SETDB1 preferentially silences evolutionarily young retroelements.

Transposable elements (TEs) are epigenetically silenced through multiple mechanisms, including heterochromatin formation and DNA methylation. Prior studies have implicated SETDB1 and the Human Silencing Hub (HUSH) complex in heterochromatin formation-based TE repression, but precise characterization of repeat subclass and locus-specific effects for SETDB1 and related mechanisms has been limited by short-read transcriptome mapping. Here, we use long-read RNA sequencing mapped to a telomere-to-telomere mouse genome assembly to resolve the specific contributions of SETDB1 to TE silencing at single-locus resolution. Knockdown of SETDB1 or core HUSH factors reveals robust and reproducible derepression of a restricted subset of long terminal repeat (LTR) retroelements distinct from those of global DNA demethylation. This derepression is heterogeneous within TE subclasses and is confined to discrete genomic loci, highlighting regulatory diversity that is obscured by aggregate, family-level analyses. Time-resolved SETDB1 depletion followed by histone 3 lysine 9 (H3K9)me3, H3K9me2, and Pol II ChIP-seq reveals a stratified derepression response: ERVK and ERV1 elements show concordant H3K9 methylation loss, Pol II gain, and RNA expression induction, consistent with direct H3K9me3-mediated silencing, whereas LINE/L1 elements show transcriptional activation without corresponding H3K9me3 loss, implicating parallel repressive mechanisms. These data support a model in which SETDB1-dependent H3K9 methylation maintains silencing of evolutionarily young LTRs, while long interspersed nuclear element (LINE) repression engages broader heterochromatic pathways not fully captured by any single chromatin readout. Collectively, our findings refine current models of heterochromatin-mediated TE control and provide a framework for dissecting how chromatin-based mechanisms cooperate to maintain subclass-selective TE repression.