Isolated DNA repeat units nucleate heterochromatin through Integrator-coupled transcriptional attenuation

Heterochromatin defines a transcription-repressive chromatin state. In mouse cells, heterochromatin is associated with underlying arrays of A/T-rich, 234 bp DNA repeat elements, called the major satellite repeats (MaSat or MSR). We have examined > 18,000 copies of MSR repeat units in mouse ES cells and identified that heterochromatin is only formed at transcriptionally competent MSR variants. To directly dissect the function of MSR DNA, we inserted isolated MSR copies into an inert genomic region that is repeat- and gene-free. Insertion of three or more intact MSR units, but not one MSR unit, induced heterochromatic histone marks, recruitment of HP1 and incorporation of histone H1. Only transcriptionally competent MSR copies, but not permutated MSR variants or other DNA repeats, such as LINE1 5'UTR elements can form de novo heterochromatin. MSR-derived transcription is bi-directional and MSR driven transcripts are attenuated by the RNA polymerase II (RNAPII) associated Integrator complex. These data link RNA processing of MSR transcripts by the Integrator with heterochromatin formation. This study resolves a long-standing paradox between heterochromatic and euchromatic transcriptional activity and uncovers a DNA/RNA based logic for the establishment of heterochromatin in the mouse epigenome. Overall design: To investigate whether Integrator complex is presented at endogenous MSR. Wild type mESCs (WT26) are fixed by double cross-linking (2 mM DSG and 1% formaldehyde) and proccessed using RELACS nuclei barcoding protocol (Arrigoni et al., 2018).