The collagen prolyl hydroxylases are novel transcriptionally silenced genes in lymphoma

Abstract

Background: Prolyl hydroxylation is a post-translational modification that affects the structure, stability and function of proteins including collagen by catalysing hydroxylation of proline to hydroxyproline through action of collagen prolyl hydroxylases3 (C-P3H) and 4 (C-P4H). Three C-P3Hs (nomenclature was amended according to approval by the HGNC symbols and names at http://www.genenames.org/ and Entrez database at http://www.ncbi.nlm.nih.gov/gene) leucineproline-enriched proteoglycan (leprecan) 1 (Lepre1), leprecan-like 1 (Leprel1), leprecan-like 2 (Leprel2) and two paralogs Cartilage-Related Protein (CRTAP) and leprecan-like 4 (Leprel4) are found in humans. The C-P4Hs are tetrameric proteins comprising a variable α subunit, encoded by the P4HA1, P4HA2 and P4HA3 genes and a constant β subunit encoded by P4HB.

Methods: We used RT-PCR, qPCR, pyrosequencing, methylation-specific PCR, western blotting and immunohistochemistry to investigate expression and regulation of the C-P3H and C-P4H genes in B lymphomas and normal bone marrow.

Results: C-P3H and C-P4H are downregulated in lymphoma. Down-regulation is associated with methylation in the CpG islands and is detected in almost all common types of B-cell lymphoma, but the CpG islands are unmethylated or methylated at lower levels in DNA isolated from normal bone marrow and lymphoblastoid cell lines. Methylation of multiple C-P3H and C-P4H genes is present in some lymphomas, particularly Burkitt's lymphoma.

Conclusions: Methylation of C-P3H and C-P4H is common in B lymphomas and may have utility in differentiating disease subtypes.

Figure 1

Leprel4 and CRTAP are down-regulated by methylation in NHL. (A) RT–PCR and MSP analysis of expression and CpG island methylation of Leprel4 and CRTAP in NHL cell lines. Expression of Leprel4, CRTAP and GAPDH was determined as described in Materials and Methods. The MSP figure shows U and M specific reactions for the indicated NHL cell lines. Also shown is control methylated (C_M) and control unmethylated (C_U) genomic DNA modified in parallel with cell line DNA samples. (B) Pyrosequencing analysis of Leprel4 and CRTAP CpG islands in NHL cell lines. The % methylation at each analysed CpG dinucleotide is represented by the intensity of shading in the circles as shown in the figure, together with the mean % methylation for each cell line. (C) Demethylation reactivates expression of Leprel4 and CRTAP. MAK-I and AK31 cells (Leprel4 and CRTAP CpG islands methylated) were grown without AZA (−) or in the presence of AZA for 24 or 48 h as indicated and expression of Leprel4 and CRTAP determined by qPCR.

Figure 2

Leprel1 and Leprel2 are frequently co-methylated in BL cell lines. (A) RT–PCR analysis of Leprel1 (upper panel), Leprel2 (middle panel) and GAPDH (lower panel) in BL cell lines and LCL. (B) Western blot analysis of Leprel1 and Leprel2 in BL cell lines. Ramos+A indicates Ramos cells treated with AZA. (C) Demethylation reactivates expression of Leprel1 and Leprel2 in BL cell lines. DG75 and Ramos cell lines were grown without AZA (−) or in the presence of AZA (+) for 48 h and expression of Leprel1 and Leprel2 determined by qPCR. (D) Quantitative analysis of methylation in Leprel1 and Leprel2 CpG islands in NHL cell lines. Cell lines were subjected to pyrosequencing analysis as described in Materials and Methods. The number denotes each individual CG dinucleotide in the amplified fragment analysed by pyrosequencing. Five levels of methylation are represented, the level of methylation increasing with increasing intensity of shading in the circles as indicated. (E) MSP analysis of Leprel1 and Leprel2 CpG island methylation in NHL cell lines. MSP was performed as described in Materials and Methods. The figure shows U and M specific MSP reactions for the indicated NHL (upper panels) and BL (lower panels) cell lines. Also shown is control unmethylated (CU) and control methylated (CM) genomic DNA modified in parallel with cell line DNA.

Figure 3

Analysis of P4H genes in BL cell lines. (A) Pyrosequencing analysis of CpG island methylation in C-P4H genes in BL and LCL (B301, LCL-K, LCL3 and CR B95.8). Pyrosequencing was performed as described in Materials and Methods. The degree of methylation at each analysed CG dinucleotide is represented by the intensity of shading in the circles as shown in the figure. (B) RT–PCR analysis of P4H gene expression in BL cell lines. Expression of P4H genes and GAPDH was determined as described in Materials and Methods. (C) qPCR analysis of P4HA3 expression. Expression is shown relative to B301.

Figure 4

Analysis of methylation in C-P4H gene CpG islands in NHL cell lines. The figure shows pyrosequencing analysis of the CpG islands of each P4H gene in NHL cells lines and LCL. The degree of methylation at each analysed CG dinucleotide is represented by the intensity of shading in the circles as indicated in the figure. Also shown in the mean % methylation of the analysed fragment and the result of expression analysis (RT–PCR or qPCR).

Figure 5

Analysis of methylation in P3H and P4H genes in primary DLBCL. (A) Representative profiles of CpG island methylation for each C-P3H and C-P4H gene in clinical DLBCL cases. Pyrosequencing was performed as described in Materials and Methods. Five levels of methylation are represented, the level of methylation increasing with increasing intensity of shading in the circles as shown in the figure. The result of simultaneous MSP analysis is indicated. (B) CpG island methylation correlates with down-regulation of expression in clinical cases of DLBCL. Scatter plots are shown of mRNA vs mean % CG methylation for Leprel1 and Leprel2 in DLBCL.

Figure 6

Analysis of the CpG islands in C-P3H and C-P4H genes in clinical lymphoma cases. (A) Examples of MSP analyses for C-P3H and C-P4H genes. For Leprel4, CRTAP, Leprel1 and Leprel2 reactions with unmethylated (U) and methylated (M) primer pairs are shown. For P4HA1, P4HA2 and P4HA3 cases are analysed with M primers only. For all genes, specificity and sensitivity of the MSP is demonstrated by the control DNA amplifications. Abbreviations: C_M=control methylated DNA; C_U=control unmethylated DNA. (B) Representativeimmunohistochemical analysis of Leprel1 and Leprel2 in B lymphomas. Cases with and without methylation in the respective CpG islands are shown.

Figure 7

Methylation in C-P3H and C-P4H CpG islands discriminates NHL from LCL and BL from DLBCL. ROC curves demonstrating utility of analysis of methylation in CpG islands of C-P3H and C-P4H genes to distinguish BL from DLBCL.