Binding of KAP1 organizes HP1alpha for transcriptional silencing [ATAC-seq]

Constitutive heterochromatin is enriched in repetitive elements, including endogenous retroviral element (ERV) sequences that must be repressed to sustain normal cell developmental programs and prevent chromosomal instability. ERVs are silenced through trimethylation of lysine 9 on histone H3 (H3K9me3) by ESET (also known as SETDB1, SET domain bifurcated 1, or KMT1E) and a co-repressor complex containing KAP1 (KRAB-associated protein 1, also known as tripartite motif-containing protein 28, Trim28) in mouse embryonic stem cells. H3K9me3 then serves as a scaffold for the recruitment of proteins that maintain ERVs in a transcriptionally silent state, including the HP1 proteins that themselves interact with the co-repressor KAP1. In this study, we provide structural insights into the KAP1-HP1 interaction. Further, we demonstrate that this interaction plays a role in maintaining inaccessible chromatin at specific ERV elements in embryonic stem cells (ESCs). Overall design: ATAC-seq analysis in mouse embryonic stem cell lines. The piggybac transposase system was used to integrate transgenic KAP1-Flag or KAP1(V488E)-Flag into KAP1-fl/fl ESCs with an integrated ERT2-Cre cassette. Exogenous expression of WT or V488E mutant KAP1 was induced with 2 µg/mL doxycycline for 48hrs and maintained in doxycycline prior to endogenous KAP1 deletion. KAP1 deletion was induced with 2 µM 4-OHT (Sigma) treatment for 48hrs. Cbx1-/-;Cbx3-fl/fl;Cbx5-fl/fl;ERT2-Cre ESCs were obtained from PMID 29795351. HP1 deletion was induced with 2 µM 4-OHT (Sigma) treatment for 48hrs or 72 hrs.