©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Tissue-specific Transcriptional Regulation of Human Leukosialin (CD43) Gene Is Achieved by DNA Methylation (*)

Shinichi Kudo , Minoru Fukuda (§)

From the (1) La Jolla Cancer Research Foundation, Cancer Research Center, La Jolla, California 92037

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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.


INTRODUCTION

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() 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.

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.


EXPERIMENTAL PROCEDURES

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.


RESULTS

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.

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.


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.




DISCUSSION

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.


FOOTNOTES

*
This work was supported by Grant CA33895 from National Cancer Institute, National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: La Jolla Cancer Research Foundation, Cancer Research Center, 10901 North Torrey Pines Rd., La Jolla, CA 92037. Tel.: 619-455-6480; Fax: 619-450-2101.

The abbreviations used are: bp, base pair(s); kb, kilobase pair(s); CAT, chloramphenicol acetyltransferase; 5-azaC, 5-azacytidine.


ACKNOWLEDGEMENTS

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.


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