Abstract
BackgroundHypoxia is pervasive in cancer and other diseases. Cells sense and adapt to hypoxia by activating hypoxia-inducible transcription factors (HIFs), but it is still an outstanding question why cell types differ in their transcriptional response to hypoxia.ResultsWe report that HIFs fail to bind CpG dinucleotides that are methylated in their consensus binding sequence, both in in vitro biochemical binding assays and in vivo studies of differentially methylated isogenic cell lines. Based on in silico structural modeling, we show that 5-methylcytosine indeed causes steric hindrance in the HIF binding pocket. A model wherein cell-type-specific methylation landscapes, as laid down by the differential expression and binding of other transcription factors under normoxia, control cell-type-specific hypoxia responses is observed. We also discover ectopic HIF binding sites in repeat regions which are normally methylated. Genetic and pharmacological DNA demethylation, but also cancer-associated DNA hypomethylation, expose these binding sites, inducing HIF-dependent expression of cryptic transcripts. In line with such cryptic transcripts being more prone to cause double-stranded RNA and viral mimicry, we observe low DNA methylation and high cryptic transcript expression in tumors with high immune checkpoint expression, but not in tumors with low immune checkpoint expression, where they would compromise tumor immunotolerance. In a low-immunogenic tumor model, DNA demethylation upregulates cryptic transcript expression in a HIF-dependent manner, causing immune activation and reducing tumor growth.ConclusionsOur data elucidate the mechanism underlying cell-type-specific responses to hypoxia and suggest DNA methylation and hypoxia to underlie tumor immunotolerance.
Highlights
Hypoxia is pervasive in cancer and other diseases
DNA methylation of hypoxia response element (HRE) sites anti-correlates with hypoxia-inducible transcription factors (HIFs) binding To investigate the role of DNA methylation in HIF binding, we stabilized HIFs in MCF7 breast cancer cells by culturing them under acute hypoxia (0.5% O2 for 16 h; Additional file 1: Fig. S1a and S2, and “Methods”), conditions that are insufficient to drive hypoxia-induced hypermethylation [1]
Model-based analysis for ChIP-seq (MACS) [16] revealed 7153 HIF1β binding peaks (Fig. 1a, Table S1). These were high-quality, bona fide HIF binding regions: they were 4.6-fold enriched for the HRE motif (RCGTG), enriched near genes involved in the hypoxia response, > 90% overlapping with peaks identified in another HIF1β ChIP-seq dataset on MCF7 cells and reproducibly detected in independent repeats (Additional file 1: Fig. S1b-d)
Summary
Hypoxia is pervasive in cancer and other diseases. Cells sense and adapt to hypoxia by activating hypoxia-inducible transcription factors (HIFs), but it is still an outstanding question why cell types differ in their transcriptional response to hypoxia. The constitutively expressed HIFα subunits are directly targeted for proteasomal degradation under normal oxygen tension (normoxia), but stabilized under limiting oxygen conditions (hypoxia), when they translocate to the nucleus to induce expression of hypoxia-responsive genes. Concordance between HIF binding sites in MCF7 breast and 786-O renal cell carcinoma cell lines is only 40–60% [9] This divergence is intriguing because HIFα paralogues are often expressed at similar levels in different cell types, and because the consensus DNA sequence that binds HIF complexes, i.e., the hypoxia response element (HRE) RCGTG, does not differ between HIFα paralogues or cell types. The concept that genes induced by hypoxia differ dramatically between cancer and cell types is wellestablished [3, 10,11,12], the reason for these divergent responses and expression programs is poorly understood
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