Known and unknown functions of TET dioxygenases: the potential of inducing DNA
modifications in Epigenetic Editing
Chen, Hui
DOI:
10.33612/diss.168496242
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Publication date: 2021
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Chen, H. (2021). Known and unknown functions of TET dioxygenases: the potential of inducing DNA modifications in Epigenetic Editing. University of Groningen. https://doi.org/10.33612/diss.168496242
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Chapter 1
Epigenetics
In mammals, when a sperm combines with an egg to form a fertilized egg, it opens a magical journey of life evolution in a real sense. After multiple divisions and programmed differentiation, the omnipotent single-cell fertilized egg has evolved into a variety of terminally differentiated cells with specific functions. These cells then form tissues and organs that can perform different physiological functions, and together form alive individuals with unique attributes. The magic of the whole development process is that although the DNA sequence of all terminally differentiated cells is identical to that of the fertilized egg at the start of development, cells differ greatly in morphology and physiological function. One of the important reasons for this difference is the regulatory role of epigenetics to induce stable gene expression profiles underlying cell fate determination.
In 1942, Waddington first proposed the concept of "epigenetics", which were further elaborated by Holliday (1, 2). Since then, with the emergence of more and more evidence on epigenetic regulation of gene expression and individual development, people have become increasingly aware of the importance of epigenetic regulation of life processes. Epigenetics is a branch of genetics. The classical definition is to study the hereditary changes of gene expression without altering the nucleotide sequence of a gene (3). This concept has, however, received a number of more or less equivalent reformulations during the past decades. Currently, from the view of molecular epigenetics, the label “epigenetic” is used to refer to any chromatin modification affecting gene activity and expression, whether it is mitotically and/or meiotically stable or not (4). Epigenetics includes DNA methylation and histone modifications which directly participate in the regulation of chromatin conformation, thus affecting gene expression. This thesis focuses on two aspects, one is DNA methylation and modifications derived from DNA methylation, and the other is the writers (enzymes) responsible for catalyzing these modifications.
DNA methylation and transcription regulation
DNA methylation exists widely in different organisms and is involved in regulating many important biological functions, including gene imprinting, X chromosome inactivation and transposon silencing (5, 6). In higher eukaryotes, DNA methylation mainly occurs on the fifth carbon atom of cytosine, i.e. in the form of 5-methylcytosine (5mC). In mammals, 5mC is mainly distributed on CpG dinucleotides, which accounts for 1% of the total human genome, and more than 70% of CpG is methylated (7, 8). In addition, most of the 5mC are located in transposon-related chromatin repeat regions, including SINE, LINE and LTR repeats, possibly related to the inhibition of transposon
activity. However, compared to the transposon elements, the distribution of 5mC for protein-coding genes is not uniform. Although more than 75% of promoter regions which containing CpG islands are hypomethylated in all cell types, there are still many genes whose expression is regulated by DNA methylation (9). In plant genomes, DNA methylation can occur in CpG, CHG or CHH (‘H’ denotes any base of A, T, G), and is mainly concentrated in the transposon-intensive heterochromatin region (10). In addition, 5mC is widely distributed in the lower eukaryotic model organism Chlamydomonas reinhardtii (C. reinhardtii), but there is still a big blank in the study of its function (11).
In mammals, 5mC is catalyzed by DNA methyltransferase (Dnmts) family proteins, which transfer the methyl group of the methyl donor S-adenosylmethionine (SAM) to the 5th carbon atom of cytosine (Figure 1). Dnmts family proteins include Dnmt1, Dnmt2, Dnmt3a, Dnmt3b, Dnmt3c and the non-catalytic Dnmt3l. Except for Dnmt2, which functions as a tRNA methyltransferase (12), the Dnmt members function as DNA methyltransferases. Dnmt3a, Dnmt3b and Dnmt3c belong to the de novo DNA methyltransferase (13), while Dnmt1, the maintenance methyltransferae, can specifically identify the hemi-methylated DNA sequence produced after DNA replication and methylate the cytosine on the newly synthesized daughter strand, thus ensuring the maintenance of methylation pattern in the process of DNA replication (Figure 1) (14). The recently identified Dnmt3c is responsible for methylating the promoters of evolutionarily young retrotransposons in the male germ line and is required for mouse fertility (15). Dnmt3l lacks methyltransferase activity, but can be recruited by unmethylated Histone 3 lysine 4 (H3K4) to subsequently recruit Dnmt3a/3b to methylate target DNA sequences (16). In the higher land plant Arabidopsis thaliana, DNMT homologous protein Domain Rearranged Methyltransferase 2 (DRM2) mediates de novo methylation. DRM2 can be recruited to target sites by a 24 nt small interfering RNA, called RNA-directed DNA methylation (RdDM) (17, 18). In methylation inheritance, the maintenance of methylation on CpG and CHG depends on DNA methyltransferase 1 (MET1) and Chromomethylase 3 (CMT3), while the maintenance of methylation on asymmetric CHH also depends on de novo methyltransferase DRM2 (18).
DNA methylation plays an important role in gene transcription regulation. The regulation of DNA methylation on genes is thus reflected in development, cell differentiation, imprinted genes, X chromosome inactivation, and disturbances in DNA methylation are associated with aging and disease (e.g. cancer). DNA methylation mediated gene silencing is considered to take place through a variety of mechanisms, which remain to be completely elucidated.
Figure 1. De novo and maintenance DNA methylation in mammals.
The de novo methyltransferases Dnmt3a/3b/3c methylate cytosine at C-5 position by transferring methyl group from SAM. The DNA methylation is maintained by Dnmt1 during DNA replication to restore the symmetrical methylation pattern. (Disclaimer: all figures contained in chapter 1 were made by PhD candidate and are not from published paper or the internet.)
On the one hand, DNA methylation in enhancer and promoter regions usually results in transcriptional inhibition. Methylation of enhancers blocks the binding of enhancers to related transcription factors, thus inhibiting the transcriptional initiation of downstream genes (19, 20). Furthermore, methylation of promoter regions recruits 5mC-binding proteins, such as the methyl-CpG-Binding proteins (MBPs) MeCP1, MeCP2, methyl-CpG-binding domain (MBD) family, etc., which cause related transcriptional repressors to bind to the target region, thereby silencing the target gene by further recruiting transcriptional inhibitors and remodeling the chromatin structure (21, 22, 23) (Figure 2a). In addition to methylation of enhancers and promoters, studies have shown that hypermethylation of the first exon is also directly related to the silencing of the gene expression (24, 25).
On the other hand, DNA methylation and histone lysine methylation in mammals are highly correlated. For example, Suv39h1/2 H3K9 methyltransferase first establishes H3K9me3 at pericentric heterochromatin through its enzyme activity (26). Subsequently, heterochromatin protein 1 (HP1) binds H3K9me3 marked heterochromatin through its chromodomain, which then recruits Dnmt3a/b (26). Interestingly, Dnmt3a/b may play a guiding role in the recognition of chromatin substrate through direct interaction with histone methyltransferase (27). For example, the Dnmt3a/b has been shown to interact with Suv39h1 and Setdb1 through its ADD domain (28, 29). In addition, it has been reported that Dnmt3a has a direct interaction with H3K9 methyltransferase G9a and H3K27 methyltransferase EZH2 through its SET domain (30, 31) (Figure 2b).
In some cases, however, such as during the early development of vertebrates, some
Figure 2. The model of DNA methylation regulating transcription.
a. The textbook model describing how DNA methylation regulates transcription. Methylation of CpG-islands (Black circle) in enhancers directly blocks the binding of enhancers to related transcription factors (Blue oval). Methylated CpG-islands in promoters recruit transcriptionally repressive methyl-CpG-Binding proteins (Plum-red hexagon), resulting in inhibition of the binding of related transcription activators (Orange oval) to the target region and further causing gene silencing by recruiting transcription inhibitors (Brown pentagon and concave quadrilateral). b. A model describing that DNA methylation and histone lysine methylation are involved in establishing patterns of gene repression in a synergistic manner. Heterochromatin protein 1 (HP1) (Orange oval) can cause the formation of heterochromatin by binding its chromodomain to H3K9-methylated marks (Yellow Flag), and it will further recruit Dnmt3a/3b to H3K9-methylated heterochromatin, resulting in blockage of the binding of transcription factors (Blue oval). Dnmt3a/b (Plum-red hexagon) has been shown to interact with H3K9 methyltransferases (Brown pentagon) and H3K27 methyltransferases (Concave quadrilateral) via the SET domain, both resulting in CpG-island methylation in promoters and in blockage of the binding of transcription factor or activators.
methylated promoters with low CpG density are actively transcribed, and the MBPs do not interact with these promoters, but the specific reason remains to be elucidated. Interestingly, histone marks H3K4me3, which indicate active transcription, have been found to be associated with some of these methylated DNA sequences with low CpG density (including enhancers and promoters). In terms of the molecular mechanism of methylation, Dnmt3l, a catalytically inactive regulatory factor of DNA methyltransferases, first recognizes and binds to histone H3 of nucleosome, and then further recruits Dnmt3a and Dnmt3b for de novo DNA methylation (32, 33). However, the direct interaction between Dnmt3l and nucleosomes is inhibited by all forms of methylation marks on H3K4 (32). Therefore, de novo methylation at these low CpG density regions in the genome may be prevented due to the presence of H3K4me3. Furthermore, H3K4me3 will recruits transcription activators and promotes the expression of corresponding genes (34). In summary, DNA methylation and histone lysine methylation are involved in establishing patterns of gene transcription activity in a coordinated manner. Certain forms of histone lysine methylation lead to the local formation of heterochromatin, which might be easy to reverse, while DNA methylation leads to stable long-term inhibition (35).
DNA demethylation and TET dioxygenase
DNA methylation profiles are not always stable, and there are some dynamic changes in DNA methylation levels. Dynamic DNA methylation patterns are essential for early embryonic development. In the process of lineage commitment, in cells undergoing differentiation the promoters of selected genes unique to other lineages are methylated to induce mitotically stable silencing. In contrast, the genes that are critical to lineage specifications remain unmethylated. For example, during mouse embryonic development, genomic DNA undergoes two times large-scale demethylation processes (36). The first occurs in the early embryonic development stage from egg fertilization to implantation. During this period, large-scale demethylation occurs most prominently in the male pronucleus, except for imprinted genes, IAP retrotransposons and heterochromatin regions near the centromere which escape this demethylation wave (37). At this stage, the epigenome undergoes widespread active and replication-dependent passive demethylation in zygotes before the first mitotic division (38, 39). The second large-scale DNA demethylation occurs during the migration of primordial germ cells (PGCs) to the genital ridge and after arrival, except for a few repetitive elements DNA sequence, methylation at almost all sites, including imprinted genes, will be erased (37). DNA demethylation in this second process is mainly accomplished by passive dilution caused by replication.
In addition to the large-scale demethylation occurring in the whole genome during
development, there are also many spatiotemporal and site-specific active DNA demethylation events in target loci. One example is that TGF-β stimulates active demethylation of the promoter of the tumor suppressor gene p15ink4b via recruitment of the DNA glycosylases thymine DNA glycosylase (TDG) (40). Normally, p15ink4b maintains transcriptional inhibition by methylation of the promoter by DNMT3A recruited by the ZNF217/CoREST complex. To examine whether the p15ink4b promoter can undergoe signal-dependent, temporal changes in DNA methylation, serum-starved HaCAT cells were treated with TGF-β for various time periods followed by sodium bisulphite sequencing and methylation-specific PCR. The results showed that in untreated cells, the core CpG island is highly methylated, and demethylation was detected within 20 min after TGF-β treatment, while nearly complete demethylation was evident by 3 hr. In addition, DNA demethylation in response to TGF-β was not inhibited by pretreatment of serum-starved HaCAT cells with the DNA replication inhibitor L-mimosine. Therefore, the rapidity of this response in quiescent cells strongly indicates that DNA demethylation is active, as opposed to passive demethylation that requires DNA replication (40). Furthermore, the study demonstrated that the mechanism of the TGF-β triggered active demethylation was achieved by recruiting TDG and base excision repair (BER) machines to the p15ink4b promoter (40). Similarly, within 20 minutes of stimulation, activated T lymphocytes undergo active demethylation at the interleukin-2 (IL2) promoter-enhancer region in the absence of DNA replication (41). In addition, it has been reported that the promoter of brain-derived neurotrophic factor (BDNF), which encodes protein products important for adult neural plasticity, also undergoes locus-specific demethylation (42). Specifically, the BDNF promoter is methylated in unstimulated neurons, allowing for the recruitment of the inhibitory factor MeCP2. However, when neurons are stimulated to depolarize with KCl, the BDNF promoter will undergo active demethylation in a short period of time, accompanied by the release of MeCP2 and upregulation of the expression of BDNF (42). These studies suggest that although DNA methylation generally functions as a long-term silencing mark, it can also play a role in the dynamic regulation of genes that need to respond quickly to specific stimuli. DNA demethylation thus includes active DNA demethylation and passive DNA demethylation. Passive demethylation refers to the process of passive dilution of methylation caused by DNA replication in the absence of maintenance DNA methyltransferase (DNMT1 deficiency or inhibition of its enzyme activity). Active DNA demethylation refers removing methyl groups directly or indirectly by the action of enzymes. Passive demethylation is widely accepted in concept, but there are still many unsolved mysteries about active DNA demethylation. Currently, there is a lack of a known biochemical mechanism for mammalian cells that can break the strong covalent
carbon-to-carbon bond between cytosine and methyl group. Initial breakthroughs in the study of active DNA demethylation came from Arabidopsis thaliana (A. thaliana), a higher terrestrial plant. In A. thaliana, active DNA demethylation is mainly accomplished by 5mC glycosylases, including ROS1, DME, DML2 and DML3, which can mediate 5mC direct removal (43). These 5mC erasers like ROS1 not only have the function of glycosylases, but also have the activity of apurinic/apyrimidinic site (AP site, also known as an abasic site) hydrolase. In the process of demethylation, ROS1 first excises 5mC to form an AP site by its glycosylases activity, then hydrolyzes the AP site, leaving a complete single nucleotide gap, which is repaired to an unmethylated C by base excision repair mechanism, completing the active demethylation process. However, no ROS1 homologous protein has been found in mammals so far. As an alternative, the removal of the methylation modification on the 5th carbon atom of DNA cytosine can be achieved by a series of chemical reactions. These reactions are all carried out by further modifying 5mC, including deamination and/or oxidation reactions, to eventually produce a product that can be recognized and removed by a specific glycosylase, and then replaced by unmodified cytosine via the BER pathway. At present, several protein factors and mechanisms have been proposed to be involved in the active DNA demethylation process in mammals (Figure 3) (44). It has been reported that Growth arrest and DNA-damage-inducible protein 45 a (Gadd45a) may play a role in active DNA demethylation via nucleotide excision repair (NER) or base excision repair (BER) pathway (45). In addition, two known deaminases, Activation Induced Deaminase (AID) and Apolipoprotein B mRNA editing enzyme, catalytic polypeptide 1 (APOBEC1), are believed to be involved in the active DNA demethylation process via deamination of the methylated cytosine into a thymine base. The resulting T/G mismatch is first recognized and excised by thymine DNA glycosylase (TDG), and then the resulting AP site is repaired through the incorporation of an unmethylated cytosine by the BER pathway (46). Interestingly, the DNA methyltransferases 3A (DNMT3A) and DNMT3B were also suggested to deaminate methylated cytosines to thymines, again resulting in incorporation of unmodified C by the BER machinery (47). But the mechanism of biochemical reaction remains to be elucidated.
Despite the indications of active DNA demethylation described above, these mechanisms of DNA active demethylation have not been well elucidated or proven so far. From a chemical point of view, to achieve active DNA demethylation, that means breakage of a carbon-carbon bond through enzymatic reactions. Although this is very difficult, studies have shown that enzymes responsible for catalyzing this reaction do exist. Warn-Cramer et al. reported that the thymine 7-hydroxylase from fungal can catalyse the conversion of thymine to iso-orotate through three consecutive oxidation reactions, with oxygen, iron and alpha-ketoglutaric acid (2-OG) as cofactors (48).
Iso-orotate can be further converted to C through a decarboxylation reaction. No homologues of thymine 7-hydroxylase have been found in mammals, two studies have revealed that the base j-binding protein (JBP), JBP1 and JBP2 derived from Trypanosoma brucei (T. brucei) have similar properties to thymine 7-hydroxylase (49, 50). These reports provided clues for Rao group to search for homologues similar to T. brucei JBP protein and with dioxygenase domain in mammalian. Using JBP domain as template, in 2009, the Rao group found that ten-eleven translocation (TET) family of proteins, a highly conserved homologous protein with JBP, exist in the human genome, and further revealed that TET had catalytic activity in oxidizing 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) (51). Then in 2011, He et al. and Ito et al. also found that TET could further oxidize 5hmC to 5-formaldehyde cytosine (5fC) and 5-carboxyl cytosine (5caC), respectively (52, 53). In addition, He et al. independently found that 5caC could be specifically identified and excised by TDG glycosylase as intermediates, and further restored to unmodified cytosine by Base Excision Repair (BER) pathway, thus enabling active DNA demethylation in a real sense (52). So far the
Figure 3. Divergent mechanisms of active DNA demethylation.
5mC could be iteratively oxidized into 5hmC, 5fC and 5caC by TET dioxygenases. Active DNA demethylation could be completed by TDG-removal of 5fC and 5caC, followed by base excision repair pathway. 5fC and 5caC might be converted into unmodified cytosine directly by enzymatic excision of formyl or carboxyl moieties. DNMT was reported to function as a demethylase, using 5mC, 5hmC or 5caC as substrates. 5hmC might be deaminated into 5hmU by AID or APOBEC proteins, initiating a TDG and BER-dependent DNA demethylation pathway.
mechanism of DNA demethylation has been well elucidated with the discovery of TET dioxygenases.
TET proteins belong to the superfamily of dioxygenases dependent on ferrous ions (Fe2+) and α-Ketoglutaric acid (2OG), each member of which has highly similar structural and catalytic characteristics. In the case of TET family proteins in mouse, all three members contain a cysteine rich domain, and a conserved double chain β-helix (DSBH) domain at the C-terminus, which contain the cofactor Fe (II) and 2-OG binding sites to form the core catalytic region (54) (Figure 4). Structural studies show that the core catalytic region preferentially binds to cytokines in a CpG context, but does not interact with surrounding DNA bases, and has little or no specificity for flanking DNA sequences (55). Unlike TET2, which has only one isoform, TET1 and TET3 have multiple isoforms. Among them, the full-length TET1 and TET3 have a CXXC domain at their amino terminal, which facilitates their binding to CpG islands in the genome. However, TET2 does not contain the CXXC domain, which may have been separated from the proteins due to genomic inversion during evolution, forming a gene named IDAX (also known as CXXC4) (54, 56). Compared with the full-length form of TET1 (TETfl), there is also an N-terminus-truncated form named TET1s (Figure 4). Current studies have shown that TET1s does not contain the CXXC domain and other N-terminal sequences, which resulted in a decreased ability to bind to chromatin and explains the weak demethylation activity compared with TET1fl (57). Interestingly, TET1fl and TET1s showed distinct expression patterns in different cell types. Specifically, TET1fl prefers to be highly expressed in totipotent or pluripotent cells/tissues, such as embryonic stem cells (ESCs), primordial germ cells (PGCs) and developing early embryos (before E5.5), while TET1s prefers to be present in somatic cells/tissues (57). TET3 has the most isoforms in the TET family, including full-length form (TET3fl) and two isoforms without CXXC domain, named TET3s and TET3o (58, 59) (Figure 4). TET3o is specifically expressed in oocytes, whereas TET3s and TET3fl are upregulated during neuronal differentiation (59).
In view of the important role of TET dioxygenase in DNA demethylation, great attention has been paid to the study of the physiological functions of its members. Although they share similar catalytic activity, genetic studies in mouse strongly suggest that the three members of TET proteins are non redundant in function (37), probably reflected by different expression levels in different cell types or tissues and distinct recruitment mechanisms of upstream and downstream interaction factors. For example, TET3o is highly expressed in mouse oocytes and early preimplantation embryos, but decreases rapidly in blastocysts as cells differentiated into inner cell mass (ICM). In contrast to TET3, TET1 and TET2 were highly expressed in ESCs and ICM, and then the expression of TET1 is gradually down regulated, while TET2 and TET3
remained constant or significantly up regulated in the process of differentiation to three germ layers. In addition, TET2 and TET3 also showed strong expression in a variety of adult tissues including hematopoietic and neuronal lineages, respectively. The issue of the relative importance of each TET member in a specific cell type will continue to be debated. To solve the controversy, one would at least need to systematically assess the absolute protein level of each TET member at different tissue and developmental stages.
Figure 4. Diagram for domain structure of mouse TET proteins.
All three TET members contain a cysteine rich domain, and a conserved double chain β-helix (DSBH) domain at the C-terminus, which contain the cofactor Fe (II) and 2-OG binding sites to form the core catalytic region. The DSBH domain contains a large low-complexity region of unknown function. TET1fl and TET3fl have a CXXC domain at their N-terminal, which facilitates the binding to CpG islands in the genome, whereas TET2 lack such domain. There is an N-terminus-truncated TET1 isoform (TET1s) and two N-terminus-truncated TET3 isoforms (TET3o and TETs) without CXXC domain has been reported.
Furthermore, the interaction partners of TET proteins may contribute to differentially regulate their activities and target specific regions of the genome, as shown in the following studies. Three studies in mouse ESCs (mESCs) reported that PR domain zinc finger protein 14 (PRDM14) (60), Polycomb repressive complex 2 (PRC2) (61) and LIN28A (62) could recruit and interact with TET proteins directly. Similarly, another study in mESCs showed that TET1 and TET2 had direct physical interaction with the pluripotency factor NANOG, and the knockout of NANOG resulted in the reduction of the binding of TET1 to the NANOG-bound regions in the genome (63). This work therefore suggests that TET1 can be recruited by NANOG to its target regions and that the complex plays a role in maintaining the pluripotency of mESCs. In
addition to pluripotent stem cells, the recruitment of TET protein also occurs in the process of cell trans-differentiation, immortal cancer cells and tissues. For instance, in the process of monocyte differentiation to osteoclast, TET2 is recruited to the target region through interaction with PU.1, resulting in demethylation of the target gene (64). Another example comes from the trans-differentiation of 3T3-L1 fibroblasts into adipocytes, TET protein-mediated DNA demethylation occurs in the methylated CpG context around the peroxisome proliferator-activated receptor-γ (PPARγ) and CCCTC binding factor (CTCF) binding sites, which is suggested to be mediated by the interaction between TET and PPARγ or between TET and CTCF (65, 66, 67). In addition, two studies on acute myeloid leukaemia (AML) showed that TET2 can be recruited by transcription factor WT1 to the target region through their direct interaction (68, 69). For TET3, a study on mouse retina identified the RE1-silencing transcription factor (REST) as an interaction partner to recruit TET3 to the target region of REST, resulting in target gene demethylation (70). In summary, these studies suggest that the interaction partners of TET proteins are the key transcription factors in many cases, which contribute to the recruitment and positioning of TET in the genome.
The function and physiological significance of TET dioxygenase in development has been investigated using genetic knockout mouse models. In 2011, Gu et al. first reported the role of TET3 in active DNA demethylation of mouse fertilized eggs. In the fertilized eggs with TET3 maternal deletion, some male prokaryotic genomic sites such as Nanog and LINE-1 could not be effectively demethylated, which eventually led to a significant increase in the probability of embryo degeneration after implantation (38). Further work showed that TET3 deletion not only affects male pronucleus, but also directly affects demethylation of some genomic sites in female pronucleus (39). TET1 knockout directly affects adult neurogenesis and cognitive process in mice (71). In TET triple-knockout (TKO) MEF cells, the demethylation of genes related to microRNA expression was blocked, resulting in the blockage of mesenchymal-epithelial transformation (MET) process, and ultimately, the MEF cells could not be effectively reprogrammed into iPS cells (72). In addition, Dai et al. found that TET TKO mouse embryos developed abnormally to E6.5. The mechanism was that demethylation of the enhancer region of Left2, a key factor in the Lefty-Nodal signaling pathway, was blocked, which resulted in a significant decrease in the expression level of Lefty2, leading to a blockade in the development of mouse embryos on E6.5 (73). The above evidence suggests that TET dioxygenase and its mediated DNA oxidative demethylation play an important role in the development of life by directly participating in a variety of physiological processes.
Target-specific DNA demethylation by Epigenetic editing
At present, there is growing evidence that in many types of diseases, epigenetic silencing is often the cause for abnormal gene inactivation (74, 75, 76, 77). This includes aberrant DNA hypermethylation around the transcription start site and exon 1, which often results in the inhibition of gene expression at this locus (24, 25). Furthermore, quantitative analysis of DNA methylation levels for a specific target has been used as a diagnostic/prognostic marker in clinical practice (78, 79). Therefore, it can be proposed that removal of aberrant DNA hypermethylation marks, at least from the target gene TSS and around exon 1, could lead to the reactivation of the expression of the epigenetically silenced gene. The methods of reactivating target gene expression by targeted DNA demethylation will provide a promising option for developing targeted epigenetic interventions in biomedical research.
Epigenetic editing refers to the introduction or removal of chromatin marks at a defined genomic region to regulate the expression of endogenous genes in a gene-specific way (80, 81). Epigenetic editing can be used as an effective platform to obtain information about the relationship between epigenetic modification and gene expression, and more importantly, such information can be used in reprogramming cell fate for basic research and therapeutic applications (82, 83). The main principle of epigenetic editing is the fusion of a gene specific DNA binding domain to an epigenetic effector domain with specific catalytic activity. Specifically, first, the epigenetic effector domain is directed to specific regions of the genome (such as near the TSS of the gene of interest) by the DNA binding domain, and then, depending on the epigenetic effector domain, to act on a substrate of the chromatin near the location, resulting in targeted writing or erasing of epigenetic marks, ultimately modulating the expression of the target gene (Figure 5).
For gene-specific DNA targeting, various binding platforms are available, including zinc finger proteins (ZFPs), triple helix forming oligonucleotides (TFOs) (84), transcription activation-like effector (TALE) domains (85) and the system of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated (Cas) proteins (86). These platforms act as customized DNA-binding domains (DBDs), which are fused to epigenetic effector domains with specific catalytic activity to manipulate epigenetic marks at specific locations in the genome. The results of epigenetic rewriting and/or erasure lead to the reconfiguration of local chromatin structure, which may cause the change of gene expression for a long time (87, 88). In this thesis, the zinc finger proteins will be selected to recognize and bind to target DNA sequences of interest, considering that zinc finger proteins have low toxicity to host cells (89). As one zinc finger recognizes three to four base pairs of DNA, six zinc fingers can be stitched together to target 18 bps, a unique sequence in the genome (90).
Figure 5. Schematic representation of Epigenetic Editing.
Epigenetic editing refers to the directed erasion (left panel) or rewriting (right panel) of DNA methylation (Black circle) or histone marks (Black five-pointed star) at specific genomic loci by using targeted Epi-Effectors, including de novo designed DNA binding domains (Irregular purple represents zinc finger, TAL effector, or modified CRISPR/Cas9 complex) and effector domains with specific catalytic activity (oval in yellow, blue, green or orange). The effector domain refers to a full-length protein or its catalytic domain that exists naturally and has specific catalytic activity. It is used to create the required epigenetic mark on the chromatin near the target site without altering the DNA sequence.
We first set out to confirm demethylase activity of some candidate demethylases to be further exploited as epigenetic effector domains with DNA demethylation activity based on the above description. These candidates include the catalytic domains of TET dioxygenase family proteins, AID, Apobec1 and Gadd45a. We were the first to assess the effect of targeted gene demethylation, resulting in re-activation or upregulation of expression of the targeted gene (91, 92). The approach of targeted demethylation has been taken to other DNA binding platforms (TALEs (93); CRISPR (94, 95, 96)) and tested in animal disease models to demonstrated therapeutic effects (97, 98, 99).
TET homologues and DNA modification in Chlamydomonas reinhardtii
In recent years, the activity of TET in lower eukaryotes Naegleria gruberi (100) and Coprinopsis cinerea (101) has been reported. They all have the ability to oxidize 5mC to 5caC. It is noteworthy that TET homologues were also found in the lower eukaryotes Chlamydomonas reinhardtii (C. reinhardtii), through alignment with TET/JBP conserved
domains (102). However, the enzymatic activity, function and catalytic mechanism of TET homologoues in lower eukaryotes have not been well elucidated.
Figure 6. A schematic of a Chlamydomonas reinhardtii cell.
Model organism Chlamydomonas is used to study various cellular processes and the response modes and mechanisms of organisms to the environment. A cartoon sketch of the longitudinal section of Chlamydomonas cells shows that it contains a large cup-shaped chloroplast, the anterior flagella rooted in basal bodies, and there is an array for intraflagellar transport (IFT) particle between the axoneme and flagellar membrane, a red eyespot, central nucleus and other organelles.
Protozoa C. reinhardtii is an eukaryotic unicellular green algae with a diameter of about 10 um, possessing photoautotrophic and heterotrophic abilities. C. reinhardtii contains a large cup-shaped chloroplast necessary for photosynthesis, and two flagella are formed at the extracellular pole to move or mate (Figure 6). Therefore, C. reinhardtii has the characteristics of both animal cells and terrestrial plant cells. It is often used as a model organism to study the mechanism of photosynthesis, flagella assembly and the pathological features of functional loss, circadian rhythm and gametogenesis. In recent years, C. reinhardtii has also been used to develop renewable bio-energy as it can accumulate triglycerides under nitrogen deficiency conditions (103). Evolutionally, C. reinhardtii branched from terrestrial plants and animals about a billion years ago (Figure 7). In 2007, Merchant et al. first reported whole genome sequencing of C. reinhardtii. It has a haploid genome consisting of 17 chromosomes, about 123-megabase (Mb) and 64% GC content (104). In addition, C. reinhardtii also contains about 80-100 copies of the chloroplast genome, each of which is about 200 kb
in size (104).
Figure 7. Phylogenic tree analysis of several model organisms.
When nitrogen sources are abundant, C. reinhardtii exists as vegetative cells and undergoes asexual reproduction through mitosis. However, when nitrogen sources are scarce, vegetative cells are induced to form gametes and reproduce sexually through meiosis (105). C. reinhardtii can be divided into two mating types, mating type plus (MT+) and mating type minus (MT-) according to the difference of genes contained in a DNA fragment of about 300 kb on chromosome 6 (106). When the MT+ and MT- gametes come into contact, the flagella become entangled gradually, and the cells fuse gradually to form a thick spore wall to resist the harsh environment. When the spore matures, if the extracellular environment is suitable (e.g. sufficient nitrogen source), it will germinate gradually and form four vegetative cells through meiosis, of which two are MT+ and two are MT- cells (Figure 8) (105). It is worth mentioning that in this process, both mitochondria and chloroplasts undergo gender-specific inheritance. Among them, the chloroplast genome in zygote comes from MT+ gamete, while the mitochondrial genome comes from MT- gamete.
There has been evidence suggesting that the uniparental inheritance of organelles in C. reinhardtii during sexual reproduction may be related to the methylation profile of organelle genome. As eukaryote, C. reinhardtii has unique DNA methylation profiles, including 5mC and N6-methyldeoxyadenosine (6mA), which are both about 0.5% of the respective base(107, 108). The 5mC content of C. reinhardtii nucleus genome is low
Figure 8. Life cycle of Chlamydomonas reinhardtii.
Haploid vegetative cells with two mating types (MT+ and MT-) are duplicated by mitosis when the supply of exogenous nitrogen is sufficient. On the contrary, when the supply of exogenous nitrogen is limited, the two mating types of vegetative cells differentiate into gametes (G+ and G-) and expression of mating type specific gamete traits. When gametes are mixed, the plus and minus agglutinins displayed on their flagellar surfaces mediate the initial adhesion reaction; adhesion generates a rise in intracellular cAMP which triggers gamete cell wall release and mating-structure activation; nuclei fuse, flagella are resorbed, and a thick cell wall is assembled around the zygote. Under laboratory conditions, the zygote will germinate in the nitrogen-containing medium with restored light after dormancy in darkness for 5 days, then release 4 haploid vegetative cells (2 MT+ and 2 MT-) by meiosis, and resume vegetative growth again when the nitrogen source is supplied.
but it remains relatively constant throughout its life cycle. The content of 5mC in chloroplast increases rapidly after gamete formation. Especially in the MT+ gametes, the content of 5mC is higher (12% vs 4%), which is also thought to be related to the uniparental inheritance of chloroplasts (107). However, follow-up studies showed that even the absence of methylation in MT+ gamete chloroplasts or hypermethylation in MT- gamete chloroplasts did not affect their uniparental inheritance (109, 110). Therefore, the detailed mechanism of this uniparental inheritance needs to be further elaborated. In addition, the methylation level of chloroplasts DNA continue to increase
after zygote formation, reaching more than 50% after spore maturation, suggesting that 5mC must play an important role in gamete induction and zygote formation. Recent studies have shown that 6mA is mainly distributed near Transcription Start Sites (TSS) and positively correlated with gene expression in C. reinhardtii (108). However, the functional studies on DNA methylation (including 5mC and 6mA) are still scarce, so it is not clear what physiological significance DNA methylation has in C. reinhardtii. In addition, there is still insufficient biochemical evidence to identify methyltransferases producing 5mC and 6mA, and investigation of DNA demethylation in C. reinhardtii is warranted. We here set out to understand the biological significance of DNA methylation in C. reinhardtii by studying the function of TET homologues and its effect on 5mC.
OUTLINE OF THIS THESIS
First, in many types of human diseases, the abnormal inactivation of genes (such as tumor suppressor genes) is often due to epigenetic silencing (74, 75, 76, 77). In view of the fact that an important feature of epigenetics is its reversibility, epigenetic editing has been put forward as novel treatment strategy. It is expected that by removing (or reducing) DNA methylation marks in promoter regions, epigenetic silenced gene expression can be reactivated.
Unfortunately, at the start of our studies no active demethylase was available and active demethylation was even though to be impossible. In order to disprove this hypothesis, in Chapters 2 and 3 of this thesis, we explored to induce expression of the epigenetically silenced ICAM-1 and EpCAM genes in ovarian cancer cells through Epigenetic Editing by targeting putative DNA demethylases candidates to the target genes. These candidates include deaminase Aid and Apobec1 and NER pathway component Gadd45a. Upon the discovery of TET proteins, we immediately also compared the three members of this family for targeted demethylation efficiency. Zinc finger proteins that specifically target the TSSes of epigenetically silenced genes ICAM-1 and EpCAM were fused to the catalytic domains of the putative “demethylases”. Subsequently, the fusion proteins were expressed in human ovarian cancer cell line A2780 and its effects were measured. So, we set out to validate the demethylase activity of enzymes, while providing a mechanism to induce targeted DNA demethylation, which would facilitate re-activation of expression of disease-associated target genes.
Second, in recent years, with the discovery of TET dioxygenase and the revelation of its catalytic mechanism and physiological function, the mechanism of DNA demethylation has started to be elucidated. These findings not only confirm the existence of a complete active DNA demethylation pathway, but also imply that besides
5mC as a stable epigenetic mark with physiological functions, its oxidized products 5hmC, 5fC and 5caC (especially 5hmC) can act as new epigenetic marks with physiological functions (111). However, current studies on the catalytic mechanism, physiological function and product function of TET dioxygenase are concentrated in higher mammals, while little is known about the catalytic properties and physiological functions of TET homologues in lower eukaryotes. Evolutionally, higher mammals evolved from lower eukaryotes. So, in chronological order, it is likely that TET homologue proteins were produced in lower eukaryotes first, and then evolved to TET dioxygenase in higher mammals. Therefore, exploring the catalytic properties and physiological functions of TET homologous in earlier evolutionary stages can not only help us to better understand the evolutionary history of these dioxygenases, but also provide a new insight into the mechanism of DNA demethylation. For these purposes, in Chapter 4, we explored the catalytic properties and physiological functions of TET homologous in C. reinhardtii, a model organism with a billion-year evolutionary history.
Third, based on bioinformatics analysis, we identified eight TET homologues in C. reinhardtii (CrTETs), all of which possess conserved HxD motif in dioxygenase for binding to Fe2+. However, their binding sites with 2-OG are not conserved, suggesting that there are some differences in the function between them and mammalian TETs. In the previous study of Chapter 4, we found that CrTET1 can modify 5mC to produce two novel stereoisomeric DNA products in vitro (called 5-glyceryl-methylcytosine, 5gmC). Therefore, it is necessary to verify that CrTET1 can also catalyze the production of 5gmC from 5mC in C. reinhardtii, and to further reveal the physiological functions of CrTET1 and its catalytic product 5gmC. In order to achieve these goals, it is urgent to obtain CrTET1 mutants as a necessary research material. However, for a long time, there has been a lack of effective targeted gene editing tools for nuclear genes in C. reinhardtii, which greatly limits the study of unknown functional genes. Although a few labs have reported that editing of known phenotypic genes can be achieved by directly delivering Cas9-gRNA ribonucleoproteins (RNPs) into C. reinhardtii (112, 113, 114), effective genetic modification to obtain mutants for non-phenotypic target genes has remained a technical hurdle to be overcomed. On the one hand, it is still difficult to accurately predict high efficient sgRNA target sites. On the other hand, differences in genomic microenvironment and repair preferences among different organisms can also lead to differences in editing efficiency. Therefore, any effort to improve the efficiency of CRISPR/Cas9-mediated gene editing is remarkable. In view of this problem, we need to establish an effective platform for target gene editing and mutant isolation in C. reinhardtii.
technique based on CRISPR/Cas9 technology to effectively obtain mutants for candidate target genes in C. reinhardtii. The specific outline is described as follows. 1), Selection of target genes that can be used as co-selection marker through literature research. 2), the mutant yield of each co-selection marker candidate gene is tested by delivering RNPs to CC-125 C. reinhardtii. A target gene with high mutant yield will be selected as co-selection marker gene. 3), Selection of genes that have been reported to produce distinct phenotypes after loss-of-function as target genes to test the efficiency of co-selection strategies. 4), Selection of genes that have not been reported and cannot predict phenotype after loss-of-function as target genes to test the feasibility of co-selection strategies. Firstly, we test whether the target gene mutants produced by Non-homologous end-joining (NHEJ) inaccurate repair pathway can be obtained, and then further test the target gene mutants produced by homology-directed repair (HDR) precise repair pathway when providing a homologous repair donor DNA for the target site.
Taken together, this thesis aims to contribute to i ) explore specifically targeted DNA demethylation for epigenetically silenced genes through epigenetic editing-mediated targeting of potential candidates of "demethylase" effector domains to the hypermethylated core promoter region of the target genes; ii ) better understand the evolutionary history of TET dioxygenases and provide a new insight into the mechanism of DNA demethylation, exploring the catalytic properties and physiological functions of TET homologues in a single-cell eukaryote C. reinhardtii; iii ) improve the efficiency of gene editing and mutant isolation in C. reinhardtii, by developing two methods based on co-selection strategy and microhomology-mediated donor DNA integration strategy with targeted integration-dependent screening processes.
Finally, the results obtained and described in this thesis are summarized and discussed in Chapter 6. In addition, considerations for future research will be also presented there.
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