AbstractThe Human Silencing Hub (HUSH) complex is necessary for epigenetic repression of LINE-1 elements. We show that HUSH-depletion in human cell lines and primary fibroblasts leads to induction of interferon-stimulated genes (ISGs) through JAK/STAT signaling. This effect is mainly attributed to MDA5 and RIG-I sensing of double-stranded RNAs (dsRNAs). This coincides with upregulation of primate-conserved LINE-1s, as well as increased expression of full-length hominid-specific LINE-1s that produce bidirectional RNAs, which may form dsRNA. Notably, LTRs nearby ISGs are derepressed likely rendering these genes more responsive to interferon. LINE-1 shRNAs can abrogate the HUSH-dependent response, while overexpression of an engineered LINE-1 construct activates interferon signaling. Finally, we show that the HUSH component, MPP8 is frequently downregulated in diverse cancers and that its depletion leads to DNA damage. These results suggest that LINE-1s may drive physiological or autoinflammatory responses through dsRNA sensing and gene-regulatory roles and are controlled by the HUSH complex.