Relationship between lysine methyltransferase levels and heterochromatin gene repression in living cells and in silico

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, trimethylation 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 knock down 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 knock-down to a ∼50% level did not stop heterochromatin formation completely. Particle-based Monte-Carlo simulations in three-dimensional space with explicit representation of key molecular processes enabled the elucidation of how SETDB1 downregulation affects the individual molecular processes underlying heterochromatin formation.