University of Groningen
Epigenetic editing
Cano Rodriguez, David
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2017
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Cano Rodriguez, D. (2017). Epigenetic editing: Towards sustained gene expression reprogramming in
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12
General Introduction
The field of epigenetics studies changes in gene expression that are independent of the underlying DNA sequence. Although all cells within an organism contain the same DNA, there are many different cell types, tissues and organs present. Different subsets of genes that are activated depending on their regulation lead to different properties and therefore to different cell types and tissues1,2. The organization of DNA and
histones into chromatin is an important aspect in gene regulation, through which the access of trans-cription complexes to the DNA can be regulated. Chromatin serves as a macromolecular scaffold in nuclei of eukaryotic cells that consists of high order structures called nucleosomes. The nucleosomal architecture is built up of octameric units of dimers of the four core histones H2A, H2B, H3 and H4 wra-pped around by 147 base pairs of DNA3. Higher-order folding of the nucleosomes can result in either
less condensed, active euchromatin or highly condensed, silent heterochromatin. Epigenetic gene regulation is mediated by several mechanisms including DNA methylation and the post-translational modifications (PTM) of the histone tails, both of which may activate or repress transcription according to the specific context4. These modifications can directly or indirectly influence chromatin structure
by modulating DNA-histone interactions and form docking sites to facilitate recruitment of proteins to the chromatin5,6. This form of epigenetic regulation is important for the maintenance of cell identity
and therefore it is implicated in processes such as proliferation, development and differentiation7,8.
DNA methylation occurs predominantly at CpG dinucleotides and is mediated by DNA methyltransferases (DNMTs). DNMTs are enzymes that catalyze the transfer of methyl groups from S-adenosyl-L-methionine to the 5’ position of cytosine, resulting in 5-methylcytosine (5mC)9,10. The DNMT family in humans
consists of DNMT3A and 3B, which catalyze de novo DNA methylation during embryonic development, and DNMT1, which is responsible for the maintenance of established DNA methylation patterns during replication (i.e. with participation of DNMT3A and 3B). Another family member, DNMT3L (DNMT3-like), resembles DNMT3 enzymes while itself lacking catalytic activity11-13.
Methylated CpG dinucleotides are not distributed evenly across the genome. There are re-petitive and foreign elements that are mainly hypermethylated, and focused locations within gene promoters that are hypomethylated. These focused locations are short (approx. 1 kilobase (kb)) CpG-rich regions called CpG islands (CGIs)14-17. The presence of 5mC within the germline is thought to lead to the loss of CpGs, because 5mC can be deaminated spontaneously or enzymatically, leading to the conversion of 5mC to thymine. CGIs are consequently thought to exist because they are never or only transiently methylated in the germline and therefore escape the pressure to be converted 18,19. Such CGIs are found in approx. 40% of promoters in the mammalian genome. Their methyla-tion has been associated with long-term transcripmethyla-tional silencing and a closed chromatin state, which is known to occur e.g. at imprinted genes, genes on the inactive X chromosome and genes that are exclusively expressed in germ cells14,18,20. Interestingly, differences in CpG-density within promoter regions have been associated with specific methylation and expression patterns. This was shown by use of methylated DNA immunoprecipitation and microarray analysis in human primary fibroblasts19. It appears that low-CpG (i.e. non-CGI) promoters are often methylated, even in the active state.
Epigenetic
gene regulation
DNA
13
Histone
modifications
The N-terminal tails of the histone proteins may be chemically modified at lysine, arginine or serine residues by for instance, acetylation, me-thylation, ubiquitination and phosphorylation5,8. The pattern of PTM’s that
occur on one or more histone tails form a so-called histone code that can be deciphered by other proteins These proteins can alter the struc-ture of higher-order chromatin and recruit effector molecules22. Histone PTM’s are reversible and
can be associated with either euchromatin or heterochromatin and therefore can control gene ex-pression. The transcriptionally inert heterochromatin state is negatively regulated by hypoacetyla-tion of H3 and H4 and tri-methylahypoacetyla-tion of lysine residues 9 and 27 on histone H3 (H3K9me3, H3K-27me3) and tri-methylation of lysine residue 20 on histone H4 (H4K20me3). In contrast, acetylation of histones H3 and H4 and tri-methylation of lysine residue 4 and 79 on histone H3 (H3K4me3, H3K79me3) situated at transcription start sites (TSS) are associated with transcriptionally active euchromatin23-28. Through these modifications, histones can regulate gene transcription by
chan-ging chromatin structure, altering electrostatic charge or by providing protein recognition sites7. The machinery that regulates histone methylation generally consists of different classes of histone methyltransferases (HMTs) and histone demethylases (HDMs)29. Histone 3 lysine 4 di- and
trimethylation (H3K4me2/3) is mainly associated with transcriptionally active genes, although in mam-mals much lower levels of this mark have also been observed at silent genes. Nevertheless, the mark is thought to positively regulate transcription by recruitment of nucleosome remodeling enzymes and histone acetylases30. The establishment of the histone mark is catalyzed by the trithorax group (trxG)
of proteins through activity of the SET1 or MLL family members, which reside within multimeric protein complexes. The first of these complexes containing SET1 was identified in yeast and was named COMPASS, for complex of proteins associated with SET131. HDMs known to abolish the H3K4me2/3 mark are KDM1/LSD1 family members (remove H3K4me1/2) and members of the JmjC-domain con-taining family (remove H3K4me3)18. H3K4me3 is one of the most studied active histone marks, and
one of the most known models for histone PTMs crosstalk. The tight regulation between monoubi-quitination of H2B and the trimethylation of H3K4 and H3K79 is a hallmark of active promoters32-36.
Histone repressive marks have been well studied, such as methylation of H3K9 and H3K27. In mammalian cells, H3K9me3 is a hallmark of heterochromatin and is also required for transcriptional silencing of genes and retroviral elements37,38. Transcriptional repression
invol-ves heterochromatin protein 1 (HP1), which specifically binds to methylated H3K9, which would, in turn, recruit DNA methyltransferases28,39-41. Between the enzymes identified to be required for
H3K9 methylation are SETDB1, SUV39H1 and G9a. Additionally, H3K27me3 marks are associa-ted with transcriptional repression, which is thought to be mediaassocia-ted by the promotion of a com-pact chromatin structure42. These marks are induced by Polycomb-group (PcG) proteins residing
within the polycomb repressive complex 2 (PRC2). PRC2 consists of Suz12, EED and EZH2, the latter being a SET1 domain-containing HMT43. The repressive H3K27me3 mark can
furthermo-re be furthermo-recognized by PRC1, which mediates the ubiquitylation of H2AK119, a mark that is thought to further maintain silencing18,43. Binding of PRC1 is thought to block the recruitment of
transcrip-tional activation factors and to prevent initiation of transcription by RNA polymerase II44. The
de-methylation of H3K27 is catalyzed by the KDM6 family, better known as UTX and JmjD318.
Different histone modifications may influence each other (e.g. H3K36 methyla-tion inhibits H3K27 methylamethyla-tion) or may influence other epigenetic modifiers or epigene-tic marks (e.g. H3K27me3 may recruit PRC1, and H3K4me3 may inhibit DNA methylation). Methylation of H3K4, H3K9 and H3K27 has been shown to mediate mitotic inheritance of linea-ge-specific gene expression patterns and accordingly have key developmental functions42. For
this reason, these epigenetic marks are important targets for epigenetic editing, in order to achie-ve sustained gene expression reprogramming. Moreoachie-ver, as cancer cells frequently remain in an undifferentiated state45, these histone marks are of particular interest in cancer research.
This implies that low concentrations of 5mC do not foreclose gene activity19,21. High-CpG (i.e CGI) promoters on the other hand are mostly unmethylated, even when inactive. This implies that the-se regions are somehow protected from methylation, not only in the germline, but also in somatic cells19. The fact that these CGI promoter genes can still be inactivated, suggests an additional le-vel of epigenetic control. Indeed, such a regulatory lele-vel is formed by the modification of histones.
14
Reading and writing
the epigenetic code
At certain stages of the cell cycle or in response to stimuli, the chromatin folding must decrease to facilitate access of appropriate factors or it must condense to render the DNA in-accessible to those factors. This level of regulation is achie-ved through the abundance and impact of specific PTM’s. This balance is regulated by the balance of enzymes that can ‘write’ or ‘erase’ a specific PTM22,46.
Writers such as acetylases, methylases and phosphorylases can introduce distinct PTM’s on his-tones. Erasers such as deacetylases, demethylases and phosphatases antagonize the function of writers by removing those histone modifications47.
Readers are proteins that contain conserved domains that can recognize histone modifications48.
These readers are for example bromodomains, chromodomains, PHD fingers or WD-40 repeats that can interpret PTMS’s to evoke specific functional outcomes such as remodeling of the chromatin, stabilization of the higher order-chromatin, further posttranslational modification (through writer or eraser) or other gene regulatory effects such as direct recruitment of RNA polymerase II machinery46.
Cancer
Epigenetics
Failure to correctly regulate chromatin condensation or decondensation can lead to an increase in DNA damage and abnormal gene expression, which on their turn can lead to genome instability. Genomic instability is implicated in many pathogenic events such as human syndromes and cancers49-51. Indeed it
is shown that there is a strong relationship between aberrant post-translational histone modifications and tumorigenesis. The gene expression abnormalities observed in cancer are frequently associated with aberrant epigenetic profiles52. For example, tumor suppressor genes can
frequently become silenced during tumor development. The promoter regions of these genes can be subject to hypermethylation, which is associated with gene silencing. In addition, a global DNA hypo-methylation is frequently involved in genomic instability and transformation into malignant cells. Mo-reover altered histone modification patterns also play a critical role in tumorigenesis of tumor suppres-sor genes and oncogenes, including an enrichment or loss of active or repressive histone marks53.
The fact that the various epigenetic marks are reversible makes it an ideal target for therapeutic intervention. Currently, US Food and drug administration (FDA)-approved epigenetic drugs, e.g. histo-ne deacetylase inhibitors and DNA demethylating agents have been demonstrated to have potent an-ti-cancer effects in clinics54-56. However, it has been demonstrated that the chemotherapeutic agents can
induce undesired off-target effects throughout the genome, e.g. induction of expression of the pro-me-tastatic gene Ezrin57. Therefore it is essential for future therapeutic intervention to target cancer in a
ge-ne-specific way. The interactions between proteins that can read and write or erase the code provide re-gulatory opportunities for the transmission of epigenetic marks along the genome, and the use of current targeting platforms has open new avenues to study the effect of epigenetic marks in health and disease.
Epigenetic Editing
An elegant method to modulate gene expression at will is depic-ted by artificial transcription factors (ATFs). ATFs that are engi-neered to bind to a promoter region of a gene of interest are ableFigure 1. Epigenetic editing. By coupling or fusing an
epigenetic effector domain to a DNA binding domain, any region in the human genome can be targeted and gene expression can be modulated
1
to specifically affect gene expression. Previous-ly, the potentials of ATF treatment have been de-monstrated for several genes involved in cancer, including SOX2, Maspin, C13ORF18 and EP-B41L358-61. In general, ATFs consist of a
DNA-bin-ding domain (DBD) and a transcriptional activator/ repressor to positively or negatively regulate trans-cription. Yet, a major disadvantage of ATF treat-ment is the transient effect on gene expression modulation. Hence, a sustained gene expression modulation can only be achieved by reprogram-ming of the cellular epigenetic context in a mitoti-cally stable manner. The most promising approach to meet this requirement is illustrated by epige-netic editing62 (Figure 1) . The goal of epigenetic
editing is to rewrite epigenetic marks at any locus at will to permanently modulate the expression of endogenous genes. Since the dynamic remodeling of the chromatin landscape (and the stable CpG dinucleotide methylation) is tightly regulated by a conglomerate of enzymes and other macro-molecules, there is a huge array of epigenetic effector domains for gene expression modulation.
15 In order to rewrite the epigenetic landscape, the catalytic domain of an epigenetic writer or an eraser is fused to a gene-specific DBD to enforce the presence of this effector domain at a particular DNA sequence. Induced epigenetic changes can be assessed by chromatin immuno-precipitation (ChIP) or bisulfite sequencing and the effect of targeting epigenetic enzymes on gene expression can be assessed by measuring gene expression levels of genes that are in close proximity of the DBD recognition site. The most used targeting DBDs used are Zinc Finger Proteins (ZFPs)63, Transcription Activator Like Effectors
(TALEs)64 and the clustered, regularly interspaced, short palindromic repeat-CRISPR associated
pro-tein (CRISPR-Cas) RNA-guided endonuclease system that can be targeted to any loci of interest 65,66.
After the introduction of the concept by us, the field of epigenetic editing has been in increa-sed development67-74. Especially the introduction of a new, cheap and flexible DNA binding platform
(CRISPR-Cas) has sparked the interest in this approach.
Several genes and epigenetic enzymes have been used to repress or reactivate gene expression at will. Most of these studies, however, have only addressed short term epigenetic reprogram-ming and are lacking sustained gene expression modulation. For this reason we have deve-loped several platforms to address stable epigenetic reprogramming after epigenetic editing.
Aims and scope
of the thesis
The aim of this thesis was to induce stable activation or repression of endogenous target genes by epigenetic editing. We develop no-vel modalities of gene expression modulation that exploits epige-netics to induce inheritable and robust states of transcriptional re-programming at desired loci. To this end, we took advantage of our expertise in developing programmable DNA Binding Domain (such as ZFPs and CRISPR/dCas9) to target the promoter of endogenous genes, and couple these to effector domains from natura-lly occurring epigenetic enzymes (e.g. histone modifying enzyme and DNA methytransferases). Previous studies showed that ATFs can be used to modulate gene transcription. However, their tran-sient activity was associated with reacquisition of the previous transcriptional state. This outcome likely reflects the inability of the chosen transcriptional regulators to recreate self-propagating epi-genetic changes at the target loci. To overcome this limitation, we hypothesized that targeting of multiple epigenetic regulators might mimic natural conditions to allow the formation of transcriptional complexes capable of creating self-sustaining epigenetic reprogramming. The hit-and-run approach of our epigenetic platform should overcome the limitations associated with the current technologies, paving the way for its therapeutic application. Finally, this epigenetic editing platform can be rapidly adopted in basic biology to interrogate the function of mammalian regulatory elements and to un-derstand the mechanistic relationship among chromatin states, gene regulation and cell phenotype. The first three chapters deal with the overall development of the field of epigenetic editing. In Chap-ter 2, we set out to present the latest efforts and achievements in the field of epigenetic editing. In this review we aimed to show the current techniques and targeting platforms used, as well as their advantages and disadvantages. We also present the most used epigenetic enzymes targe-ted at different regulatory regions, the clinical application and the future perspectives. Chapter 3 aims to provide a clear protocol for the field covering the methodology to create Zinc Finger Pro-teins and to target the promoter of a hypermethylated gene (ICAM-1) using epigenetic editing to de-methylate DNA using the catalytic domain of Tet2. In Chapter 4, we aimed to increase effective-ness of epigenetic editing by identifying main barriers in targeting genes and subsequent epigenetic reprogramming. As DNA methylation at CpG islands has been shown to be a hurdle when targe-ting loci, addressing this issue will help in achieving the optimal possibilities for genome targetarge-ting. The next chapters serve as an example for the potential use of epigenetic editing in clinical research and basic fundamental biology research. Chapter 5 explores the possibility of targeting two different promoters of the RASSF1 gene, with the aim to demonstrate that two transcripts from the same gene, but regulated from different promoters, have opposing effects in cancer. It has already been proven by exogenous overexpression or silencing that the two transcript act differently in cells, and we aimed to show that endogenous regulation of the two promoters by epigenetic editing has the same results.
16
While the first promoter generates a transcript that has tumor suppressive activity, the shortest iso-form, transcribed from an inner promoter is thought to be an oncogene. In Chapter 6, we use epi-genetic editing to show the potential function of the NCO2 gene, which is overexpressed in cancer. The aim of this chapter was to identify the function of this gene by using epigenetic editing to repress its expression in cancer cells and evaluate their behavior. With these two chapters we aimed to show that some genes could serve not only as biomarkers but can also provide therapeutic targets. The last chapters depict the possibility to achieve sustained gene expression reprogramming with epigenetic editing. In Chapter 7 we aim to accomplish sustained transcriptional repression of a can-didate gene dysregulated in Chronic Obstructive Pulmonary Disorder (COPD) by means of compa-rison between the transcriptional repressor Super Krab Domain (SKD) and epigenetic enzymes. In Chapter 8, we investigate the capability of H3K4me3 to induce gene expression, and whether there is dependency on the chromatin microenvironment targeted. We also set out to unravel requirements that need to be achieved in order to stably activate gene expression from hypermethylated genes. In Chapter 9, the most important findings of this thesis are summarized and discussed in the context of existing literature. We also show the potential for future research from the findings of this thesis.
17
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