DOI: 10.6084/m9.figshare.13181440
title: Additional file 1 of DNA hydroxymethylation is associated with disease severity and persists at enhancers of oncogenic regions in multiple myeloma

description: Additional file 1. Figure S1: Agilent BioAnalyzer profile of a 5hmC-seq Illumina library. Figure S2: MS quantification of 5mC in genomic plasma cell DNA is independent of age and sex. (A) Dot plot of 5mC global quantification by MS in normal plasma cells from healthy donors (N=5), and of myeloma cells of patients at diagnosis (N=40). (B) Dot plot of 5mC global quantification by MS by disease stage (ISS I N=9; ISS II N=17; ISS III N=13; NA=1). 5mC (C) and 5hmC (D) dot plot of MS quantification depending on the sex of the patients. 5mC (E) and 5hmC (F) dot plot of MS quantification depending on the age of the patients. Figure S3: Survival course depending on DNA methylation (5mC) level-based separation of two risk groups of NDMM (n=20 and 20). Figure S4: 5hmC association with expression and criteria of 5hmC peaks to merge in 5hmC peak clusters. (A) Average level of 5hmC in all genes normalized to the same body length. Red line stands for average 5hmC in genes with high expression level (greater than 100 Reads per Kilobase Million (RPKM)). Orange line stands for medium expression level (between 10 and 100 RPKM). Green line represents lowly expressed genes (between 1 and 10 RPKM), and blue line stands for very lowly expressed genes (below 1 RPKM). (B) Stitching of 5hmC into 5hmC-enriched domains. The y-axis represents the number of peaks left after merging. The x-axis represents the distance between peaks to merge. Each 5hmC sample was analyzed (one color per patient). The distance 12.5 kb was chosen to stitch 5hmC peaks into the 5hmC-enriched domains that we describe in this study. (C) Fraction of overlap between 5hmC-enriched domains of this study and CpG from the Illumina 450K chip. The red bar represents overlap with hypermethylated CpGs in B cell-specific enhancers that were described by Agirre and colleagues (see Additional file 3: Methods). Blue bars represent random CpGs from the same chip. Figure S5: 5hmC allows the identification of a putative CCND2 enhancer. (A) Correlation between CCND2 expression, 5hmC at CCND2 gene body and 5hmC at the putative 5hmC enhancer across the 40 MM patients. (B) Hi-C contact map in lymphoblastoid cells (GM12878 cell line) at the CCDN2 locus showing the spatial interaction between CCND2 gene and its putative enhancer. (C) Expression of core transcription factors predicted to orchestrate core regulatory circuitries with 5hmC and RNA expression genomic data. Figure S6: MM 5hmC-enriched domains associate with H3K27ac super-enhancers. Rank ordering of the 100 strongest 5hmC-enriched domains on average in the cohort (A), in the MMSET group (B), in the CCND1 group (C) and in the hyperdiploid group (D). Color highlights domains present in only one of the ROSE plots by group. Figure S7: 5hmC signal levels at WNT5B-associated domain are increased at relapse in MM07. (A) Normalized 5hmC enrichment at WNT5B-associated domain. Point shapes match replicates. Fold change=1.3, p=0.003, FDR&gt;0.1. (B) Gene expression levels in RPKM measured by RNA-seq at diagnosis and relapse for three genes surrounding the WNT5B-associated domain. (C) 5hmC genomic signal around WNT5B-associated domain. Colors match those of (A) and (B). 5hmC domain is depicted under signal tracks (hg38: chr12:1,517,750-1,621,200).