The collagen prolyl hydroxylases are novel transcriptionally silenced genes in lymphoma.

Non-Hodgkin lymphomas (NHLs) are a heterogeneous group of lymphoid neoplasms that display distinct cytogenetic and molecular genetic features (Piva et al, 2010; Gutierrez-Garcia et al, 2011; Salaverria and Siebert, 2011; Visco et al, 2012). Studies of recurrent chromosome aberrations in NHL have provided significant insight into genetics of lymphomagenesis. Most common translocations are t(14;18) in follicular lymphoma (FL), t(11;14) in mantle cell lymphoma (MCL), t(3;14) in diffuse large B-cell lymphoma (DLBCL) and t(8;14) in Burkitt's lymphoma (BL) (Ong and Le Beau, 1998; Chaganti et al, 2000). In addition to structural changes, transcriptional silencing is another major mechanism of tumour suppressor gene inactivation in human cancer, including haematological malignancies (Lee et al, 2010; Eberle et al, 2011). A number of mechanistically important genes have been identified as targets for methylation-dependent transcriptional silencing in lymphomas, including the cyclin-dependent kinase inhibitors CDKN2A and CDKN2B, the p53 homologue TP73, the DNA damage-activated protein kinase DAPK and other genes with putative tumour suppressor functions (Belaud-Rotureau et al, 2008; Guan et al, 2010; Murray et al, 2010). Using suppression subtraction PCR, we previously identified the Polo-like kinase PLK2 to be transcriptionally silenced in NHL, with a particularly high frequency of methylation in BL (Syed et al, 2006). However, epigenetic changes in some NHL types, for example FL and MCL remain to be elucidated. Prolyl hydroxylation is an important post-translational modification that affects the structure and function of proteins. A major substrate of prolyl hydroxylation is collagen and appropriate post-translational processing of collagen requires both C-P3H and C-P4H, respectively. Three C-P3H enzymes have been identified in humans: leucineproline-enriched proteoglycan (leprecan) 1 (Lepre1), leprecan-like 1 (Leprel1) and leprecan-like 2 (Leprel2) (Wassenhove-McCarthy and McCarthy, 1999); Vranka et al, 2004). Prolyl 3-hydroxylation typically occurs in the Gly–3Hyp–4Hyp sequence of collagen and P3H modified residues are abundant in basement membrane collagens (Gryder et al, 1975). Initially, Leprel1 was shown to localise predominantly in the ER and Golgi, but more recently has been detected in tissues rich in basement membranes and shown to participate in the hydroxylation of type IV collagen (Jarnum et al, 2004; Tiainen et al, 2008). We have previously shown that Leprel1 and Leprel2, but not Lepre1, are subject to methylation-dependent transcriptional silencing in breast cancer. Further, whereas methylation in Leprel2 was also present in multiple tumour types, methylation in Leprel1 appeared specific to breast cancer as the CpGisland was unmethylated in several other carcinomas (Shah et al, 2009). The human genome contains two paralogs of the P3H genes, namely Cartilage-Related Protein (CRTAP) and leprecan-like 4 (Leprel4). Although CRTAP is not enzymatically active, it is required in vivo for collagen prolyl 3-hydroxylation (Morello et al, 2006). The biological importance of prolyl hydroxylation in the post-translational modification of collagen is demonstrated by the observation that germ-line mutations in CRTAP and Lepre1 cause recessive forms of osteogenesis imperfecta and loss or decrease of type I collagen prolyl 3-hydroxylation (Cabral et al, 2007; Baldridge et al, 2008) and mutation in Leprel1 is associated with inherited myopia (Mordechai et al, 2011). Prolyl 4-hydroxylation is essential for the biosynthesis of collagens because 4-hydroxyproline residues are critical for stability of the collagen triple helix. The human C-P4Hs are tetrameric isoenzymes composed of two β subunits (encoded by P4HB) and two (catalytic) α subunits. There are three different α subunits encoded by P4HA1, P4HA2 and P4HA3. The β subunit is identical to the protein duslphideisomerase. In contrast to the C-P3Hs, no disorders of collagen synthesis have thus far been associated with heritable change in genes encoding the C-P4Hs. As well as collagen, both C-P3Hs and C-P4Hs may have additional substrates that contain collagen-like domains. Here, we have investigated expression and regulation of the collagen prolyl hydroxylase genes in lymphoma.