KRAB-Induced Heterochromatin Effectively Silences PLOD2 Gene Expression in Somatic Cells and is Resilient to TGFβ1 Activation.

Abstract

Epigenetic editing, an emerging technique used for the modulation of gene expression in mammalian cells, is a promising strategy to correct disease-related gene expression. Although epigenetic reprogramming results in sustained transcriptional modulation in several in vivo models, further studies are needed to develop this approach into a straightforward technology for effective and specific interventions. Important goals of current research efforts are understanding the context-dependency of successful epigenetic editing and finding the most effective epigenetic effector(s) for specific tasks. Here we tested whether the fibrosis- and cancer-associated PLOD2 gene can be repressed by the DNA methyltransferase M.SssI, or by the non-catalytic Krüppel associated box (KRAB) repressor directed to the PLOD2 promoter via zinc finger- or CRISPR-dCas9-mediated targeting. M.SssI fusions induced de novo DNA methylation, changed histone modifications in a context-dependent manner, and led to 50%-70% reduction in PLOD2 expression in fibrotic fibroblasts and in MDA-MB-231 cancer cells. Targeting KRAB to PLOD2 resulted in the deposition of repressive histone modifications without DNA methylation and in almost complete PLOD2 silencing. Interestingly, both long-term TGFβ1-induced, as well as unstimulated PLOD2 expression, was completely repressed by KRAB, while M.SssI only prevented the TGFβ1-induced PLOD2 expression. Targeting transiently expressed dCas9-KRAB resulted in sustained PLOD2 repression in HEK293T and MCF-7 cells. Together, these findings point to KRAB outperforming DNA methylation as a small potent targeting epigenetic effector for silencing TGFβ1-induced and uninduced PLOD2 expression.

• Authors: Rutger A F Gjaltema, Désirée Goubert, Christian Huisman, Consuelo Del Pilar García Tobilla, Mihály Koncz, Pytrick G Jellema, Dandan Wu, Uilke Brouwer, Antal Kiss, Pernette J Verschure, Ruud A Bank, Marianne G Rots
• Journal: International journal of molecular sciences (Int J Mol Sci) Vol. 21 Issue 10 (May 21 2020) ISSN: 1422-0067 [Electronic] Switzerland
• PMID: 32455614 (Publication Type: Journal Article)

Chemical References

• Heterochromatin
• Kruppel-Like Transcription Factors
• Transforming Growth Factor beta
• PLOD2 protein, human
• Procollagen-Lysine, 2-Oxoglutarate 5-Dioxygenase
• DNA modification methylase SssI
• DNA-Cytosine Methylases

Topics

• Adult
• Cells, Cultured
• DNA-Cytosine Methylases (genetics, metabolism)
• Epigenesis, Genetic
• Gene Silencing
• HEK293 Cells
• Heterochromatin (metabolism)
• Humans
• Kruppel-Like Transcription Factors (genetics, metabolism)
• MCF-7 Cells
• Procollagen-Lysine, 2-Oxoglutarate 5-Dioxygenase (genetics, metabolism)
• Promoter Regions, Genetic
• Transcriptional Activation
• Transforming Growth Factor beta (metabolism)