Extended Data Figure 6: Local chromatin context at the alternative transcription initiation (ATI) site.
From: Alternative transcription initiation leads to expression of a novel ALK isoform in cancer
a, UCSC Genome Browser view of the ATI site. The RepeatMasker track shows transposable elements at the ATI region, including a long-terminal repeat (LTR) in intron 19 (LTR16B2) and a long interspersed element (LINE) in intron 18. The ENCODE tracks reveal a DNase I hypersensitivity cluster and H3K4me1 enrichment, but no H3K27ac enrichment. b, The methylation status of the ALK locus was assessed by custom capture of the ALK locus, followed by bisulfite treatment and next-generation sequencing. Bisulfite sequencing results of H3122 (top) and MM-15 (bottom) are displayed in the CG-bisulfite mode of IGV. The red colour denotes ‘C’ (cytosine) corresponding to methylated cytosine, which is preserved during the bisulphite reaction. The blue colour denotes ‘T’ (thymine) corresponding to unmethylated cytosine, which is converted to uracil in the bisulfite reaction, and subsequently amplified to thymine during PCR. c, Methylation level at CpG sites in ALKATI-expressing tumour samples (MM-15 and ATC-28) and non-ALKATI-expressing control cells (H3122, a lung cancer cell line with EML4–ALK expression and SKMEL-28, a melanoma cell line without ALKATI expression). d, Comparison of the methylation status of CpG sites adjacent to the ATI site in ALKATI-expressing tumour samples (MM-15 and ATC-28) and non-ALKATI-expressing control cells (H3122 and SKMEL-28). The regions flanking LTR16B2 have significantly lower CpG methylation levels in ALKATI-expressing samples than controls; red dots indicate a statistically significant difference (P < 0.05; Mann–Whitney test) between ALKATI-expressing and non-expressing samples. Black dots indicate no statistically significant difference. e, ChIP-seq profile of H3K27ac at the ALKATI locus. The 17 blue profiles were retrieved from ENCODE, the 5 red profiles are original data from our lab. Only the 3 melanoma samples (MM-15, SKMEL-28, and SKMEL-524; bottom), but not the 19 non-melanoma cell lines, show H3K27ac enrichment at the ATI site. f, ChIP–qPCR validation for the H3K27ac enrichment at the ATI site in 6 melanoma cell lines. Error bars show mean ± s.e.m.; n = 3 technical replicates. g, Luciferase reporter assay of LTR16B2 in melanoma cell lines (red) and lung cancer cell lines expressing EML4–ALK (green). Error bars show mean ± s.d.; n = 9 (3 biological replicates combined from 3 independent experiments).
