Gene-Specific Targeting of H3K9 Methylation Is Sufficient for Initiating Repression In Vivo

Abstract

Covalent modifications of chromatin have emerged as key determinants of the genome's transcriptional competence. Histone H3 lysine 9 (H3K9) methylation is an epigenetic signal that is recognized by HP1 and correlates with gene silencing in a variety of organisms. Discovery of the enzymes that catalyze H3K9 methylation has identified a second gene-specific function for this modification in transcriptional repression. Whether H3K9 methylation is causative in the initiation and establishment of gene repression or is a byproduct of the process leading to the repressed state remains unknown. To investigate the role of HMTs and specifically H3K9 methylation in gene repression, we have employed engineered zinc-finger transcription factors (ZFPs) to target HMT activity to a specific endogenous gene. By utilizing ZFPs that recognize the promoter of the endogenous VEGF-A gene, and thus employing this chromosomal locus as an in vivo reporter, we show that ZFPs linked to a minimal catalytic HMT domain affect local methylation of histone H3K9 and the consequent repression of target gene expression. Furthermore, amino acid substitutions within the HMT that ablate its catalytic activity effectively eliminate the ability of the ZFP fusions to repress transcription. Thus, H3K9 methylation is a primary signal that is sufficient for initiating a gene repression pathway in vivo.

Towards Sustained Silencing of HER2/neu in Cancer By Epigenetic Editing

“…Here, we added that also on irrelevant genes no enrichment of the fusion protein could be detected and interestingly genome-wide data showed that the Her2/neu ZFP is preferentially bound to Her2/neu gene. The domains of G9a and SUV39-H1 used in our ZFP fusion complexes have been previously reported to result in H3K9 methylation of the promoter of VEGF-A gene and in downregulation of this gene (30). We showed here that the approach is also suitable for downregulation of an overexpressed oncogene.…”

“…In this study, catalytic domains of 2 histone methyltransferases were cloned as reported previously (30). For G9a, the N-terminal domain (amino acid 1-829) was not included (excluding most of the ankyrin repeats); for SUV39-H1, the C-terminal region encoding amino acids 76-412 was amplified, lacking the N-terminal HP1 interaction domain.…”

“…For G9a, the N-terminal domain (amino acid 1-829) was not included (excluding most of the ankyrin repeats); for SUV39-H1, the C-terminal region encoding amino acids 76-412 was amplified, lacking the N-terminal HP1 interaction domain. Primers were derived from Snowden and colleagues, 2002, with introduced AscI and PacI restriction sites allowing swapping with VP64 (30). Catalytic mutant G9a (ref.…”

“…Targeted DNA methylation to repress gene expression was recently reported for three endogenous genes (28,29). Inducing repressive histone modifications has been reported for one gene; upon targeting of catalytic domains of histone methyltransferase enzymes (G9a, SUV39-H1) to the VEGF-A promoter, repressive histone marks were induced and result in downregulation of gene expression (30).…”

Linking the epigenetic ‘language’ of covalent histone modifications to cancer

“…We and others favour the view that histone deacetylation, histone methylation, DNA methylation and the production of small RNAs, all work together in various orders to bring about an efficient silent state (Grewal and Moazed, 2003). Remarkably, these epigenetic silencing mechanisms can be targeted to ectopic genes, thereby marking chromatin in such a way that this silencing state is inherited over many cell generations (Snowden et al, 2002;Ayyanathan et al, 2003).…”