Principal promotor de la música de percusión

in oesophageal CAMs and ATMs

For comparative purposes genome–wide differential DNA methylation analysis was performed between oesophageal CAMs and patient-matched ATMs at both individual CpG loci and genomic regions. Figure 3.8 summarises the results from differential DNA methylation analysis at the individual CpG loci, while results from differential DNA methylation analysis at genomic regions is provided in Supplementary Figure S3.4.

In total, 3611 differentially methylated CpG loci were identified in oesophageal CAMs compared to ATMs, including 2826 hypomethylated and 785 hypermethylated CpG loci. Overall, differentially methylated loci were distributed throughout the genome.

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3.3.3.1

Characterization

of

identified

differentially

methylated loci in oesophageal CAMs compared to ATMs

Differentially methylated CpG loci were compared to the CpG distribution in the Illumina 450k array and subjected to GREAT analysis in order to assign genomic localisation.

In the case of oesophageal CAMs, hypomethylated loci were preferentially located in shelves and seas rather than islands whereas hypermethylated loci were preferentially located in CpG islands and shores (Figure 3.9A). The GREAT analysis

of these data revealed that 45 CpG loci (including 44 hypomethylated and 1 hypermethylated) were not associated with any genes, 1206 CpG loci (including

967 hypomethylated and 239 hypermethylated) were associated with only one gene, 2351 CpG loci (including 1809 hypomethylated and 542 hypermethylated) were associated with 2 genes and 9 CpG loci (including 6 hypomethylated and 3 hypermethylated) were associated with 3 genes (Figure 3.9B). The analysis of the distance to transcription start site (TSS) of the differentially methylated CpG loci associated with genes showed that most of the CpG loci are located 50–500kb upstream of TSS and that hypomethylated CpG loci are overrepresented in each TSS distance category (Figure 3.9C).

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Figure 3.8 Differentially methylated CpG loci identified in oesophageal CAM vs ATM comparison. A. Volcano plot represents differentially methylated CpG sites |Δβ| > 0.1;

dashed lines |Δβ| > 0.2, p-value <0.05. B. Heatmap represents differentially methylated CpG sites |Δβ|>0.2, p-value <0.05. To tal arr ay Dif fere nti ally m eth ylate d Hy po me thy late d Hy pe rm eth ylate d 0 % 5 0 % 1 0 0 % 1 5 0 % Is la n d S h o r e S h e lf S e a P ro p o ti o n o f C p G s 0 1 2 3 0 % 2 0 % 4 0 % 6 0 % 8 0 % G e n o m ic r e g io n s N u m b e r o f a s s o c ia te d g e n e s < -50 0 -50 0 t o - 50 -50 to -5 -5 t o 0 0 t o 5 5 t o 5 0 50 to 50 0 >5 00 0 % 1 0 % 2 0 % 3 0 % R e g io n -g e n e a s s o c ia ti o n s T S S D is ta n c e to T S S ( k b ) H y p o m e th y la te d H y p e r m e th y la te d A . B . C .

Figure 3.9 Genomic region and gene associations of differentially methylated CpG loci in oesophageal CAMs vs ATMs. A. Distribution of differentially methylated CpG

loci in CpG islands, shores, shelves and seas. B. Number of associated genes per identified CpG site given in percentages (total 3611 sites, including 2826 hypomethylated and 785 hypermethylated in CAMs). C. Distance to transcription start site (TSS) of identified CpG loci that were associated with genes; blue – hypomethylated CpG sites in CAMs;

red – hypermethylated sites in CAMs.

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3.3.3.2 Gene ontology enrichment analysis for differentially

methylated loci in oesophageal CAMs compared to ATMs

To investigate the potential biological relevance of identified differentially methylated CpG loci in oesophageal CAMs, gene ontology (GO) enrichment analysis was again performed using gometh() function from the Bioconductor package missMethyl (Maksimovic, Gordon et al. 2012, Phipson and Oshlack 2014), using a total of 424355 CpG sites as background.

The enrichment analysis of: (i) GO biological processes (BP) identified 116 GO terms, (ii) GO molecular functions (MF) identified 19 GO terms and (iii) GO cellular component (CC) identified 15 GO terms with p-value < 0.01. Table 3.2 shows list of all GO terms with FDR p-value < 0.05. Other interesting GO biological process terms

with p-value < 0.01 include: cell communication (GO:0007154, 741 genes,

p = 4.13 x 10-5), regulation of signalling (GO:0023051, 415 genes, p = 2.9 x 10-4), regulation of signal transduction (GO:0009966, 371 genes, p = 6.22 x 10-4), synaptic transmission (GO:0007268, 124 genes, p = 2.84 x 10-3), cell differentiation (GO:0030154, 48 genes, p = 2.88 x 10-3), regulation of cytokine biosynthetic process (GO:0042035, 18 genes, p = 4.20 x 10-3) and negative regulation of secretion by cell (GO:1903531, 26 genes, p = 5.18 x 10-3).

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Table 3.2 Gene ontology (GO) enrichment analysis for differentially methylated CpG

loci identified in oesophageal CAMs compared to ATMs. BP - biological process;

MF - molecular function; CC - cellular component; N - number of genes in the GO term; DE - number of genes that are differentially methylated

GO ID GO Term Ontology N DE p-value FDR

GO:0007156

homophilic cell adhesion via plasma membrane adhesion

molecules

BP 135 53 3.85E-10 7.46E-06

GO:0098609 cell-cell adhesion BP 188 57 1.43E-07 9.21E-04 GO:0098742 cell-cell adhesion via plasma-

membrane adhesion molecules BP 187 57 1.29E-07 9.21E-04 GO:0044707 single-multicellular organism

process BP 6017 822 1.21E-06 5.89E-03 GO:0032501 multicellular organismal

process BP 6238 843 1.89E-06 7.32E-03 GO:0022610 biological adhesion BP 1303 227 6.61E-06 2.13E-02 GO:0007155 cell adhesion BP 1297 224 1.60E-05 3.45E-02 GO:0048731 system development BP 3783 574 2.50E-05 4.85E-02 GO:0005509 calcium ion binding MF 645 123 1.34E-05 3.25E-02 GO:0071944 cell periphery CC 4511 614 1.25E-05 3.25E-02