number(s)
L16840[GenBank].
Volume 271,
Number 9,
Issue of March 1, 1996 pp. 5131-5142
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
1,4-Galactosyltransferase in Somatic Cells
ANALYSIS OF A GENE THAT SERVES BOTH A HOUSEKEEPING AND A MAMMARY
GLAND-SPECIFIC FUNCTION (*)
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
1,4-Galactosyltransferase (4-GT) is a constitutively
expressed enzyme that synthesizes the
4-N-acetyllactosamine structure in glycoconjugates. In
mammals, 4-GT has been recruited for a second biosynthetic
function, the production of lactose which occurs exclusively in the
lactating mammary gland. In somatic tissues, the murine 4-GT gene
specifies two mRNAs of 4.1 and 3.9 kilobases (kb), as a consequence of
initiation at two different start sites 200 base pairs apart. We
have proposed that the region upstream of the 4.1-kb start site
functions as a housekeeping promoter, while the region adjacent to the
3.9-kb start site functions primarily as a mammary gland-specific
promoter (Harduin-Lepers, A., Shaper, J. H., and Shaper, N. L.(1993) J. Biol. Chem. 268, 14348-14359).
4-GT gene to accommodate the
recruited role of 4-GT in lactose biosynthesis.
1,4-Galactosyltransferase (4-GT) (
)is a
trans-Golgi resident, type II membrane-bound glycoprotein that is
widely distributed in the vertebrate kingdom. It catalyzes the transfer
of galactose to N-acetylglucosamine residues, forming the
4-N-acetyllactosamine (Gal4-GlcNAc) or
poly-N-acetyllactosamine structure found in glycolipids and
the N- and O-linked side chains of glycoproteins and
proteoglycans(1) . Since glycoconjugate biosynthesis occurs in
essentially all tissues, it can be considered a housekeeping function.
In mammals, 4-GT has been recruited for an additional
tissue-specific biosynthetic function, which is the production of
lactose (Gal4-Glc) in the lactating mammary gland
(LMG)(2) .4-GT and 
-lactalbumin. The net result of this
association is to lower the K of glucose
for
4-GT about three orders of magnitude, thus making glucose an
effective acceptor substrate at physiological concentration.
-Lactalbumin is synthesized exclusively in the epithelial cells of
the mammary gland beginning in late pregnancy(3) . Enzymatic
levels of 4-GT also increase in the mammary gland beginning in
mid-pregnancy, in preparation for lactose biosynthesis(3) . The
expression of both -lactalbumin and 4-GT is positively
influenced by the lactogenic hormones, insulin, hydrocortisone, and
prolactin(3) .4-GT genes specify two mRNAs of 4.1 and
3.9 kb in somatic cells. The two transcripts are generated as a
result of initiation at two different start sites located on exon 1,
and separated by
200 bp. The main difference between the two mRNAs
is the length and extent of predicted secondary structure present in
the respective 5`-untranslated region(6) . Because each start
site is positioned either upstream of the first two in-frame ATGs (4.1
kb) or between these two in-frame ATGs (3.9 kb), translation of the two
mRNAs results in the synthesis of two functional, structurally related
protein isoforms that differ only in the lengths of their
NH
-terminal cytoplasmic domain (reviewed in Shaper and
Shaper(7) ).4-GT enzyme for
lactose biosynthesis. These observations, combined with a promoter
deletion analysis using 4-GT/CAT hybrid constructs, led us to
propose a model for transcriptional and translational regulation of the
4-GT gene in which the distal region upstream of the 4.1-kb start
site functions as a housekeeping promoter in all somatic cells, while
the proximal region upstream of the 3.9-kb start site serves primarily
as a mammary gland-specific promoter. In addition, we proposed that a
putative negative regulatory region identified adjacent to the 3.9-kb
start site, down-regulates transcription from this start site in all
somatic tissues except the mid- to late pregnant and lactating mammary
gland. The key feature of our model is that mammals have evolved a
two-step mechanism to generate the elevated levels of 4-GT
enzymatic activity required for lactose biosynthesis. First, there is
an up-regulation of the steady state levels of 4-GT mRNA by the
predominant synthesis of the transcript (3.9 kb) that is regulated by
mammary gland-specific factors. Second, the 3.9-kb 4-GT transcript
with its short (20 nucleotides), less structured 5`-untranslated
region is translated more efficiently compared to its housekeeping
counterpart (4.1 kb) which has a long (
200 nucleotides), highly
structured 5`-untranslated region(6) .
4-GT gene. We have used
DNase I protection and electrophoretic mobility shift assays (EMSAs) to
identify specific cis-acting elements and the corresponding
trans-acting factors potentially involved in the expression of the
4.1-kb and the 3.9-kb 4-GT transcripts. We show that the distal
promoter region immediately upstream of the 4.1-kb start site is bound
primarily by the ubiquitous transcription factor Sp1. In contrast, the
proximal promoter region adjacent to the 3.9-kb start site is a target
for binding by multiple proteins which include a candidate negative
regulatory factor, Sp1, a mammary gland-specific form of CTF/NF1 and
the tissue-restricted factor, AP2.
Materials
Reagents for molecular biology and
tissue culture were from Life Technologies, Inc. P-Labeled
radioisotopes were from Amersham Corp. All protease inhibitors, formic
acid (99%), and piperidine were from Sigma. Protein assay dye reagent
was from Bio-Rad. Poly(dI-dC), proteinase K, and calf intestinal
alkaline phosphatase were from Boehringer Mannheim. DNase I was from
Cooper Biomedical. Mid-pregnant Swiss Webster mice were obtained from
Harlan Sprague-Dawley Laboratory Animals. Purified Sp1 protein and
anti-Sp1 and anti-AP2 antibodies were from Santa Cruz Biotechnology
Inc. Anti-CTF/NF1 antiserum was a kind gift from Dr. N. Tanese (New
York University Medical Center).
Cells and Cell Culture
Mouse L-cells were obtained
from ATCC and maintained in Dulbecco's modified Eagle's
medium supplemented with 10% horse serum, 100 units/ml penicillin, and
50 mg/ml streptomycin at 37 °C in 5% CO.Preparation of Nuclear Extracts
Nuclear extracts
from mouse L-cells (90% confluent) were prepared according to the
method of Dignam et al.(8) and from mouse brain and
LMG by the combination of methods of Roy et al.(9) and Dignam et al.(8) . Briefly,
frozen tissue (2 g) was pulverized under liquid nitrogen to a fine
powder, using a mortar and pestle, and transferred to an ice-cold
Dounce homogenizer (type B pestle) containing 10 ml of NE1 buffer (250
mM sucrose, 15 mM Tris-HCl, pH 7.9, 140 mM NaCl, 2 mM EDTA, 0.5 mM EGTA, 25 mM KCl, 2 mM MgCl
, 0.15 mM spermine,
0.5 mM spermidine, and 1 mM dithiothreitol). The
number of strokes required to lyse the cells depended on the individual
tissue, and this step was monitored by checking aliquots of the lysate
with a phase-contrast microscope. The homogenate was centrifuged at
1000 
g for 10 min. The nuclear pellet was washed once
with the same buffer and resuspended in 1 packed cell volume of NE2
buffer (NE1 buffer containing 350 mM KCl). The extracted
nuclei were centrifuged at 180,000 g for 90 min, and
the supernatant (nuclear extract) was collected, dialyzed against
buffer D(8) , aliquoted, and stored at -70 °C. All of
the steps were carried out at 4 °C, and the buffers were
supplemented with a mixture of the following protease inhibitors: 0.5
mM phenylmethylsulfonyl fluoride (added from anhydrous stock
immediately before use), 1 µg/ml each of leupeptin, chymostatin,
and pepstatin, 2 µg/ml antipain, 10 µg/ml benzamidine, and 1
unit/ml aprotinin. Protein concentrations of the extracts, which ranged
from 2 to 5 mg/ml, were estimated by the method of
Bradford(10) .Oligonucleotide Probes for Electrophoretic Mobility Shift
Assays
Single-stranded oligonucleotides were synthesized by
Integrated DNA Technologies, and complementary strands were annealed
before use. Each double-stranded oligonucleotide contained a recessed
3`-end which was filled in with [-P]dCTP
and the remaining dNTPs using the Klenow enzyme. The
P-labeled probes were separated from the unincorporated
nucleotides by chromatography on Sephadex G-25 (fine) packed in a
9-inch disposable Pasteur pipette and equilibrated with 10 mM Tris-HCl, pH 8.0, and 1 mM EDTA. The DNA sequence of the
oligonucleotides used is shown in Table 1.
Electrophoretic Mobility Shift Assays
EMSAs were
performed essentially as previously described(11) . Briefly, 5
µg of each nuclear extract was incubated with 20,000 cpm of P-labeled, double-stranded probe (5-10 fmol) and 1
µg poly(dI-dC) in a 20-µl reaction mixture containing 20 mM Hepes-NaOH, pH 7.9, 50 mM KCl, 5 mM MgCl
, 1 mM EDTA, 1 mM dithiothreitol, 10% glycerol, and 4% Ficoll at room temperature
for 20 min. For competition experiments, a 50-500-fold molar
excess of unlabeled, doubled-stranded probe was incubated with the
nuclear extract for 20 min, prior to the addition of the labeled probe.
To identify specific transcription factors in a protein-DNA complex, 2
µl (1 mg/ml IgG) of antibodies against a known transcription factor
were included in the binding reaction, and the mixture was incubated at
4 °C for 60 min. The samples were subjected to electrophoresis on a
5% nondenaturing, polyacrylamide gel in 40 mM Tris acetate, pH
8.0, 1 mM EDTA at 10 V/cm at room temperature. The gel was
dried and exposed to x-ray film at -70 °C with intensifying
screens.Probes for DNase I Protection Assays
Restriction
digests, fragment isolation and purification, and 3`- and 5`-end
labeling with Klenow enzyme or T
polynucleotide kinase,
respectively, were performed using standard
techniques(12, 13) . The cDNA clone,
MGT-P5(4) , harboring the mouse 4-GT sequence from
-172 to +187, was digested with HindIII and BamHI, or EcoRV and BamHI, and the
respective fragment was isolated. The former was labeled at the 3`-end
and digested with MaeIII, and the 299-bp single-end-labeled HindIII-MaeIII fragment (containing 17 bp of the
vector sequence and 4-GT region from -172 to +110) was
purified on a 4% polyacrylamide gel. To generate a probe labeled on the
complementary (coding) strand, the EcoRV-BamHI
fragment was digested with MaeIII and labeled at the 3`-end,
and the 293-bp EcoRV-MaeIII fragment (with 11 bp of
the vector sequence and the 4-GT sequence from -172 to
+110) was isolated. Two additional probes prepared from the
-474/+55 CAT-En construct (6) were labeled at the
3`-end on the noncoding strand: (i) HinfI-HindIII
fragment (containing the 4-GT sequence from -295 to +55
and 13 bp of vector DNA) and (ii) EcoRI-HindIII
fragment (with 4-GT sequence from -474 to +55 and 20
and 10 bp of vector sequence at each end). Finally, a 379-bp AvaII-EcoO109I fragment containing the 4-GT
sequence from -828 to -449 was isolated from the
-1897/+55 CAT plasmid (6) and 5`-end-labeled on the
noncoding strand.DNase I Protection Assays
The protein-DNA binding
reactions were performed as described for EMSA above, except that
10,000 cpm of P-, single-end-labeled DNA fragment was
incubated with 25 µg of bovine serum albumin (BSA) or 25-50
µg of nuclear extract in the presence of 2 µg of poly(dI-dC).
Following incubation at room temperature for 20 min, 1 µl of DNase
I, diluted from a 5 mg/ml stock solution in 10 mM Hepes-NaOH,
pH 7.6, and 25 mM CaCl
, was added to the binding
mixture, and digestion was allowed to proceed for 2 min at room
temperature. Dilutions of DNase I used were 1:1500 for BSA, 1:150 for
L-cell nuclear extract, and 1:20 for brain and LMG nuclear extracts.
The reaction was stopped by the addition of 80 µl of a solution
containing 20 mM Tris-HCl, pH 8.0, 20 mM EDTA, 250
mM NaCl, 0.5% SDS, 10 µg of sonicated salmon sperm DNA,
and 10 µg of proteinase K. The samples were incubated at 45 °C
for 60 min, extracted once with phenol/chloroform (1:1), and
precipitated with ethanol. The pellets were resuspended in 80%
formamide dye and electrophoresed on an 8% polyacrylamide, 8 M urea sequencing gel. An aliquot of the same end-labeled DNA
fragment was also subjected to the A + G chemical sequencing
reaction (14) and electrophoresed on the same gel to determine
the position and the sequence of the protected regions. The gel was
dried and exposed to x-ray film at -70 °C with an
intensifying screen.
4-GT enzymatic activity correlates with the transcriptional start
site used (6) . In the majority of mouse somatic tissues,
including the mammary gland from virgin mice, ()and
established cell lines derived from somatic tissues (e.g. L-cells), the 4.1-kb start site is predominantly used (the ratio
of the 4.1- to the 3.9-kb transcript is 5:1). However, in brain
tissue, the N18TG2 neuroblastoma cell line, and spermatogonia, the
steady state levels of
4-GT mRNA are 10-fold lower relative
to most somatic tissues and L-cells, and the 4.1-kb start site is
exclusively used. Additionally, in the mid- to late pregnant and
lactating mammary gland, the steady state
4-GT mRNA levels are
10-fold higher compared to most somatic tissues and L-cells, and
the 3.9-kb start site is preferentially used (the ratio of the 4.1- to
the 3.9-kb transcript is
1:10). This differential utilization of
the two start sites suggested that housekeeping and mammary
gland-specific transcription factors, binding to different promoter
elements, regulated the use of the 4.1- and the 3.9-kb start sites,
respectively. Therefore, to experimentally verify this prediction, the
DNA sequence flanking the two start sites was analyzed for protein
binding by DNase I footprinting and EMSAs using nuclear extracts
prepared from L-cells, brain tissue, and LMG, which represent the three
patterns of
4-GT mRNA expression described above.Identification of Nuclear Factor Binding Sites in the Region
Adjacent to the 3.9-kb Transcriptional Start Site (-172 to
+110): Evidence for Tissue-specific Binding
Promoter
deletion analysis using 4-GT-CAT hybrid constructs transfected
into L-cells showed that the DNA fragment just upstream of the 3.9-kb
start site (-172 to -13) had promoter activity. However,
inclusion of additional sequence from -13 to +55 in this
construct reduced this activity about 90-fold, suggesting the presence
of a negative regulatory element within this 68-bp region. An
examination of the sequence from -172 to +55 revealed
potential binding sites for positive ubiquitous and mammary
gland-specific transcription factors, as well as a putative negative
element(6) .4-GT sequence from -172 to +110 was subjected to DNase
I footprinting analysis using nuclear extracts from mouse L-cells,
brain tissue, and LMG. Five protected regions, designated FP-1 to FP-5,
were seen on the noncoding strand (Fig. 1A), and four
protected regions corresponding to FP-1 to FP-4, were observed on the
coding strand (Fig. 1B). The sequence of each protected
region was subsequently compared against the entries in the
transcription factor data base (15) . The combined results of
these analyses are summarized in Fig. 2.
4-GT sequence from
-172 to +110 was labeled at the 3`-end of the noncoding
strand, incubated with BSA (lane 1) or nuclear extract from
L-cells (L, lane 2), brain (Br, lane
3), and lactating mammary gland (LMG, lane 4),
and treated with DNase I. An A + G chemical sequencing reaction (lane 5) performed on the same probe was run in parallel with
the samples on an 8% sequencing gel. The nucleotide numbering is
relative to A (+1) of the first in-frame ATG (Fig. 2). The
areas protected from DNase I digestion are marked by brackets and designated FP-1 to FP-5. The DNase I
hypersensitive sites are indicated by arrows. B, identical to A except that the DNA fragment (-172 to +110) was
labeled at the 3`-end of the coding strand. C, identical to A except that an overlapping DNA fragment (-295 to
+55) 3`-end labeled on the noncoding strand was used. Footprints
FP-3 to FP-7 are shown.
4-GT gene. The sequence of the 4-GT gene (-850
to +60) is shown; numbers are relative to A (+1) of the first
in-frame ATG. The first two in-frame ATGs are underlined. The
clusters of upward bent arrows designate the transcriptional
start sites of the 3.9-kb (+14 to +24), the 4.1-kb
(-190 to -145) and the male germ cell-specific (Gc, -732) transcripts. The sequences protected from
DNase I digestion on the coding and the noncoding strands are overlined and underlined, respectively, and are
labeled FP-1 to FP-15. Protein binding motifs,
identified by comparison to a transcription factor data base, are boxed. In the case of FP-2, FP-4, FP-7, and FP-8, the
protected region extends further than the designated Sp1 site and may
well contain an additional Sp1 site. The binding of each indicated
nuclear factor was experimentally established by
EMSA.
Characterization of the Nuclear Protein Binding to the
FP-1 Site: Identification of a Putative Negative Regulatory
Factor
Based on our previous data, a negative regulatory element
involved in repressing transcription from the 3.9-kb start site is
predicted to reside between -13 and +55(6) . A
potential candidate for the negative regulatory factor is the
protein(s) that interacts at the FP-1 site (+36 to +60) and
the hypersensitive site at +29. To characterize this factor, oligo
1 (+20 to +59, Table 1), containing both GC-rich
elements, was analyzed by EMSA. An equal amount of total protein (5
µg) was used per reaction in order to compare the relative binding
activity of this factor in each of the three nuclear extracts.
Proteins Binding to the FP-2 and FP-4 Sites Are Members
of the Sp1 Family
The protected region FP-2 contains an inverted
GT box (CCCACCC), which is similar to the inverted GC box (CCCGCCC)
recognized by Sp1. Recently, several novel Sp1-related factors, that
also bind GC and GT boxes, have been
described(17, 18, 19) . Therefore, the
strategy we used to identify the protein interacting with the FP-2 site
included experiments to assess the involvement of Sp1 or a related
family member. Oligo 2 which spans FP-2 (-47 to -10, Table 1), and oligo Sp1 were analyzed by EMSA using nuclear
extracts from L-cells, brain tissue, and LMG. Two protein-DNA complexes
(I and II) (
)were seen when nuclear extract from either
L-cells (Fig. 4A, lane 2) or LMG (lane
4) was incubated with oligo 2. This binding activity was very low
in brain and only a weak band, corresponding to complex I, was observed (lane 3). When purified Sp1 protein was incubated with oligo
2, an intense upper band that comigrated with complex I was observed (Fig. 4A, lane 5); the lower band resulted
from nonspecific binding (data not shown). The binding of Sp1 to this
GT box suggests that it, or a related protein, is responsible for the
observed protein-DNA complexes.Complex Interactions at the FP-3 Site: Binding of Mammary
Gland-specific and Ubiquitous Transcription Factors
Even though
DNase I footprinting analysis showed that FP-3 was most prominent with
the LMG extract and therefore likely resulted from binding of
LMG-specific factors, the sequence within this protected region
contains recognition sites for tissue-restricted as well as ubiquitous
transcription factors (Fig. 2). To determine the nuclear factors
that interact with this complex region, oligo 3 (-82 to
-37, Table 1) was analyzed by EMSA. Three distinct
protein-DNA complexes (I-III) were observed upon incubation of
labeled oligo 3 with the L-cell nuclear extract (Fig. 5A, lane 2), whereas only a single band,
comigrating with complex III, was seen with the brain extract (lane
3). As pointed out earlier, it was not totally unexpected to
detect protein binding with the L-cell and brain extracts using EMSAs,
in the absence of clear footprints with the same extracts using the
DNase I protection assay, as the former is a more sensitive technique.
With the LMG extract, a major unique band of higher mobility (complex
IV) was observed in addition to a band corresponding to complex III and
two very weak bands corresponding to complexes I and II (lane
4).
)GCCA) than the half-site (TGGC) present
in oligo 3(24, 25) . Alternatively, competition may
occur between multiple factors binding to overlapping sites in this
region. As noted earlier, MG-C/N also has a greater affinity for the
full site than the half-site (Fig. 5B), but it appears
to bind to the half-site (in the context of the FP-3 site) better than
the ubiquitous form of CTF/NF1.
The Tissue-restricted Transcription Factor AP2 Binds to
Both the FP-5 and FP-6 Sites
Footprints FP-5 and FP-6 were seen
only with the nuclear extract from the LMG and therefore it was
expected that the factor binding to these sites would be restricted in
its tissue distribution. Consistent with this, a recognition motif for
the tissue-restricted transactivator, AP2, was identified in each of
the footprinted regions. To determine if AP2 binds to the FP-5 site,
oligo 5 (-162 to -127, Table 1) was analyzed by EMSA.
As seen in Fig. 6A, only the extract from the LMG
exhibited a prominent protein-DNA complex (lane 4). Complex
formation was abolished by the addition of a 50- or a 250-fold molar
excess of unlabeled oligo 5 (Fig. 6B, lanes 2 and 3). Since only partial inhibition was observed using
the same molar excess of oligo AP2 (lanes 4 and 5),
the binding protein appears to have a greater affinity for the AP2
motif within the FP-5 site than the consensus AP2 binding sequence.
However, this nuclear protein was unequivocally shown to be AP2 (or
related to AP2) as incubation with anti-AP2 antibodies caused a
supershift of the specific complex (Fig. 6C, lane
3).The Ubiquitous Transcription Factor Sp1 Binds to Multiple
GC Boxes Upstream of the 4.1-kb Start Site
We have previously
shown that the 4.1-kb 4-GT transcript is ubiquitously expressed at
similar levels in all somatic tissues, except brain tissue, where the
levels are 10-fold lower. The sequence upstream of the 4.1-kb
start site (at
-190) shares features in common with other
housekeeping genes, including the lack of a consensus TATA box,
multiple start sites, high GC content, and multiple putative binding
sites for the transcription factor, Sp1(26) . Transfection
studies in L-cells and Drosophila SL2 cells show that a 287-bp
(-474 to -187) DNA fragment immediately upstream of the
4.1-kb start site has promoter activity and that some or all of the Sp1
binding sites within this region are functional(6) .
4-GT gene sequence from -474 to
+55 was labeled at the 3`-end of the noncoding strand, incubated
with BSA (lane 1) or nuclear extract from L-cells (L, lane 2), brain (Br, lane 3), and lactating
mammary gland (LMG, lane 4) and digested with DNase
I. An A + G sequencing reaction performed on the same probe was
run in parallel with the samples on an 8% polyacrylamide-urea gel (lane 5). The regions protected from DNase I digestion are
marked by brackets and labeled FP-7 to FP-12. The
DNase I hypersensitive sites are indicated by the arrows.
10-fold lower steady
state levels of the 4.1-kb mRNA compared to L-cells and LMG, also shows
the lowest level of Sp1 binding activity, whereas L-cells and LMG which
have comparable amounts of the 4.1-kb transcript, have similar levels
of Sp1 binding activity ( Fig. 4and Fig. 6). The relative
Sp1 binding activity most likely reflects the amount of Sp1 protein
present in each tissue, as the study by Saffer et al.(27) shows that Sp1 protein levels are very low in the
brain tissue.
Analysis of the Region between -474 to -805
for Nuclear Factor Binding
Even though the above analyses show
that regulatory elements necessary for expression of the 3.9- and
4.1-kb 4-GT transcripts reside between -474 and +55, we
examined additional upstream sequence for nuclear factor binding, since
DNA sequence analysis identified putative AP2 binding sites upstream of
-474. When the sequence from -828 to -449 was
subjected to DNase I footprinting analysis, three footprints (FP-13,
FP-14, and FP-15) were observed (Fig. 8). FP-13 was seen with
nuclear extracts from all three tissues and contained a full
palindromic CTF/NF1 recognition sequence (TGGCGGAGCGCCA; Fig. 2). As expected, the factor binding to this site was
identified as CTF/NF1 by EMSA (data not shown). It should be noted that
both the ubiquitous and the mammary gland-specific forms of CTF/NF1
were found to bind to the FP-13 site with the LMG extract, as seen
earlier with oligo C/N (Fig. 5D, lane 3).
Footprints FP-14 and FP-15 were specific to the mammary gland and were
shown to bind AP2. Therefore, CTF/NF1 and AP2 binding sites, found in
the proximal promoter region and implicated in high level expression of
the 3.9-kb transcript, are present further upstream and may function in
an enhancer-like capacity.
4-GT gene sequence from -828 to -449 was 5`-end
labeled on the noncoding strand and incubated with BSA (lane
1) or nuclear extract from L-cells (L, lane 2),
brain (Br, lane 3), and lactating mammary gland (LMG, lane 4) and treated with DNase I. An A + G
sequencing reaction performed on the same probe was run in parallel
with the samples on an 8% polyacrylamide-urea gel (lane 5).
The regions protected from DNase I digestion are marked by brackets and designated FP-13 to FP-15.
The organization of the 5`-end of the murine
4-GT: One Gene, Three Transcriptional Start Sites,
and Three Promoters4-GT gene is unusual in that three transcriptional start sites are
embedded within an 800-bp contiguous piece of DNA ( Fig. 2and 9). The most distal start site (relative to the
translation initiation codon) is male germ cell-specific (Gc)
and it is used exclusively in late pachytene spermatocytes and round
spermatids(28) . The ``middle'' start site (4.1 kb)
is predominantly used in all somatic cell types examined (6) as
well as spermatogonia(29) . The proximal start site (3.9 kb) is
preferentially used in the mid- to late pregnant and lactating mammary
gland(6) . The differential use of the three start sites
suggests the presence of both tissue-specific and housekeeping
promoters, each regulating the expression of the respective mRNA
species.
-galactosidase, exclusively to
the late pachytene spermatocytes and round spermatids of transgenic
mice(30) . This fragment contains several motifs including two
CRE (cAMP-responsive element)-like elements, that have been noted in
the promoters of other genes expressed during the later stages of
spermatogenesis (see discussion in Shaper et
al.(30) ). CRE-motifs have been shown to bind a unique
form of the CRE binding protein (CREM) expressed only
in postmeiotic male germ cells (31) .
4-GT expression in somatic cells, our previous promoter deletion
studies in L-cells revealed two potential promoter regions; one
upstream of the 4.1-kb start site that contained binding sites for the
ubiquitous factor Sp1, and the other adjacent to the 3.9-kb start site
that contained motifs for several positive factors (CTF/NF1, mammary
gland activating factor (MAF), Sp1) and a negative factor. Based on
these initial studies we proposed a model of transcriptional regulation
of the 4-GT gene in which expression of the 4.1-kb transcript is
governed by a housekeeping promoter, whereas expression of the 3.9-kb
transcript is regulated by a tissue-specific promoter(6) .
4-GT gene sequence between -800 to +100 are
shown. The upward bent arrows indicate the location of the
3.9- and the 4.1-kb start site; increasing thickness of the
arrow depicts increasing transcriptional activity. The GCBF is shown
tightly bound to the site downstream of the 3.9-kb start site in the
brain, somewhat displaced in L-cells and completely displaced in the
LMG. The low level of Sp1 in brain is indicated by lightly shaded
ovals compared to higher Sp1 levels in L-cells and LMG, indicated
by dark ovals. The CTF/NF1 binding indicated by the asterisk at -500 may not be functionally important
in L-cells and brain. See text for a more detailed
discussion.
Expression of the 4.1-kb mRNA
Our previous
promoter deletion analysis using 4-GT/CAT constructs transfected
into L-cells and Drosophila SL2 cells (6) combined
with the current data showing that the six Sp1 sites immediately
upstream of the 4.1-kb start site bind Sp1, confirm that this
transcription factor is the primary modulator of 4.1-kb mRNA expression
in essentially all somatic cells. Clustering of Sp1 sites in close
proximity to the transcriptional start site is typical of TATA-less
promoters(26) , and it has been suggested that multiple binding
sites are required for synergistic activation of the
promoter(20) . The direct correlation between tissue levels of
Sp1 and 4.1-kb mRNA levels further supports the conclusion that
expression of this mRNA is governed by Sp1.4-GT/CAT construct
containing both this motif and the cluster of Sp1 sites
(-805/-187), has CAT activity similar to the construct
lacking the CTF/NF1 site (-474/-187). The tissue-restricted
distribution of AP2 rules out any role for this protein in 4.1-kb mRNA
expression.Expression of the 3.9-kb mRNA
The data presented
confirm that the regulation of the 3.9-kb transcript is complex and
involves positive (both ubiquitous and tissue-restricted) and negative
trans-acting factors. Cooperation between tissue-specific and
ubiquitous factors is commonly observed for tissue-specific promoters (33, 34, 35, 36, 37) .
Genes expressed in a tissue-specific manner are also known to use
negative control mechanisms to prevent expression in inappropriate
tissues mediated by the binding of ubiquitous factors
alone(36, 38, 39) . Therefore, our findings
are consistent with the conclusion that expression of the 3.9-kb mRNA
is primarily mammary gland-specific.Role of the Negative Regulatory Factor
The initial
identification of a 68-bp region (-13 to +55) that
down-regulates expression of the 3.9-kb mRNA in L-cells was one of the
key findings that led to our hypothesis that this mRNA species is
regulated by a tissue (mammary gland)-specific promoter. We predicted
that a protein binds a motif within this 68-bp region resulting in
reduced transcription from the 3.9-kb start site. GCBF is the candidate
protein for such a role and the data suggest that 3.9-kb steady state
mRNA levels are determined by the balance between this negative factor
and the positive factors, MG-C/N, AP2, and Sp1. For example, brain
tissue which lacks the 3.9-kb mRNA, has high levels of GCBF and low
levels of Sp1. L-cells make low levels of this transcript and contain
moderate amounts of both GCBF and Sp1. The LMG which synthesizes high
levels of the 3.9-kb mRNA, contains moderate amounts of GCBF and Sp1,
but high levels of the mammary gland-enriched factors, MG-C/N and AP2.
These findings suggest that the binding of positive factors to sites
adjacent to the GCBF binding site displaces GCBF, thus allowing
transcription from the 3.9-kb start site.4-GT/CAT constructs
(-172 to -13) previously analyzed(6) . Therefore,
the sequence context of the GC-rich element may determine whether GCBF
acts as a negative or positive regulator. Examples of transcription
factors exhibiting dual function are YY1(40) ,
Egr-1(41) , and WT-1(42) .Role of CTF/NF1
CTF/NF1 constitutes a family of
proteins which bind to the palindromic sequence,
TGG(C/A)(N)GCCAA, or with lower affinity to the half-site,
TGG(C/A)(24, 25, 43, 44) . Although
generally considered to be a ubiquitous factor, CTF/NF1 has been
associated with liver-(35, 37) , brain-(45) ,
and adipocyte- (46) specific gene expression, and
tissue-specific molecular forms have been reported in liver (47) and brain(48) . Relevant to our studies is the
fact that CTF/NF1 has also been implicated in the mammary
gland-specific expression of MMTV and several milk protein genes
including
-lactalbumin(32, 49, 50, 51, 52) .
Moreover, a mammary gland-specific form of CTF/NF1, similar to the one
we observed (MG-C/N), has been described in rat (51) and bovine (53) LMG. However, it is not known if this size variant
represents a unique gene product, a spliced variant, or a partially
degraded form.4-GT/CAT constructs, that contained or lacked this
sequence, exhibited similar CAT activities(6) . However,
binding at this site may be important for 3.9-kb mRNA expression in the
LMG, as it is juxtaposed between two AP2 sites ( Fig. 2and Fig. 9).Role of AP2
AP2 was first identified in Hela cells
as a transcription factor that binds to GC-rich motifs in the enhancer
regions of SV40 and the human metallothionein genes(23) . It
was shown to be tissue-restricted (23, 54) and has
been implicated in the control of gene expression in the neural crest (54) and epidermal cell(55, 56) lineages.
Recently, AP2 was found to be involved in MMTV expression in the
mammary gland(57) . The 5` enhancer of the MMTV long terminal
repeat contains four elements, AP2, CTF/NF1, ``F12,'' and
``mp4.'' While mutation of any one motif decreases enhancer
activity, the most significant reduction results from mutation of the
AP2 site.Transcription Factors Involved in the Expression of Genes
in the Mammary Gland
The primary function of the mammary gland
is to synthesize and secrete a group of milk specific proteins which
include caseins, whey acidic protein, -lactoglobulin, and
-lactalbumin, a variety of lipids and carbohydrates (e.g. lactose) required by the newborn. While the milk proteins are
abundantly expressed exclusively in the mammary gland, different
members of this group are expressed asynchronously, beginning in
mid-pregnancy and continuing throughout lactation.4-GT transcript is predominantly expressed in the mid- to
late pregnant and lactating mammary gland, therefore, it was of
interest to compare the regulatory elements involved in its expression
with those of the milk protein genes. CTF/NF1 has been implicated in
the expression of -lactalbumin (49) and
-lactoglobulin (50) and has been shown to be functionally
important for the expression of whey acidic protein(52) . MMTV,
which is expressed primarily in the late pregnant and lactating mammary
gland, also contains a functional CTF/NF1 site (32) in addition
to a functional AP2 site(57) . Binding sites for the mammary
gland-enriched factor, MGF, are found in all milk protein genes
(reviewed in Groenen and van der Poel(58) ) and have been shown
to be functionally involved in the expression of
-casein(59) , whey acidic protein(52) , and
-lactoglobulin(60) . However, this site is not present in
the 4-GT gene sequence analyzed.Recruitment of
The evolutionary route, resulting in the formation of
lactose synthetase (a heterodimer between 4-GT for Lactose Biosynthesis in
Mammals-lactalbumin and
4-GT) in mammals, is both remarkable and unique. -Lactalbumin
and lysozyme are homologous proteins that have evolved from a common
ancestral gene. It has been estimated that the -lactalbumin gene
line diverged from the lysozyme gene line about 400 million years ago,
prior to the divergence of tetrapods and fishes (61) to emerge
in mammals as a milk protein gene. In contrast, the 4-GT gene has
been recruited from the non-mammalian vertebrate pool of
constituitively expressed genes. This is evidenced by the fact that
4-GT from non-mammalian vertebrate species, such as chicken, ()can functionally interact with -lactalbumin in
vitro, indicating that the -lactalbumin binding domain in
4-GT predates the rise of mammals.4-GT for lactose biosynthesis, the problem arose as to how to
increase the levels of this enzyme in the LMG, while maintaining the
relatively low levels of constituitively expressed enzyme in all
somatic tissues. Based on our analysis of the structure and regulation
of the murine 4-GT gene, we would argue that this was achieved by
the generation of the 3.9-kb start site and its accompanying
tissue-restricted regulatory elements. It is interesting to note in
this regard that both AP2 and GCBF, two of the transcription factors
implicated in the regulation of transcription from the 3.9-kb start
site, bind to GC-rich sequence motifs, which could have been generated
by mutations in the GC-rich regions flanking the 4.1-kb start site. 4-GT gene is a direct consequence of the recruitment of 4-GT
for the mammary gland-specific biosynthesis of the uniquely mammalian
disaccharide, lactose.
)4-GT,
1,4-galactosyltransferase (UDP galactose: N-acetyl--D-glucosaminylglycopeptide
1,4-galactosyltransferase, EC 2.4.1.38); LMG, lactating mammary
gland; kb, kilobase(s); bp, base pair(s); CAT, chloramphenicol
acetyltransferase; EMSA, electrophoretic mobility shift assay; BSA,
bovine serum albumin; GCBF, GC binding factor; MAF, mammary gland
activating factor; MMTV, mouse mammary tumor virus; MGF, mammary gland
factor; CRE, cAMP-responsive element.)))
We thank Michael Collector for assistance in obtaining
mouse organs, Dr. Naoko Tanese for the kind gift of anti-CTF/NF1
antibodies, Dr. Yoshikuni Nagamine for critical reading of this
manuscript, and Ann Larocca for editorial assistance.
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
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