University of Groningen
Epigenetic editing
Cano Rodriguez, David
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Publication date:
2017
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Cano Rodriguez, D. (2017). Epigenetic editing: Towards sustained gene expression reprogramming in
diseases. University of Groningen.
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CHAPTER 10
Summary
Epigenetic marks such as histone modifications and DNA methylation have been correlated with gene expression. Besides genetic mutations, aberrant epigenetic modifications (epigenetic mutations) are linked to several diseases such as cancer. Since epigenetic factors are reversible, they provide attracti-ve targets for noattracti-vel therapeutic approaches. Moreoattracti-ver, these reattracti-versible epigenetic marks are stable and will be inherited to daughter cells. Many initiatives have been launched to inhibit epigenetic enzymes, but these approaches encounter different limitations, including their genome-wide effect. The concern of modulating the expression of unintended genes prompted us to develop gene-specific targeting approa-ches to modulate gene expression. The aim of this thesis was to re-write epigenetic modifications on target genes using Epigenetic Editing in order to permanently modulate their expression.
After a general introduction in chapter 1, the first part of the thesis deals with the general tools to use Epigenetic Editing. First, we reviewed in chapter 2 the most recent advances in targeting epigenetic effector domains to different regions in the genome, in order to alter gene expression. Indeed, several researchers have been able to show that targeted Epigenetic Editing is a powerful tool to address se-veral questions in the field of epigenetics. Sese-veral targeting domains have been developed In order to direct epigenetic enzymes to several loci. The first domains used were Zinc Finger Proteins, derived from mammalian transcription factors. Then, the discovery of Transcription Activators-Like Effectors or TALEs, increased the possibilities of targeting. Finally, with the leverage of the innovative CRISPR-Cas system, the field has grown to be one of the most promising in the last decade. In chapter 3 we show the general protocol to use Zinc Finger targeted DNA demethylation using the TET2 enzyme. Finally in chapter 4, we reviewed the limitations in targeted activation of endogenous genes with different platfor-ms, due to the complexity of the epigenome. Indeed, one of the limitations in targeting seems to be the chromatin microenvironment, with nucleosomes as the main player.
The second part of this thesis shows the uses of Epigenetic Editing in modulating gene expression in diseases. Chapter 5 we focus on the RASSF1 gene to show the possibilities of addressing Epigenetic Editing in two different regions. We constructed tools to address the dual role of this gene in cancer by targeting the two promoters that render two transcripts (A and C) with contrasting features. On one hand, overexpression of the first promoter generates the RASSF1A transcripts that exhibits a tumor suppressive activity. On the other hand, the inner promoter creates a smaller transcript (RASSF1C), which seems to be involved in stemness. In chapter 6, we describe the identification of a novel gene overexpressed in several types of cancers. By using Epigenetic Editing we were able to show that this gene has oncogenic features (TCTN2), and that this provides a novel therapeutic target
Finally, the last part of this thesis deals with the possibility of achieving sustained gene expression modulation by using Epigenetic Editing. In chapter 7, we use this technique to accomplish sustained transcriptional repression of a candidate gene dysregulated in Chronic Obstructive Pulmonary Disorder (COPD) by means of comparison between the transcriptional repressor Super Krab Domain (SKD) and epigenetic enzymes. We showed that by using H3K9 methylating enzymes, the effect of repression is sustained over cell passages. Chapter 8 demonstrates the capability of H3K4me3 to induce gene expression depending on the effect of the chromatin microenvironment that is targeted. We showed some of the requirements that need to be achieved in order to stably activate gene expression from hypermethylated genes.
In chapter 9, a general discussion on the research in this thesis is provided and we also describe some important factors, which influence the efficacy of Epigenetic Editing technology.
