DNA methylation repels binding of hypoxia-inducible transcription factors to maintain tumor immunotolerance

Abstract

Background: 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. Results: We 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. Conclusions: Our data elucidate the mechanism underlying cell-type-specific responses to hypoxia and suggest DNA methylation and hypoxia to underlie tumor immunotolerance.

Hypoxia and Inflammation: Insights From High-Altitude Physiology

“…DNA methylation patterns are also altered by hypoxia, in part due to the impact on ten-eleven translocation (TET) enzyme expression and activity ( Thienpont et al, 2016 ). The HIF consensus binding site also contains a CpG dinucleotide, indicating that expression of HIF-pathway genes may be dependent on DNA methylation ( Wenger et al, 2005 ; Chen et al, 2020 ; D’Anna et al, 2020 ). Given that immune cell phenotypes, including inflammatory macrophage phenotypes, can be regulated by epigenetic modifications, hypoxia has the potential to significantly alter immune cell function via its impact on epigenetics ( Davis and Gallagher, 2019 ).…”

Naturalistic Stress Hormone Levels Drive Cumulative Epigenomic Changes along the Cellular Lifespan

“…The observation that prolonged stress hormone exposure affects an exponentially increasing number of CpG sites warrants further examination. Because DNA methylation patterns, once established, can influence local chromatin accessibility [14,17,19,31], it can be hypothesized that the initial methylation changes induced by cortisol at susceptible CpG sites could facilitate subsequent additional changes at proximally located CpGs. To address this "epigenetic seeding and spreading hypothesis", analyses examined the likelihood of additional cortisol-induced methylation changes to emerge at late passage ("spreading") at CpG sites located within varying windows (1, 10, or 100 kb) from the closest CpG affected already at middle passage ("seeding").…”

Intratumor heterogeneity of breast cancer detected by epialleles shows association with hypoxic microenvironment

“…We obtained four sets of breast cancer cell line expression data from GEO (GSE71401 34 , GSE85353 35 , GSE149132 36 and GSE111653 37 ) under hypoxic conditions and normal oxygen conditions. Differential expression analysis was performed with “edgeR” package on four datasets, respectively.…”