Restriction-Modification and CRISPR-Cas Systems: Cooperation Between Innate and Adaptive Immunity in Prokaryotes

Bacteria have evolved numerous mechanisms to resist the constant assault of viruses (called bacteriophages, or simply phages) that can infect and kill them. Restriction-modification (RM) systems represent one such strategy. Generally, these systems provide defense by coordinating the activities of two distinct enzymes: a restriction endonuclease and a methyltransferase. Both enzymes recognize the same short DNA sequences. The methyltransferase modifies these target sites in the host chromosome, which prevents the restriction endonuclease from cleaving the host’s own DNA. In contrast, foreign phage DNA is usually not methylated at these sequences. Consequently, upon injection into the host, the viral DNA is recognized and cleaved by the restriction endonuclease, preventing the progression of the phage’s life cycle. Therefore, RM systems are considered a part of the innate immune response because they can provide defense against any phage, including ones that have never been encountered previously, as long as they harbor RM target sites. Clustered regularly interspaced short palindromic repeats (CRISPR) loci and their associated genes (cas) form another defense system that destroys foreign DNA. The CRISPR array consists of a series of repetitive DNA sequences separated by unique DNA sequences known as spacers. During phage infection, short DNA fragments are taken from the viral DNA and integrated into the CRISPR locus to form new spacers. These sequences are then transcribed into CRISPR RNAs (crRNAs). In type II-A CRISPRCas systems, the crRNAs guide the Cas9 nuclease to a matching viral DNA target for cleavage. As such, unlike RM systems, CRISPR-Cas systems represent an adaptive immune response because they require an initial exposure to a virus in order to become successfully immunized through the acquisition of new spacer sequences. CRISPR-Cas and RM are two of the most prevalent types of defense systems found in bacteria and often co-exist together in a single host. Yet, how they may interact with each other in the context of immunity during bacteriophage infection is poorly understood. Here, in my thesis work, I investigate the interplay between RM and type II-A CRISPR-Cas systems. First, I demonstrate that RM systems provide a weak and temporary protection that stimulates CRISPR spacer acquisition, enabling the cells to survive the viral infection. Then, I go on to show that the restriction activity of the RM system is critical for this process and that the rate of spacer acquisition is correlated to the number of RM target sites in the phage genome. To further uncover the mechanistic link between restriction and the acquisition of new spacers, I implement next-generation sequencing to demonstrate that spacers are preferentially extracted at the dsDNA breaks (DSBs) generated by the restriction endonuclease. Additionally, I show that the host DNA repair complex, AddAB, can process these breaks, which further enhances spacer acquisition. Finally, I follow the dynamics between RM and CRISPR-Cas during the chain of events that occur upon viral infection. I demonstrate that although the RM system provides an immediate line of defense due to its ability to recognize a broad range of foreign invaders, it is ultimately overcome by the rapid emergence of methylated phages, resulting in the death of much of the bacterial population. However, the early RM immune response creates substrates for spacer acquisition by the CRISPR-Cas system in a subset of cells. By using these newly acquired spacers which specify the viral sequences for lethal cleavage by Cas9, these cells can now extinguish the methylated phages, resulting in the survival and regrowth of the population. Collectively, my thesis reveals the molecular mechanisms connecting RM and CRISPR-Cas systems in providing a synergistic anti-phage defense. Reminiscent of eukaryotic immunity, I demonstrate that RM systems provide an initial, short-lived innate immune response, which stimulates a secondary, more robust adaptive immune response by CRISPR-Cas. This work highlights an example of cooperation between RM and CRISPR-Cas, which are two of the most common bacterial defense systems. However, prokaryotes have been shown to harbor a multitude of other putative antiphage defense systems, which can often exist together in a single host. I predict that future studies will likely uncover many more fascinating instances of immune interaction among other sets of defense systems

Mechanisms of DNA Modification-Dependent Regulation in Gram-Positive Bacteria

The presence of DNA modifications is pervasive among both prokaryotic and eukaryotic species. In bacteria, the study of DNA methylation has largely been in the context of restriction-modification systems, where DNA methylation serves to safeguard the chromosome against restriction endonucleases that are intended to cleave invading foreign DNA. There has been a growing recognition that the methyltransferase component of restriction-modification systems can also function in the regulation of gene expression. Outside of restriction modification systems, DNA methylation from orphan methyltransferases, which lack cognate restriction endonucleases, have been shown to regulate critical cellular processes. The majority of research articles focuses on the epigenetic regulatory roles of bacterial DNA methylation in the context of Gram-negative bacteria, with particular bias towards Escherichia coli, Caulobacter crescentus, and related Proteobacteria. Despite the critical functions of DNA methylation in Gram-negative bacteria, far less is known about how DNA methylation contributes to epigenetic regulation of gene expression in Gram-positive bacteria. In this thesis I investigated the effects of DNA modifications in Gram-positive bacteria. I showed that DNA methylation from an active Type I restriction-modification system in Streptococcus pyogenes also functions in the epigenetic regulation of a small subset of virulence genes, all of which are significantly down regulated in the absence of DNA methylation. Moreover, I showed that the methylation-dependent decrease in gene expression results in attenuated virulence of an S. pyogenes clinical isolate, implicating DNA methylation as an important contributor to S. pyogenes pathogenesis. I also characterized the methylomes for two strains of the Gram-positive Firmicute Bacillus subtilis and demonstrated that DNA methylation regulates the expression of a small subset of genes involved in chromosome structure and maintenance. I further identified a methylation-sensitive transcriptional regulator, providing some of the first insight into the mechanisms of methylation-dependent gene regulation in Gram-positive bacteria. Finally, I identified a previously uncharacterized gene, rnhP, which is a plasmid encoded RNase HI. I found that RnhP contributes to genome maintenance in B. subtilis NCIB 3610 by removing RNA-DNA hybrids with four or more ribonucleotides embedded in DNA. I showed that RnhP does not contribute to plasmid maintenance or hyper-replication. Importantly, I showed that RnhP contributes to genome maintenance by allowing DNA replication forks to progress through the terminus region. Together, my work highlights the importance of DNA modifications and noncanonical nucleotides in Gram-positive bacteria and provides a framework for future studies of epigenetic regulation by RM systems in bacterial pathogenesis and development.PHDMolecular, Cellular, and Developmental BiologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/163073/1/tnye_1.pd

Towards modification of Medicago truncatula epigenome: genome editing with engineered nucleases

Periodic drought is the primary limitation of plant growth and crop yield. The rise of water demand caused by the increase in world population and climate change, leads to one of the biggest challenges of modern agriculture: to increase food and feed production. De novo DNA methylation is a process regulated by small interfering RNA (siRNAs), which play a role in plant response and adaptation to abiotic stress. In the particular case of water deficit, growing evidences suggest a link between the siRNA pathways and drought response in the model legume Medicago truncatula. As a first step to understand the role of DNA methylation under water stress, we have set up several bioinformatics and molecular methodologies allowing the design of Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 systems and the assembly of TALENs (transcription activator-like effector nucleases), to target both dicer-like 3 (MtDCL3) and RNA-Dependent RNA polymerase (MtRDR2), enzymes of the RNA-directed DNA methylation pathway. TALENs efficiency was evaluated prior to plant transformation by a yeast-based assay using two different strategies to test TALENs activity: Polyacrylamide gel electrophoresis (PAGE) and Single strand conformation polymorphisms (SSCP). In this assay, yeast cells triple transformation emerged as good and rapid alternative to laborious yeast mating strategies. PAGE analysis might be a valuable tool to test TALENs efficacy in vivo if we could increase TALENs activity. SSCP-based approach proved to be ineffective due to the generation of several false positives. TALENs and CRISPR/Cas9 system constructed and designed in this work will in the future certainly enable the successful disruption of DCL3 and RDR2 genes and shed the light on the relationship between plant stress resistance and epigenetic regulation mediated by siRNAs in M.truncatula

Development of SNAP-tag-based fusion proteins targeting HIV-1 viral reservoirs

Background Globally, the HIV/AIDS epidemic has cost over 35 million lives and approximately a further 37 million people are currently infected with HIV. In South Africa alone, more than 7 million people are HIV positive. Since the initiation of combination antiretroviral therapy (cART), viral replication can be supressed below the limit of detection by conventional testing. There is, however, no approved therapy for the cure of HIV. This is because HIV establishes viral reservoirs in memory CD4+ T-cells, where replication is low or arrested, allowing prolonged survival. Since there is little or no replication, a therapeutic strategy which targets the viral production and replication becomes ineffective and upon cessation of antiretroviral therapy a dramatic viral relapse occurs. The eradication of HIV, therefore, requires the targeted killing of the reservoir cells, or latency reversal followed by the prevention of further infection using cART. Targeting of cell-surface antigens for therapeutic purposes is the basis of immunotherapy. FDA-approved monoclonal antibodies such as Trastuzumab have been used to treat breast cancer via the human epidermal growth factor 2 (HER2) receptor. Immunotoxins (ITs) composed of an antibody fragment fused to apoptosis-inducing protein toxins targeting cellsurface antigens have been used for therapy of refractory leukaemia. The anti-CD22 recombinant IT Moxetumomab pasudotox based on Pseudomonas aeruginosa exotoxin A (ETA) has been FDA approved to treat hairy cell leukaemia. Moxetumomab pasudotox targets the antigen CD22 found on the surface of tumour cells. The HIV neutralizing VHH-nanobody J3, isolated from an immunised Llama has demonstrated anti-HIV properties against more than 95 % of HIV strains in vitro. As part of an ongoing project to develop a J3-ETA IT, this work sought to produce a J3-SNAP fusion protein by osmotic stress expression in the presence of compatible solutes in the periplasmic space of E. coli. SNAP-tag is a self-labelling protein that covalently binds benzylguanine (BG)-modified substrates in a 1:1 stoichiometric ratio. When recombinantly fused to any protein of interest, SNAP-tag allows the stable labelling of the protein of interest of in vitro and in vivo imaging. The periplasmic space of bacteria has been reported as a dedicated compartment to express functional proteins of interest. Furthermore, osmotic stress expression in the presence of compatible solutes has been reported to result in up to a thousand-fold increase in protein yield for difficult to express proteins. This study ultimately aimed to understand whether a functional J3-SNAP or J3-ETA can be expressed under osmotic stress in the presence of compatible solutes, in the periplasmic space of E. coli. 11 Experimental work In this study, a SNAP-tag-based fusion protein and an ETA-based IT were designed using J3, an anti-HIV-1 Env VHH-nanobody isolated from an immunised llama. Using the SnapGene® software (v.5.0.8, GSL Biotech LLC, USA), in silico design and cloning of an ETA-based IT J3-ETA and SNAP-tag-based fusion protein J3-SNAP was performed. Molecular cloning of designed open reading frames (ORFs) was performed into appropriate bacterial expression plasmid vectors. Plasmid vectors confirmed to contain the required ORFs by Sanger sequencing were transformed into E. coli BL21-DE3. Histidine-tagged J3-SNAP was expressed by osmotic stress in the presence of compatible solutes. J3-SNAP was purified by IMAC and assessed by SDS-PAGE and Western blot analysis. To ascertain the binding of J3- SNAP to cells expressing HIV-1 Env in vitro, recombinant Env protein was transiently transfected into HEK293T-cells to generate an Env expressing cell line. Cell-surface binding of SNAP-Surface® Alexa Fluor® 488 -conjugated J3-SNAP on Env expressing HEK293Tcells was assessed by confocal microscopy analysis. Results Successful expression of J3-SNAP in E. coli BL21-DE3 was confirmed by SDS-PAGE and Western blot analysis. The J3-SNAP fusion protein was subsequently purified by IMAC. Purified J3-SNAP was conjugated to the benzyl guanine-modified fluorophore SNAPSurface® Alexa Fluor® 488 and full-length conjugated protein was confirmed by combinations of SDS-PAGE and Western blot analysis. Cell-surface binding of J3-SNAP to HIV-1 Env-expressing HEK293T-cells was demonstrated in vitro by confocal microscopy analysis. These results prompted the generation of the IT, J3-ETA, by replacing SNAP-tag with ETA. Conclusion Successful binding studies suggest using J3 to target HIV-1 Env. Accessing patient probes would allow for the confirmation of these results for future human applications. Future in vitro studies would need to confirm the selective elimination of Env expressing T-cells by J3-ETA and thereafter confirmed on Env-positive patient probes

Common patterns in type II restriction enzyme binding sites

Restriction enzymes are among the best studied examples of DNA binding proteins. In order to find general patterns in DNA recognition sites, which may reflect important properties of protein–DNA interaction, we analyse the binding sites of all known type II restriction endonucleases. We find a significantly enhanced GC content and discuss three explanations for this phenomenon. Moreover, we study patterns of nucleotide order in recognition sites. Our analysis reveals a striking accumulation of adjacent purines (R) or pyrimidines (Y). We discuss three possible reasons: RR/YY dinucleotides are characterized by (i) stronger H-bond donor and acceptor clusters, (ii) specific geometrical properties and (iii) a low stacking energy. These features make RR/YY steps particularly accessible for specific protein–DNA interactions. Finally, we show that the recognition sites of type II restriction enzymes are underrepresented in host genomes and in phage genomes

Systematic prediction of DNA shape changes due to CpG methylation explains epigenetic effects on protein–DNA binding

Background DNA shape analysis has demonstrated the potential to reveal structure-based mechanisms of protein–DNA binding. However, information about the influence of chemical modification of DNA is limited. Cytosine methylation, the most frequent modification, represents the addition of a methyl group at the major groove edge of the cytosine base. In mammalian genomes, cytosine methylation most frequently occurs at CpG dinucleotides. In addition to changing the chemical signature of C/G base pairs, cytosine methylation can affect DNA structure. Since the original discovery of DNA methylation, major efforts have been made to understand its effect from a sequence perspective. Compared to unmethylated DNA, however, little structural information is available for methylated DNA, due to the limited number of experimentally determined structures. To achieve a better mechanistic understanding of the effect of CpG methylation on local DNA structure, we developed a high-throughput method, methyl-DNAshape, for predicting the effect of cytosine methylation on DNA shape. Results Using our new method, we found that CpG methylation significantly altered local DNA shape. Four DNA shape features—helix twist, minor groove width, propeller twist, and roll—were considered in this analysis. Distinct distributions of effect size were observed for different features. Roll and propeller twist were the DNA shape features most strongly affected by CpG methylation with an effect size depending on the local sequence context. Methylation-induced changes in DNA shape were predictive of the measured rate of cleavage by DNase I and suggest a possible mechanism for some of the methylation sensitivities that were recently observed for human Pbx-Hox complexes. Conclusions CpG methylation is an important epigenetic mark in the mammalian genome. Understanding its role in protein–DNA recognition can further our knowledge of gene regulation. Our high-throughput methyl-DNAshape method can be used to predict the effect of cytosine methylation on DNA shape and its subsequent influence on protein–DNA interactions. This approach overcomes the limited availability of experimental DNA structures that contain 5-methylcytosine

KEY ACHIEVEMENTS IN GENE THERAPY DEVELOPMENT AND ITS PROMISING PROGRESS WITH GENE EDITING TOOLS (ZFN, TALEN, CRISPR/CAS9)

Gene therapy concept is based on introduction of the wild-type allele into a patient’s genome in order to reverse a specific mutation. It is designed to treat hereditary diseases as well as the other diseases occurring later in life. Gene therapy was first mentioned in the 1960s and 70s, whereupon a series of studies was carried out, and in 1990 the first successful gene therapy was conducted. Since then about 2 600 clinical trials based on this concept were completed or are in progress. The two biggest issues are introduction of an exogenous DNA to target tissue, and its controlled integration in the genome. Until recently, the exogenous DNA sequences were incorporated randomly in the patient’s genome. Even though most of these treatments gave positive results, there was always a possibility of insertional mutagenesis. Controlling the integration place has rapidly progressed with the development of gene editing tools: ZFN, TALEN and CRISPR/Cas9. Although they have been used in only several clinical studies, gene editing tools are a small step away from clinical usage. In this review, we will give historical overview of gene therapy development and describe recent tools that can be used in precision medicine

Chromatin and siRNA pathways cooperate to maintain DNA methylation of small transposable elements in Arabidopsis

BACKGROUND: DNA methylation occurs at preferred sites in eukaryotes. In Arabidopsis, DNA cytosine methylation is maintained by three subfamilies of methyltransferases with distinct substrate specificities and different modes of action. Targeting of cytosine methylation at selected loci has been found to sometimes involve histone H3 methylation and small interfering (si)RNAs. However, the relationship between different cytosine methylation pathways and their preferred targets is not known. RESULTS: We used a microarray-based profiling method to explore the involvement of Arabidopsis CMT3 and DRM DNA methyltransferases, a histone H3 lysine-9 methyltransferase (KYP) and an Argonaute-related siRNA silencing component (AGO4) in methylating target loci. We found that KYP targets are also CMT3 targets, suggesting that histone methylation maintains CNG methylation genome-wide. CMT3 and KYP targets show similar proximal distributions that correspond to the overall distribution of transposable elements of all types, whereas DRM targets are distributed more distally along the chromosome. We find an inverse relationship between element size and loss of methylation in ago4 and drm mutants. CONCLUSION: We conclude that the targets of both DNA methylation and histone H3K9 methylation pathways are transposable elements genome-wide, irrespective of element type and position. Our findings also suggest that RNA-directed DNA methylation is required to silence isolated elements that may be too small to be maintained in a silent state by a chromatin-based mechanism alone. Thus, parallel pathways would be needed to maintain silencing of transposable elements