Regulatory evolution of innate immunity through co-option of endogenous retroviruses

Abstract

Endogenous retroviruses (ERVs) are abundant in mammalian genomes and contain sequences modulating transcription. The impact of ERV propagation on the evolution of gene regulation remains poorly understood. We found that ERVs have shaped the evolution of a transcriptional network underlying the interferon (IFN) response, a major branch of innate immunity, and that lineage-specific ERVs have dispersed numerous IFN-inducible enhancers independently in diverse mammalian genomes. CRISPR-Cas9 deletion of a subset of these ERV elements in the human genome impaired expression of adjacent IFN-induced genes and revealed their involvement in the regulation of essential immune functions, including activation of the AIM2 inflammasome. Although these regulatory sequences likely arose in ancient viruses, they now constitute a dynamic reservoir of IFN-inducible enhancers fueling genetic innovation in mammalian immune defenses.

Fig. 1. Dispersion of IFNG-inducible regulatory elements by ERVs

A) Age distribution (left) and enrichment within ChIP-Seq datasets (right) of 27 TE families that were enriched within binding sites for IFNG-stimulated cells (18). Estimated primate/rodent divergence time (82 My) from (34). B) Frequency histogram of absolute distances from each ERV to the nearest ISG, for CD14+ cells. The background expectation is from the genome-wide ERV distribution (18). Statistical significance of the observed enrichment within the first 10 kb of the nearest ISG assessed by binomial test. C) Heatmap of CD14+ ChIP-Seq signals centered across STAT1 peak summits within MER41B elements. Bottom metaprofiles represent average normalized ChIP signal across bound elements. D) Schematic of the MER41B LTR consensus sequence. Triangles indicate Gamma Activated Site (GAS; TTCNNNGAA) motifs predicted to bind STAT1 in response to IFNG (13). Heatmap depicts the presence of GAS motifs across 728 extant STAT1-bound MER41B copies in HeLa cells (18). Bottom metaprofile represents average presence of STAT1 motifs relative to the MER41 consensus sequence, overlain with normalized STAT1 ChIP-Seq density across the same elements.

Fig. 2. A MER41 element is essential for AIM2 inflammasome activation

A) Genome browser view of AIM2. ChIP-Seq tracks are normalized per million reads. The “Uniqueness” track displays genome-wide short-read alignability. B) qPCR of AIM2 levels in wild-type and ΔMER41.AIM2 HeLa cells after 24 hrs IFNG treatment. C) Western blot of AIM2 in wild-type and ΔMER41.AIM2 cells after IFNG treatment. D) Luciferase reporter assays of MER41.AIM2, MER41.AIM2 with mutations in the predicted STAT1 sites, and primate orthologs of MER41.AIM2 (see Fig S7A). E) Western blot of caspase-1 from supernatants of wild-type and ΔMER41.AIM2 cells infected with vaccinia virus (18). * p < 0.05, Student's t-test.

Fig. 3. Multiple MER41 elements have been co-opted to regulate the IFNG response

A) Genome browser views of MER41 elements located near APOL1, IFI6, and SECTM1. ChIP-Seq data is depicted as normalized signal per million reads. B) qPCR of each gene comparing IFNG-inducible levels in wild-type HeLa cells and MER41 deletion mutants. * p < 0.05, Student's t-test.

Fig. 4. IFNG-inducible ERVs are pervasive in mammalian genomes

A) A consensus mammalian species phylogeny overlain with boxplots (median and 25^th/75^th percentiles) depicting the estimated age of MER41-like amplifications (18). Triangles depict conserved GAS motifs. B) Luciferase reporter assays of MER41-like LTR consensus sequences from cow and dog (18). C) Heatmap of ChIP-Seq signals centered across STAT1 peak summits within mouse RLTR30B elements. BMM: bone marrow-derived macrophages. Bottom metaprofiles represent average normalized ChIP signal across bound elements. D) Rodent phylogeny overlain with a boxplot depicting the amplification of RLTR30B, as in (A). ISRE: Interferon Stimulated Response Element motif (TTTCNNTTTC) predicted to bind STAT1 in response to IFNB (13). E) Luciferase reporter assay of RLTR30B consensus sequence, as in (B). Time-calibrated phylogenies in (A) and (D) are from (34). * p < 0.05, Student's t-test.