TNRC18 recognizes H3K9me3 to mediate transposable elements silencing at ERV regions [CUT&RUN]

Trimethylation of histone H3 lysine 9 (H3K9me3) is critical for regulation of gene repression and heterochromatin formation, cell-fate determination, and organismal development. In particular, H3K9me3 provides an essential mechanism for silencing various transposon elements (TEs). However, previous studies showed that the canonical H3K9me3 readers (e.g. HP1 and MPP8) play rather limited roles in silencing endogenous retroviruses (ERVs), one of major TE classes in the mammalian genome. Here, we report that Trinucleotide Repeat Containing 18 (TNRC18), a previously under-studied chromatin regulator, recognizes H3K9me3 and directly binds ERVs, mediating ERV silencing. Our biochemical, biophysical and structural studies identified the C-terminal Bromo Adjacent Homology (BAH) domain of TNRC18 (TNRC18BAH) as a H3K9me3-specific reader, and its N-terminal segment as a platform for direct recruitment of co-repressors such as TRIM28 and HDAC-Sin3-NCoR complexes, thereby enforcing optimal repression of the H3K9me3-demarcated ERVs. Point mutagenesis that disrupts the TNRC18BAH-mediated H3K9me3 engagement caused neonatal lethality in mice and, in multiple mammalian cell models, led to de-repressed expression of ERVs, affecting the landscape of cis-regulatory elements and thus gene-expression programs. Collectively, this study describes a previously-unknown TNRC18BAH-directed H3K9me3-sensing pathway, which operates to epigenetically silence the evolutionarily young ERVs, thereby exerting profound impacts on host genome integrity, transcriptomic regulation, immunity, and development. We used CUT&RUN to assess the genome-wide binding of TNRC18 (with either anti-GFP antibody for GFP-tagged TNRC18 or endogenous antibody for TNRC18), H3K9me3, H3 acetylation, HDAC2, TRIM28 and SETDB1 in HEK293 cells, either parental lines or those with the engineered knock-in (KI) mutation at TNRC18?s H3K9me3-binding BAH domain. IgG CUT&RUN served as the negative control. TNRC18 binding was also assessed in HEK293 cells with the SETDB1 knockout (KO) versus WT cells.