From the
The expression of human leukosialin (CD43), a major
sialoglycoprotein on the surface of hematopoietic cells, is regulated
in cell lineage-specific as well as differentiation stage-specific
manners. We have shown previously that transcription from the TATA-less
promoter is mediated by the transcription factor Sp1, which binds to
repeats of a GGGTGG motif in the 5`-flanking sequence. This regulatory
region is ubiquitously functional in mammalian cells, providing a high
transcriptional potential. No cis-acting element responsible
for the specificity of this gene expression was revealed by extensive
studies using transient as well as stable expression systems. Here, we
demonstrate that DNA methylation plays a key role in leukosialin gene
expression. Southern blot analysis of genomic DNAs from various human
cell lines with methylation-sensitive and -insensitive restriction
enzymes showed a tight correlation between gene activity and
demethylation state of the 5`-region of the leukosialin gene.
Consistent results were obtained from the same analysis of genomic DNAs
from various human tissues. In addition, in vitro DNA
methylation of the 5`-region drastically reduced transcriptional
activity in a transient expression system. These results indicate that
DNA methylation around the 5`-region of the leukosialin gene is
required to shut off a high level of transcription. Thus, the
tissue-specific expression of the leukosialin gene is constitutively
achieved by alteration of DNA methylation.
Human leukosialin (CD43) is a major sialoglycoprotein expressed
in hematopoietic cells
(1, 2, 3, 4) . In
addition to the fact that this molecule prevents cell aggregation by
placing a charge on the cell membrane, it has been demonstrated that
leukosialin is involved in cell adhesion, interacting with
intercellular adhesion molecule 1
(5) . Leukosialin may also play
a role in signal transduction since addition of a leukosialin-specific
antibody to cells induces T-cell proliferation
(6) and activates
natural killer cells
(7) and monocytes
(8) . Leukosialin
is present in T-lymphocytes, granulocytes, monocytes, platelets, and
hematopoietic stem cells but is absent from
erythrocytes
(3, 7, 9, 10) . In the
erythroid cell lineage, its expression is observed only at an early
stage of differentiation then decreases during cell maturation (11). In
the B-cell lineage, leukosialin is expressed in pre-B cells but is
absent in resting B cells. Once B cells are activated to plasma cells,
leukosialin is re-expressed
(10) .
We have shown previously
that the 5`-regulatory region of leukosialin is a guanine-rich sequence
on the sense strand and possesses high transcriptional
activity
(12) . A general transcriptional factor Sp1 binds to the
GGGTGG motif located about 40 bp
The above results
also suggest that the transcription of leukosialin may be
down-regulated in non-expressing cells. To determine how the expression
of leukosialin is governed in a tissue-specific manner, we examined the
methylation status of cytosine residues in CpG dinucleotides present in
the leukosialin promoter. Our results show that the leukosialin
promoter sequence is not methylated in cells synthesizing leukosialin
while it is methylated in those cells lacking leukosialin. We also
found that leukosialin can be synthesized by 5-azacytidine treatment of
non-producer cells. Regulation of leukosialin gene expression is thus a
typical example of how DNA methylation determines the tissue-specific
expression of a gene.
Leukosialin transcripts were faintly detected
in HT1080 fibrosarcoma cells as shown in Fig. 2B. In
this cell line, the majority of hybridizing bands with HpaII
digestion were shifted to a higher molecular weight, but a certain
fraction of an unmethylated fragment was also observed
(Fig. 2A). We analyzed the transcription and the
methylation state in subclones derived from HT1080 cells (Fig. 3,
A and B). Subclone H4, which has highly methylated
sites, did not produce the transcripts. Subclone QT5, which showed the
demethylation state at these sites, did produce a larger amount of
transcripts compared with those of HT1080 cells (Fig. 3, A and B). The low level of transcription and partial
demethylation state seen in original HT1080 cells thus appeared to be
due to heterogeneous populations in this cell line. When we treated the
non-expressing subclone H4 with 5-azacytidine, an inhibitor of
methyltransferase
(20) , the unmethylated fraction of the
hybridizing band was increased, and transcription of the endogenous
leukosialin gene was also induced (Fig. 3, A and
B). This result also indicates DNA methylation is involved in
leukosialin gene expression in cultured cells.
The leukosialin gene is expressed in tissue-specific and
differentiation stage-specific manners. In this study, we showed that
DNA methylation plays a key role in regulation of leukosialin gene
expression. We obtained a clear correlation between transcription and
the demethylation of the regulatory region in various cultured cell
lines. Furthermore, endogenous leukosialin gene expression was induced
when a subclone of non-expressing HT1080 cells was incubated with
5-azaC methyltransferase inhibitor (Fig. 3). Correlation between
gene expression and demethylation of the leukosialin gene was also
demonstrated by the analysis of genomic DNA derived from various human
tissues. Thymus, spleen, and leukocytes, which are the major expressing
tissues and cell types, showed hypomethylation of the regulatory region
(Fig. 4). These results indicate that the state of DNA
hypomethylation is clearly associated with leukosialin gene expression.
We demonstrated that in vitro methylation of the
leukosialin regulatory sequence in CAT reporter plasmids dramatically
suppressed the transcriptional potential. This repression is not due to
an effect of methylation on the CAT reporter gene or on plasmid DNA
because the pCAT control containing the SV40 enhancer and promoter was
barely affected by this treatment (Fig. 5). It is thus unlikely
that the strength of the promoter is a critical factor in determining
the influence of methylation. Although it was reported that the density
of CpG dinucleotides in a promoter correlated with the extent of
methylation inhibition in a stable expression system
(21) , CpG
dinucleotides are not particularly abundant in the leukosialin
regulatory region of LS5CAT. Therefore, it is conceivable that the
pattern of CpG dinucleotides distributed in a promoter is rather an
important factor for the transcription inhibition by DNA methylation.
The leukosialin gene lacks TATA boxes, but its transcription starts
from a single site. It is noteworthy that the sequence CCAGTCT from
-2 to +5 fits into the consensus sequence of an initiator
(PyPyANT/APyPy), which can provide a basal transcriptional level and
define a single start site by RNA polymerase II, located at the start
site
(22) . Our previous study of transient expression with the
Drosophila Schneider line 2, which does not possess endogenous
Sp1 activity, showed that the leukosialin regulatory region could
provide a weak transcriptional activity without cotransfection of the
Sp1 expression vector
(13) . This activity might represent the
basal transcriptional level conferred by an initiator. In agreement
with a previous observation that Sp1 can activate transcription through
an initiator element
(23) , cotransfection of the Sp1 expression
vector led to a 10-fold increase of leukosialin transcriptional
activity in Drosophila Schneider line 2
(13) . Thus, it
appears that the transcription of the leukosialin gene is principally
directed by these two regulatory elements. Sp1 is reported to be able
to bind methylated DNA and activate
transcription
(24, 25) . In accordance with these
observations, Sp1 could enhance transcriptional activity in
Drosophila Schneider cells, even when the leukosialin-CAT
constructs were methylated (data not shown), since methyl binding
proteins are presumably absent in the cells
(26) . These results
suggest that DNA methylation can prevent transcription through
interaction of methyl binding proteins with the regulatory region.
The molecular mechanism involved in modulating gene activity by
methylation has been elucidated in several instances. Sequence-specific
binding of some transcription factors is inhibited by cytosine
methylation of the recognition
sites
(27, 28, 29, 30, 31) . On
the other hand, transcription is thought to be blocked by binding of
methyl CpG binding proteins such as MeCP-1 and
MeCP-2
(32, 33) . It has also been demonstrated that DNA
methylation alters chromatin structure, which leads to inactivation of
a gene
(34) . Recently, it was demonstrated that an Sp1 element
is involved in the demethylation of a CpG island of a housekeeping
gene, adenine phosphoribosyltransferase gene
(35, 36) .
It remains to be addressed whether the Sp1 binding site of the
leukosialin promoter could play a role in demethylation as well as
transcriptional activation.
Among different hematopoietic cells,
leukosialin expression correlates well with B cell development. At the
stage of large B cell progenitors, leukosialin is expressed, but
maturation to small pre-B cells down-regulates its expression, and most
mature B cells do not express leukosialin. Once B cells are activated
to differentiate into B lymphoblasts, leukosialin is
re-expressed
(10, 37) . Moreover, transgenic mice that
continuously express leukosialin in mature B cells produced increased
numbers of B cells, and those B cells exhibited decreased
susceptibility to apoptosis
(38) . These results clearly indicate
that turning on and turning off of the leukosialin gene is critical for
proper maturation of B cells. It is thus significant to determine
whether DNA methylation or demethylation accounts for the inactivation
or activation of the leukosialin gene in B cell differentiation.
Leukosialin gene regulation thus provides an excellent system to
understand how DNA methylation can influence gene transcription.
We thank Drs. Michiko Fukuda and Craig Hauser for
helpful discussions, Anthony Sher for excellent technical assistance,
Andrew Magnet for editing the manuscript and helpful suggestions, and
Bobbi Laubhan for secretarial assistance.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
(
)
upstream from
the transcription start site, and this factor strongly activates
transcription
(13) . This regulatory element, however, does not
conform the tissue-specific expression of leukosialin. Moreover,
extensive analysis with the CAT reporter gene did not reveal a
cis-acting element responsible for cell type-specific
expression within the 11-kb genomic region encompassing the leukosialin
coding region and 5`- and 3`-flanking sequences
(13) . The
results indicate that these regulatory sequences do not exist within
the 11-kb segment of the gene analyzed to date.
Cell Lines, Tissues, and Preparation of Nucleic
Acids
Human cell lines were obtained from American Type
Culture Collection. Hematopoietic cell lines, HL60 (promyelocytic),
K562 (erythroid), Jurkat (T-lymphocytic), HuNS-1 (B-lymphoblastic),
Raji (B-lymphocytic), and Daudi (B-lymphocytic) were maintained in RPMI
1640 medium supplemented with 10 or 20% fetal calf serum, 2 mM
glutamine, penicillin (500 units/ml), and streptomycin (100 µg/ml).
Non-hematopoietic cell lines, PA-1 (teratocarcinoma), Hela
(epithelial), Hep3B (hepatocytic), HT1080 (fibrosarcoma), and WI38
(fibroblastic) were maintained in modified Eagle's medium with
10% fetal calf serum and the same other supplements. Human tissues were
obtained from the Tissue Bank at the University of California, San
Diego, and leukocytes were isolated from peripheral blood of normal
individuals. High molecular weight DNAs were prepared from these cells
and tissues as described
(14) . Total RNAs were prepared from
cultured cells by the guanidinium thiocyanate method, and
poly(A) RNAs were isolated by oligo(dT)-cellulose
column chromatography
(14) .
Southern and Northern Blot
Hybridization
High molecular weight genomic DNAs (10
µg) were digested with MspI or HpaII restriction
endonucleases, resolved on 1% agarose gel, and transferred onto a nylon
filter. A 560-bp MspI fragment, whose sequence corresponds to
the first exon and the 5`-flanking region (see Fig. 1) was
prepared from the leukosialin genomic clone LeuS-2
(12) and used
as a probe after labeling with [-
P]dCTP.
Figure 1:
Map of leukosialin
gene. Nucleotide sequence reported in the previous paper (12) is
numbered. Distributions of CpG and GpC dinucleotides and
MspI/HpaII (CCGG) sites are shown. E1 and
E2 denote exon 1 and exon 2. MspI DNA fragment (560
bp) used for a hybridization probe is
indicated.
Poly(A) RNAs (5 µg) derived from various human
cell lines were separated by 1% agarose, formaldehyde gel
electrophoresis and transferred onto a nylon filter. Multiple tissue
blots containing several human tissue poly(A)
RNAs
were obtained from Clontech. The 0.9-kb DNA fragment prepared by
EcoRI and ApaI digestion of the leukosialin cDNA
PEER3
(15) was used as a probe. Hybridization was carried out in
the reaction conditions previously described
(16) . A human
-actin DNA probe was used as a control.
Treatment with 5-Azacytidine
The HT1080
derivative subclone, H4, was provided by Dr. S. M. Fritsch (at our
institute), and QT5, another subclone of HT1080, was established in our
laboratory. Treatment of subclone H4 with 5-azacytidine (5-azaC) was as
follows. H4 cells were cultured in modified Eagle's medium
containing 10 µM 5-azaC (Sigma), and the medium was
changed every 2 days. After 6 days of culture, the cells were
harvested, and genomic DNA and poly(A) RNAs were
prepared as described above.
In Vitro DNA Methylation
The construction
of leukosialin promoter-CAT reporter plasmids, PSCAT
(-1793/+90) and LS5CAT (-91/+90) has been
previously described
(12) . pCAT control containing the SV40
promoter and enhancer was obtained from Promega. These CAT plasmids (15
µg) were treated with 10 units of SssI methylase (for CG
methylation) or HpaII methylase (for CCGG methylation) (both
from New England Biolabs) at 37 °C for 5 h. Complete methylation at
CCGG sites in treated plasmids was confirmed by HpaII
restriction enzyme digestion.
DNA Transfection and Transient Expression
Assay
Hela cells (1.0 10
cells/100-mm
dish) were cotransfected with 10 µg of CAT constructs and 1 µg
of the
-galactosidase expression vector Lk4lac (provided by Dr. R.
Oshima at our institute) by the Lipofectin method
(17) . After 48
h, cellular extracts were subjected to the CAT assay as described by
Gorman et al.(18) . The transfection efficiency was
standardized by an assay of the
-galactosidase activity.
Leukosialin Gene Activity Is Correlated with the
Demethylation State of the Regulatory Region in Human Cell
Lines
Methylation status of the regulatory region and its
flanking region of the leukosialin gene was analyzed by Southern blot
hybridization using methylation-sensitive HpaII and
insensitive MspI restriction enzymes. Various human cell lines
derived from hematopoietic and non-hematopoietic origins were examined.
As a hybridization probe, we used the 0.56-kb MspI DNA, which
occupies the sequence between two CCGG sites positioned at -493
and +68 relative to the transcription start site
(12) . This
region covers the entire first exon of 70 bp and the 5`-flanking
sequence, including the regulatory sequence (Fig. 1). Comparison
of hybridizing signals between MspI- and
HpaII-digested lanes indicated that genomic DNAs were
practically unmethylated at these CCGG sites in HuNS-1
(B-lymphoblastic), HL60 (promyelocytic), Jurkat (T-lymphocytic), and
K562 (erythroid) cell lines (Fig. 2A). This result
reflects hypomethylation of the regulatory region in these
hematopoietic cell lines.
Figure 2:
Correlation between transcription and the
demethylation state of the 5`-region of leukosialin gene in various
human cell lines. A, methylation state of leukosialin gene in
human cell lines. Genomic DNAs (10 µg) derived from indicated cell
lines were digested with the methylation-sensitive restriction enzyme,
HpaII (H) or the methylation-insensitive restriction
enzyme, MspI (M) and separated by 1% agarose gel
electrophoresis. The blotted filter was hybridized with the 0.56-kb
MspI fragment of the 5`-region of the leukosialin gene, shown
in Fig. 1. B, leukosialin transcripts in human cell lines.
Poly(A) RNAs (5 µg) derived from the indicated
cell lines were separated on 1% agarose, formaldehyde gel. The
transferred filter was hybridized with the EcoRI-ApaI
DNA fragment (0.9 kb) of the leukosialin cDNA PEER3 (upperpanel). The same blot was rehybridized with a
-actin
probe (lowerpanel).
To interpret the relationship between
methylation state and the gene activity, Northern blot analysis of
mRNAs from the same cell lines was performed in parallel. The analysis
revealed two distinct bands, 2.1 and 8 kb in size, in these cells as
shown in Fig. 2B. These two transcripts are probably the
result of an alternative transcription termination
(19) . The
other hematopoietic cell lines derived from Burkitt lymphoma, Raji, and
Daudi showed the methylated patterns with these restriction enzymes
(Fig. 2A). In Raji, Daudi, and Hela cells, MspI
digestion produced two hybridizing bands. The upper band, indicated by
an arrowhead in Fig. 2A, appeared to be
generated by the polymorphic difference of the CCGG sequence at
+68 from the size of this fragment. By contrast, HpaII
digestion of genomic DNA produced distinct bands, which were shifted to
a higher molecular weight, and no signal was revealed at the sizes
obtained with MspI digestion. This methylated state is
associated with the non-transcription of the leukosialin gene in these
cells (Fig. 2, A and B). Similarly, genomic
DNAs derived from other non-hematopoietic cells were largely resistant
to HpaII digestion at these sites (Fig. 2A). A
high degree of methylation of CCGG sites in these cell lines was also
observed with double digestions using these restriction enzymes
together with HindIII restriction enzyme, which produces
6.8-kb genomic fragment (data not shown). In most of these cell lines
except for HT1080, no transcript was detected (Fig. 2B).
These results demonstrate that there is a clear positive correlation
between active transcription and demethylation of the regulatory region
in these cell lines.
Figure 3:
Relationship between methylation state and
transcription in HT1080 subclones and induction of endogenous
leukosialin gene with 5-azaC. A, Southern blot analysis as
described in Fig. 2A was performed with Jurkat, HT1080, and
its derived cells. HT1080 H4 and QT5 are subclones of HT1080, showing
different methylation states. Genomic DNA of H4 cells treated with
5-azaC is indicated as H4-5AZ. B, leukosialin
transcripts were detected by Northern blot hybridization as described
in Fig. 2B (upperpanel). -Actin
transcript is shown as a control (lowerpanel).
Relationship between Leukosialin Gene Activity and
Methylation State in Human Tissues
To better understand the
relationship between DNA methylation and leukosialin gene
transcription, similar analyses were applied to various human tissues.
First, a high level of transcription was detected in thymus,
leukocytes, and spleen by Northern blot analysis (data not shown).
These tissues are thought to contain a large amount of hematopoietic
cells expressing leukosialin. We then tested the methylation status of
four leukocyte DNAs obtained from different healthy individuals and
found that mostly the CCGG sites were unmethylated (Fig. 4).
T-cells are highly abundant in thymus, and the demethylated state of
those sites was also demonstrated (Fig. 4). In addition, spleen,
which is dominantly occupied by hematopoietic cells, showed the
demethylated state as well. In contrast, other tissues showed a much
lower level of demethylation at these sites (Fig. 4). It is also
likely that residual demethylated DNA bands were detected in other
tissues as a result of blood cells in those tissues. These results
establish that the methylation state is associated with leukosialin
gene activity in human tissues.
Figure 4:
Methylation state of leukosialin gene in
various human tissues. Genomic DNAs (10 µg) isolated from the
indicated tissues were digested with the methylation-sensitive
restriction enzyme, HpaII (H), or the
methylation-insensitive restriction enzyme, MspI (M).
Four leukocyte DNAs were isolated from different individuals. The
blotted filter was hybridized as described in Fig.
2A.
Effect of in Vitro Methylation on the Transcriptional
Potential
To investigate whether methylation of the
regulatory region of the leukosialin gene can affect transcriptional
potential, we carried out a transient expression assay with CAT
constructs after in vitro methylation. In this study, we used
PSCAT(-1793/+90) and LS5CAT (-91/+90), the latter
of which showed maximum promoter activity
(12) . When these
constructs were methylated with HpaII methylase and
transfected into leukosialin non-expressing Hela cells, a 2-fold
reduction of transcriptional activity was detected in comparison with
that of untreated PSCAT, and no reduction was observed in LS5CAT
(Fig. 5). In contrast, a drastic reduction of transcriptional
activity by both constructs was observed when CpG sites of these
constructs were methylated with SssI methylase (Fig. 5).
This treatment had, however, much less influence on the transcriptional
activity of the control vector, where the CAT gene is under control of
the SV40 enhancer and promoter (Fig. 5). Therefore, the
5`-regulatory region of the leukosialin gene is susceptible to
inhibition by methylation. Taken together, these results demonstrate
that the high transcriptional level of the leukosialin gene is
down-regulated by DNA methylation.
Figure 5:
Effect of in vitro methylation of
the 5`-region on transcriptional activity. The CAT constructs were
in vitro methylated with HpaII methylase
(HpaII) or SssI methylase (CpG) and transfected into
Hela cells. Relative CAT activities compared with that of each
unmethylated construct are presented. The value is an average of three
independent experiments. AcCM, acetylchloramphenicol;
CM, chloramphenicol.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.