Mythylation of human HLA-D/DR genes: derangement in chronic lymphocytic leukemia

HLA-DR antigens are expressed as differentiation markers in certain human leukemias. To investigate whether DNA methylation plays a role in expression of DR genes in leukemia, we analyzed methylation patterns of the DR-alpha and D/DR-beta genes in the DR antigen-positive and -negative B-cell lines, in normal adults and in chronic lymphocytic leukemia (CLL) patients using Southern blot hybridization of DNA digested with Msp I and Hpa II. The DR-alpha and D/DR-beta genes of a DR antigen positive B-cell line, T5-1, were heavily methylated, while those of DR antigen-negative variant, 6.1.6, were hypomethylated. Blood cells collected from four normal adults contained different levels of DR-alpha and D/DR-beta mRNAs, but their relative amounts were about the same among the individuals. By contrast, the relative amounts of these mRNAs in CLL cells varied widely, indicating aberrant expression of one or both of these genes in CLL. The DR-alpha gene in four normal adults and six CLL patients produced only a 3 kb hybridizable band after Msp I digestion. Normal adult DR-alpha genes were resistant to Hpa II digestion, suggesting that all Hpa II sites are methylated. In contrast, digestion of CLL DNA with Hpa II yielded various bands of larger sizes which differed among the CLL patients, suggesting that Hpa II sites are differentially methylated in the CLL DNA. In the case of D/DR-beta genes, normal adult DNA gave Msp I bands which were slightly polymorphic among four individuals tested. In contrast, CLL DNA showed a high degree of restriction fragment length polymorphism (RFLP) on Msp I digestion. We speculate that the high RFLPs in the CLL DNA may result from differential methylation in CpG clusters in the D/DR-beta genes, and that this characteristic may be of use for diagnosis of CLL.     

Transcriptional regulation of HLA class II and invariant chain genes

Class II (Ia) antigens are coded for by a family of genes located in the human MHC (HLA). These genes are regulated in a complex manner, being constitutively expressed, inducibly expressed, or not expressed, depending on the cell type examined. 6.1.6 is a variant of a normal B lymphoblastoid line that has lost expression of all class II molecules and has previously been shown to have a defect in the regulation of class II genes. In this report, we have examined those genes by Southern and Northern blotting and have found that 6.1.6 is severely deficient in mRNA for all class II genes examined, although the genes are structurally intact. P30, a partial revertant of 6.1.6, re-expresses mRNA for a subset of class II genes. mRNA for the class II-associated invariant chain is substantially reduced but not absent in 6.1.6.     

Mutations in the<Emphasis Type="Italic"> HLA</Emphasis> class II genes leading to loss of expression of HLA-DR and HLA-DQ in diffuse large B-cell lymphoma

Loss of expression of human leukocyte antigen (HLA) class II molecules on tumor cells affects the onset and modulation of the immune response through lack of activation of CD4+ T lymphocytes. Previously, we showed that the frequent loss of expression of HLA class II in diffuse large B-cell lymphoma (DLBCL) of the testis and the central nervous system (CNS) is mainly due to homozygous deletions in the HLA region on chromosome band 6p21.3. A minority of cases showed hemizygous deletions or mitotic recombination, implying that mutation of the remaining copy of the class II genes might be involved. Here, we studied three DLBCLs with loss of HLA-DQ expression for mutations in the DQB1 and DQA1 genes and three tumors with loss of HLA-DR expression for mutations in the DRB1 and DRA genes. In one case, a point mutation in exon 2 of the DQB1 gene, leading to the formation of a stop codon, was detected at position 47. In a second case, a stop codon was found at position 11 due to a deletion of 19 bp in exon 1 of the DRA gene. No mutations were found in the promoter sequences of the DRA, DQA1 and DQB1 genes. We conclude that both homozygous deletions and hemizygous deletions or mitotic recombination with mutations of the remaining allele may lead to loss of expression of the HLA class II genes, which is comparable to the mechanisms affecting HLA class I expression in solid cancers.     

DO beta: a new beta chain gene in HLA-D with a distinct regulation of expression

The HLA-D region of the human major histocompatibility complex encodes the genes for the alpha and beta chains of the DP, DQ and DR class II antigens. A cDNA clone encoding a new class II beta chain (designated DO) was isolated from a library constructed from mRNA of a mutant B-cell line having a single HLA haplotype. Complete cDNA clones encoding the four isotypic beta chains of the DR1, DQw1, DPw2 and putative DO antigens were sequenced. The DO beta gene was mapped in the D region by hybridization with DNA of HLA-deletion mutants. DO beta mRNA expression is low in B-cell lines but remains in mutant lines which have lost expression of other class II genes. Unlike other class II genes DO beta is not induced by gamma-interferon in fibroblast lines. The DO beta gene is distinct from the DP beta, DQ beta and DR beta genes in its pattern of nucleotide divergence. The independent evolution and expression of DO beta suggest that it may be part of a functionally distinct class II molecule.     

DNA methylation and the regulation of aldolase B gene expression

DNA methylation was studied as a potential factor for the regulation of tissue-specific and developmentally specific expression of the rat aldolase B gene. We examined cytosine methylation in the HpaII and HhaI recognition sequences in the aldolase B gene in aldolase expressing and nonexpressing tissues and cells. Out of the 15 methyl-sensitive restriction sites examined, the sites in the 3'-half and 3'-flanking regions were found to be heavily methylated in all the tissues or cells, regardless of the level of aldolase B gene expression. However, the methylation pattern in the region immediately upstream and in the 5'-half of the gene exhibited tissue-specificity: the site located about 0.13 kb upstream of the cap site (just next to the CCAAT box), and the sites in the first intron (intron 1) were heavily methylated in nonexpressing cells and tissues (ascites hepatoma AH130 and brain), whereas those in an expressing tissue (liver) were considerably less methylated. These results suggest that cytosine methylation at the specific sites in the 5'-flanking and 5'-half regions of the gene is associated with repression of the gene activity. However, the gene is still substantially methylated in the fetal liver on day 16 of gestation, when it is in a committed state for rapid activation in the period immediately afterwards (Numazaki et al. (1984) Eur. J. Biochem. 152, 165-170). This suggests that demethylation of the methylated cytosine residues in the specific gene region is not necessarily required before activation of the gene during development, but it may occur along with or after the activation.     

DNA Methylation Profile of the Mouse Skeletal α-Actin Promoter during Development and Differentiation

Genomic levels of DNA methylation undergo widespread alterations in early embryonic development. However, changes in embryonic methylation have proven difficult to study at the level of single-copy genes due to the small amount of tissue available for assay. This study provides the first detailed analysis of the methylation state of a tissue-specific gene through early development and differentiation. Using bisulfite sequencing, we mapped the methylation profile of the tissue-specific mouse skeletal α-actin promoter at all stages of development, from gametes to postimplantation embryos. We show that the α-actin promoter, which is fully methylated in the sperm and essentially unmethylated in the oocyte, undergoes a general demethylation from morula to blastocyst stages, although the blastula is not completely demethylated. Remethylation of the α-actin promoter occurs after implantation in a stochastic pattern, with some molecules being extensively methylated and others sparsely methylated. Moreover, we demonstrate that tissue-specific expression of the skeletal α-actin gene in the adult mouse does not correlate with the methylation state of the promoter, as we find a similar low level of methylation in both expressing and one of the two nonexpressing tissues tested. However, a subset of CpG sites within the skeletal α-actin promoter are preferentially methylated in liver, a nonexpressing tissue.     

Differential methylation of the ornithine carbamoyl transferase gene on active and inactive mouse X chromosomes.

Ornithine carbamoyl transferase (Oct) is an X-linked gene which exhibits tissue-specific expression. To determine whether methylation of specific CpG sequences plays a role in dosage compensation or tissue-specific expression of the gene, 13 potentially methylatable sites were identified over a 30-kilobase (kb) region spanning from approximately 15 kb upstream to beyond exon II. Fragments of the Mus hortulanus Oct gene were used as probes to establish the degree of methylation at each site. By considering the methylation status in liver (expressing tissue) versus kidney (nonexpressing tissue) from male and female mice, the active and inactive genes could be investigated on active and inactive X-chromosome backgrounds. One MspI site, 12 kb 5' of the Oct-coding region, was cleaved by HpaII in liver DNA from males but not in kidney DNA from males and thus exhibited complete correlation with tissue-specific expression of the gene. Six other sites showed partial methylation, reflecting incomplete correlation with tissue-specific expression.     

Tissue- and cell-specific casein gene expression. II. Relationship to site-specific DNA methylation

The relationship between DNA methylation and the expression of the gamma- and beta-casein genes was investigated in both expressing and nonexpressing tissues and in isolated tumor cell subpopulations displaying differential casein gene expression. MspI/HpaII digestions of DNA isolated from liver, a totally nonexpressing tissue, indicated that specific sites of hypermethylation existed in these genes as compared to the DNA isolated from casein-producing lactating mammary gland. The positions of these sites were mapped in the gamma-casein gene by comparing total genomic DNA Southern blots to the restriction digests of several overlapping phage clones constituting the gamma-casein gene. In contrast, the methylation status of the HhaI sites in the gamma-casein gene was found to be invariant regardless of the expression status of the gene. The inverse correlation between the hypermethylation of certain MspI/HpaII restriction sites in the casein genes and their potential expressibility was further substantiated by studies in 7,12-dimethylbenz(a)anthracene- and N-nitrosomethylurea-induced mammary carcinomas, which have an attenuated casein gene expression, and in cell subpopulations isolated from the 7,12-dimethylbenz(a)-anthracene tumor which were either depleted or enriched in casein-producing cells. Analysis of total tumor DNAs indicated that the casein genes were hypermethylated at the same sites observed in liver. However, a very faint hybridization signal was observed in the HpaII digests, suggesting cell-specific methylation differences. We have confirmed the hypomethylation of at least two of these MspI/HpaII sites within the subpopulation containing the casein-producing cells at a level consistent with the relative enrichment in that fraction. These results demonstrate differential site-specific casein gene methylation not only between tissues but also between cell subpopulations within a single tissue.     

Two distinct genetic loci regulating class II gene expression are defective in human mutant and patient cell lines

Heterokaryons were prepared and analyzed shortly after cell fusion using two mutant class-II-negative human B cell lines (RJ 2.2.5 and 6.1.6) and a cell line (TF) from a patient with a class-II-negative Bare Lymphocyte Syndrome. The resulting transient heterokaryons were analyzed by using an anti-HLA-DR monoclonal antibody to assess the cell surface expression of HLA-DR (the major subtype of class II antigens) by immunofluorescence microscopy and by using uniformly 32P-labeled SP6 RNA probes in Northern blots and RNase protection assays to assess mRNA synthesis. We find that class II gene expression in a B cell line from a Bare Lymphocyte Syndrome patient (TF) is rescued by a B cell line which expresses class II antigens indicating that this disease, at least in part, is caused by a defect(s) in a genetic locus encoding a factor(s) necessary for class II gene expression. Secondly, reciprocal genetic complementation was demonstrated in the heterokaryons 6.1.6 x RJ 2.2.5 and TF x RJ 2.2.5 (but not in TF x 6.1.6) by detection of cell surface DR by immunofluorescence microscopy and by a novel class II mRNA typing technique which allows characterization of distinct class II alleles. Thus, the two mutants generated in vitro have defects at two different genetic loci encoding specific regulatory factors necessary for human class II gene expression. One of these mutant cell lines, but not the other, complements the defect in the patient cell line, TF.