Epigenetic landscape of the H3K27me3 mark in macrophages transformed by Theileria annulata

Significance statement

Tropical theileriosis is a major constraint to livestock production worldwide. Infection with Theileria annulata induces the transformation of bovine leukocytes that subsequently exhibit similarities to human leukemia. Long-term in vitro culture of transformed leukocytes attenuates their tumorigenic properties. The molecular changes underlying this process are currently unclear. We focused on H3K27me3, a key histone tail post-translational modification that regulates chromatin structure and gene expression, and compared its genomic landscapes in virulent, highly disseminating macrophages versus attenuated macrophages with reduced dissemination potential that are used as a live vaccine against tropical theileriosis. Western blot and ChIP-seq revealed higher global levels of H3K27me3 and a striking broadening of its genomic distribution in attenuated macrophages. To our surprise, this reconfiguration is not associated with major changes in gene expression and attenuated cells appear to be less responsive to PRC2 inhibition, suggesting that large-scale broadening of H3K27me3 does not systematically translate into increased PRC2-mediated silencing activity. Despite these changes, H3K27me3 maintains the silencing of key tumor suppressor genes in both virulent and attenuated macrophages. These findings shed light on the interplay between infection, the evolution of oncogenic potential and reconfiguration of the chromatin landscape following attenuation of virulence.

Theileria annulata and T. parva are obligate intracellular parasites that induce bovine leukocyte transformation, leading to uncontrolled proliferation and heightened dissemination of infected leukocytes, thus amplifying the parasite and enhancing its transmission. Early passage T. annulata -transformed macrophages are virulent, but virulence decreases with long-term passage and infected macrophages become attenuated for dissemination. Loss of dissemination is restored when TGF-β2-mediated signaling is re-established in attenuated macrophages, suggesting that genetic and/or epigenetic changes occurring within macrophages contribute to the attenuated phenotype. To elucidate the relationship between attenuation of dissemination and epigenetic dysregulation of T. annulata -transformed macrophages, we focused on one of the important repressive histone marks, tri-methylated lysine 27 of histone H3 (H3K27me3) catalysed by the Polycomb Repressive Complex 2 (PRC2). We found that the global level of H3K27me3 of T. annulata -infected macrophages is increased in attenuated macrophages compared to virulent macrophages. Chromatin immunoprecipitation sequencing (ChIP-seq) revealed that the genomic distribution of H3K27me3 is heavily remodeled from virulent to attenuated macrophages. In attenuated macrophages, many genes transition from a focal peak of H3K27me3 around the transcription start site to larger chromatin domains. But this transition is not paralleled by large-scale transcriptional downregulation. In addition, RNAseq analysis following PRC2 inhibitor treatment reveals that fewer genes are derepressed in attenuated macrophages than in virulent macrophages, suggesting that broader H3K27me3 profiles do not systematically translate into increased gene silencing activity. Our findings shed light on the mechanisms underlying the dysregulation of epigenetic modifications in Theileria- induced leukocyte transformation and contribute to a better understanding of the attenuation of tumor dissemination.