Supplementary Figure 12: DUX4 induces ERGalt expression.

(a) Analysis of previously published myoblast DUX4 ChIP–seq and transcriptome sequencing data²³, depicting the region of ERG flanking the first exon of ERGalt and showing expression of canonical ERG transcripts in cells not expressing DUX4 and expression of ERGalt transcripts (red bracket) exclusively in cells expressing DUX4. (b) ChIP–PCR data confirming binding of DUX4 at ERG exon 6 alt in NALM-6 but not Reh cells. Primers for PCR at the DUX4 binding site amplify chr. 21: 39,764,747–39,764,881 (hg19), and negative-control primers amplify chr. 21: 39,769,872–39,769,942 (primers are listed in Supplementary Table 16). (c) DUX4 ChIP–seq and ATAC–seq data at CD200R1 in NALM-6 and Reh cells, showing peaks of DUX4 binding corresponding to regions of open chromatin (identified by ATAC–seq) in the DUX4/ERG cell line NALM-6 but not the ETV6-RUNX1 cell line Reh. This corresponds to overexpression of this gene in DUX4/ERG ALL. (d) Lentiviral expression of the rearranged DUX4 allele encoding Glu415*, but not empty vector, results in expression of ERGalt in three individual cord blood pools, as demonstrated by RT–PCR. Representative Sanger sequencing electropherograms confirming expression of ERGalt are shown below the RT–PCR gel image.