Abstract
Gene regulation plays essential roles in all multicellular organisms, allowing for different specialized tissue types to be generated from a complex genome. Heterochromatin-driven gene repression, associated with a physical compaction of the genome, is a pathway involving core components that are conserved from yeast to human. Posttranslational modification of chromatin is a critical component of gene regulation. Specifically, tri-methylation of the nucleosome component histone 3 at lysine 9 (H3K9me3) is a key feature of this pathway along with the hallmark heterochromatin protein 1 (HP1). Histone methyltransferases are recruited by HP1 to deposit H3K9me3 marks which nucleate and recruit more HP1 in a process that spreads from the targeting site to signal for gene repression. One of the enzymes recruited is SETDB1, a methyltransferase which putatively catalyzes posttranslational methylation marks on H3K9. To better understand the contribution of SETDB1 in heterochromatin formation, we downregulated SETDB1 through knockdown by a dCas9-KRAB system and examined heterochromatin formation in a chromatin in vivo assay (CiA-Oct4). We studied the contribution of SETDB1 to heterochromatin formation kinetics in a developmentally crucial locus, Oct4. Our data demonstrate that SETDB1 reduction led to a delay in both gene silencing and in H3K9me3 accumulation. Importantly, SETDB1 knockdown to a ∼50% level did not stop heterochromatin formation completely. Particle-based Monte Carlo simulations in 3D space with explicit representation of key molecular processes enabled the elucidation of how SETDB1 downregulation affects the individual molecular processes underlying heterochromatin formation.