Page 263 - Haematologica March 2020

The hydroxymethylome of multiple myeloma
enriched region showed higher expression than genes which had no such region in their vicinity (Figure 2A). We next considered the presence of these 5hmCpG in regions harboring different chromatin states (ChromHMM) and defined for the lymphoblastoid cell line GM12878 (a cell line used as a model of normal B cells) by the ENCODE consortium. In accordance with a proposed global opening of chromatin in MM cells and as already described for open chromatin regions in MM,16 and for MM hypomethylated CpG,17 MM 5hmCpG mainly reside in regions of hete- rochromatin in GM12878 cells (Online Supplementary Figure S2A). During differentiation of NBC into PB, genomic sites undergoing 5mC oxidation often bear histone marks of either commissioned (H3K4me1) or active (both, H3K27ac and H3K4me1) enhancers.14,15 To investigate the relation- ship between 5mC oxidation and enhancers in MM, we next analyzed enrichment in H3K4me1 and H3K27ac from NBC, PC and MM cells around 5hmCpG detected in MM patients. Most MM 5hmCpG were found in PC and NBC H3K4me1-premarked genomic sites that become active in MM (Figure 2B). We next investigated the presence of MM 5hmCpG in active enhancers and super-enhancers (SE) identified in the MM1.S HMCL.34 Most notably, 89.3% (275 of 308) of the active MM1.S SEs and 36% (2949 of 8285) of all active enhancers were enriched in 5hmCpG in MM (Online Supplementary Figure S2B), strongly suggesting a role for 5mC oxidation in the control of SE activity. Similar results were obtained when analyzing the overlap
between 5hmCpG and U-266 enhancers and SE defined on the H3K27ac ChIP-seq signal from the Blueprint consor- tium (not shown). However, as exemplified for the KLF13 SE (Figure 2C), the vast majority (98.5%, 271 of 275) of these MM1.S active SE were already marked with 5hmC in PB, indicating that these SE probably maintain a cell-of-origin identity in MM cells. Nonetheless, four SE showed a MM- specific hydroxymethylation profile, including 2 DEPTOR SE, MYC SE and GAS2 SE (Figure 2C). DEPTOR and MYC are known to promote MM cell proliferation and sur- vival4,35 and, in support of our data, their SE were found to be specifically active in MM cells.16 GAS2 has not been associated with MM yet but it has been shown to be upregulated and to favor survival in chronic myeloid leukemia cells in correlation with hypomethylation of its promoter region.36,37 When examined during NBC to PC differentiation (Online Supplementary Figure S2C) as well as in patients of the Arkansas MM cohort (Online Supplementary Figure S2D), GAS2 mRNA levels were detected in bone marrow plasma cells (BMPC) but found to be higher in MM cells. Furthermore, GAS2 overexpres- sion impacted patient survival (Figure 2C). Since GAS2 inhibits calpain proteases which degrade TET enzymes,38 it could thus play a central role in sustaining 5mC oxidation levels in MM cells. Overall, this analysis indicates that 5mC oxidation in MM occurs mainly in intronic enhancers and participates in the establishment of a myeloma-specif- ic gene expression program.
AB
C
Figure 2. Genomic and epigenomic characterization of sites of 5-methylcytosine (5mC) oxidation in multiple myeloma plasma cells (MMPC). (A) CEAS31 analysis of the distribution of high confidence multiple myeloma (MM) 5hmCpG (n=86,591) and box plot comparing expression levels of genes associated or not with 5-hydrox- ymethylcytosine (5hmC) peaks in MM patients. (B) Heatmap representation of H3K4me1 and H3K27ac ChIP-seq signals at MM 5hmCpG. NBC and PC H3K4me1 data were from the Blueprint Consortium (http://dcc.blueprint-epigenome.eu/#/files), and MM H3K27ac data were downloaded from the European Nucleotide Archive (PRJEB25605). (C) Integrated genome browser (IGB) visualization of H3K4me1 from normal plasma cells (PC H3K4me1), 5hmC from in vitro naive B cells (NBC) and plasma cells (PC) differentiated plasmablasts (PB 5hmC), MM 5hmCpG and SCL-exo signal from E10025 at selected super enhancers active in MM1.S cells. The survival of patients from the Arkansas cohort classified as high (n=71) or low (n=343) GAS2 expression is shown on the right.
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