CmC(A/T)GG DNA methylation in mature B cell lymphoma gene silencing

Abstract

DNA methylation has been linked to gene silencing in cancer. Primary effusion lymphoma (PEL) and myeloma are lymphoid malignancies that arise from terminally differentiated B cells. Interestingly, PEL do not express immunoglobulins or most B lineage-specific genes. The B cell-specific B29 (Igbeta/CD79b) gene is silenced in PEL and some myelomas but is expressed in other normal and malignant B cells. B29 expression was reactivated in PEL by demethylating and histone deacetylase inhibiting treatments. Bisulfite sequencing revealed two types of DNA methylation in silenced B29 promoters: at conventional CpG and at CC(A/T)GG B29 promoter sites. The pattern of methylated CpG ((m)CpG) and C(m)C(A/T)GG B29 promoter methylation observed was similar to that recently reported for epigenetic silencing of an integrated retrovirus. Methylation of C(m)C(A/T)GG sites in the B29 promoter significantly repressed in vivo transcriptional activity. Also, methylation of a central conserved C(m)CTGG B29 promoter site blocked the binding of early B cell factor. This methylated motif formed DNA-protein complexes with nuclear extracts from all cell types examined. Therefore, C(m)C(A/T)GG methylation may represent an important type of epigenetic marker on mammalian DNA that impacts transcription by altering DNA-protein complex formation.

Figure 1

DNA demethylation and HDAC inhibition activates endogenous B29 gene expression detected by reverse transcription–PCR. Products (750 and 438 bp) correspond to the normal and alternately spliced forms of B29 mRNA (48). Lanes: (1) BJAB untreated; (2) BCBL-1 + 5-aza-2′-deoxycytidine (5-aza); (3) BCBL-1 + 5-aza/TsA; (4) BCBL-1 + TsA; (5) BCBL-1 + sodium butyrate; (6) BCBL-1 untreated; and (7) water blank.

Figure 2

Genomic bisulfite sequencing of the endogenous human B29 promoter. Compiled results of bisulfite sequencing from B29-expressing (A) and nonexpressing (B) cell lines. The CpG positions analyzed are labeled A-T in alphabetic order (see supplemental data). The cell lines (Left) list the B29 promoter clones examined. Data for BC-3 cells are from two independent bisulfite conversions on distinct days. The span sequenced for each DNA clone is indicated by the extent of the horizontal line. A vertical slash indicates a ^mCpG at that position; a darkened box indicates a methylated C^mC(A/T)GG at that position. X = C to G polymorphism. The plasmid is the B29 minimal promoter that was bisulfite sequenced after passage through dcm+ DH5α bacteria and demonstrates positions of C^mC(A/T)GG sites.

Figure 3

Effect of C^mC(A/T)GG methylation on B29 promoter activity in B29 expressing BL-41 B cells. Renilla luciferase B29 promoter, SV40 promoter and promoter-less (null) reporter constructs were propagated in dcm(+) DH5α [CH3(+)] or dcm(−) scs110 [CH3(−)] E. coli. Renilla luciferase activities are SV40 firefly luciferase normalized (pGL3 control) with the ±SD of at least three transfections by using three preparations of DNA. The data are presented as percent activity relative to the scs110-propagated B29 Renilla luciferase construct, [B29 CH3(−)]. The activity of SV40 constructs, which contain no CC(A/T)GG sites, is statistically equivalent whether passed through dcm+ or dcm− bacteria (Student's two-sided t test, P > 0.50). *, statistical significance, P < 0.05.

Figure 4

Methylation of C⁻⁹¹ in C^mCTGG dramatically alters transcription factor binding and results in a unique DNA–protein complex. Symmetrically ^mC⁻⁹¹-methylated or unmethylated dsDNA oligonucleotide probes were analyzed by EMSA with 20 μg of nuclear extracts from (A) Ramos, a B29-expressing B cell line, (B) BC-3, a B29 nonexpressing PEL cell line, and (C) HeLa, a B29 nonexpressing carcinoma cell line. Oligonucleotide cold competitor (500-fold) was added as indicated. EMSA banding patterns were similar in all the B cell extracts tested (data not shown). Unlabeled arrows denote specific complexes. NS, nonspecific complex.

Figure 5

Methylation negative genomic DNA does not compete for the ^mC⁻⁹¹ DNA–protein complex. Symmetrically ^mC⁻⁹¹-methylated dsDNA oligonucleotide probe was analyzed by EMSA with 20 μg of Ramos B cell nuclear extracts. One microgram of genomic DNA cold competitor was added, as indicated. Unlabeled arrows denote specific complexes.

Figure 6

Methylation of C⁻⁹¹ in C^mCTGG blocks EBF binding. Symmetrically ^mC⁻⁹¹-methylated or unmethylated dsDNA oligonucleotide probes were analyzed by EMSA with human IVT EBF (lanes 2–6 and 9–13), control IVT luciferase (lanes 7 and 14), or 20 μg Ramos nuclear extracts (lanes 15 and 16). Oligonucleotide cold competitor (250-fold) was added, as indicated.