Fig. 1

ID4 binds to chromatin in BLBC cell lines and PDX models and does not regulate transcription. a Immunofluorescence analysis of ID4 protein expression in the HCC70 BLBC cell line, blue; nuclear marker DAPI, magenta; cytoskeletal marker phalloidin, green; ID4. 20 μm scale. b Three technical replicates of ID4 ChIP-seq analysis in HCC70 cell line. IgG ChIP-seq and Input controls are shown for comparison. ID4 binding to (i) the genomic region encoding the long non-coding RNAs NEAT1 and MALAT1 and (ii) the protein coding gene ELF3. c ID4 binding to tRNA_Leu (uc021yth.1) in HCC1954 cell line measured by ChIP-exo. Identification of ID4 binding enrichment identified by MACS peak-calling algorithm. Chromosome location, transcription start site (TSS) and Refseq information tracks displayed. Reads have been aligned to the human reference genome Hg19 and peaks called using MACs peak calling algorithm (v2.0.9) [38]. Images contain ChIP-seq coverage data and the peaks called for each ID4 technical replicate and the consensus peaks called for all three ID4 ChIP-seq technical replicate for selected gene regions. ID4 binding is shown in comparison to IgG and Input data for the same region. Data visualised using IGV [56, 57]. Transcription Start Site (TSS) indicated with black arrow. d Alignment of ID4 ChIP-exo peaks with DNAse hypersensitivity clusters, Transcription Factor ChIP and histone marks H3K27Ac and H3K4Me3 ChIP-seq at NEAT1 and MALAT1, UCSC Genome Browser. e Box and whisker plot of ID4 ChIP-qPCR analysis in HCC70 cells. Multiple primers were designed to tile across the large ID4 binding sites. ID4 binding normalised to input DNA and to a region not bound by ID4 (negative region) and represented as fold-change over IgG control. Ratio paired t test, *p < 0.05. Whiskers indicate min to max. n = 4. f qRT PCR analysis of mRNA transcript expression of ID4-bound genomic regions following depletion of ID4 in HCC70 cells using a lentiviral, doxycycline-inducible short hairpin RNA #2 (SMARTChoice). Data normalised to B2M housekeeping gene and to HCC70 cells treated with vehicle control. One-way ANOVA, multiple comparisons test of control primer B2M with each primer set. ***p < 0.001, ns = non-significant. Error bars represent standard error. n = 4