Name: targeted in situ mutagenesis of histone genes in budding yeast
Uploaded: 2017-01-26T15:00:00
Description: Watch this Scientific Journal Video about Targeted in Situ Mutagenesis of Histone Genes in Budding Yeast at JoVE.com

We thank Reine Protacio for helpful comments during the preparation of this manuscript. We express our gratitude to the National Science Foundation (grants nos. 1243680 and 1613754) and the Hendrix College Odyssey Program for funding support.

NOTE: The experimental strategy for targeted in situ histone gene mutagenesis includes several steps (summarized in Figure 1). These steps include: (1) Replacement of the target histone gene with the URA3 gene, (2) Generation and purification of PCR products corresponding to two partially overlapping fragments of the target histone gene using primers harboring the desired mutation(s), (3) Fusion PCR of the two partially overlapping fragments to obtain full size PCR products for integrat...

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The high level of sequence homology between the two non-allelic genes that code for each of the four core histone proteins in haploid S. cerevisiae cells can represent a challenge for investigators who wish to specifically target one of the two genes for mutagenesis. Previously described yeast mutagenesis methodologies, including the Delitto Perfetto, site-specific genomic (SSG) mutagenesis, and cloning-free PCR-based allele replacement methods5,6</su...

The four core histone proteins H2A, H2B, H3, and H4 play central roles in the compaction, organization, and function of eukaryotic chromosomes. Two sets of each of these histones form the histone octamer, a molecular spool that directs the wrapping of ~147 base pairs of DNA around itself, ultimately resulting in the formation of a nucleosome1. Nucleosomes are active participants in a variety of chromosome-based processes, such as the regulation of gene transcription and the formation of euchromatin and heterochromatin across chromosomes, and as such have been the focus of intense research over the course of the past several decades. A number of mechanisms have been described by which nucleosomes can be manipulated in ways that can facilitate execution of specific processes &#8211; these mechanisms include posttranslational modification of histone residues, ATP-dependent nucleosome remodeling, and ATP-independent nucleosome reorganization and assembly/disassembly2,3.
The budding yeast Saccharomyces cerevisiae is a particularly powerful model organism for the understanding of histone function in eukaryotes. This can be largely attributed to the high degree of evolutionary conservation of the histone proteins throughout the domain eukarya and the amenability of yeast to a variety of genetic and biochemical experimental approaches4. Reverse-genetic approaches in yeast have been widely used to study the effects of specific histone mutations on various aspects of chromatin biology. For these types of experiments it is often preferable to use cells in which the mutant histones are expressed from their native genomic loci, as expression from autonomous plasmids can lead to abnormal intracellular levels of histone proteins (due to varying numbers of plasmids in cells) and concomitant alteration of chromatin environments, which can ultimately confound the interpretation of results.
Here, we describe a PCR-based technique that allows for targeted mutagenesis of histone genes at their native genomic locations that does not require a cloning step and results in the generation of the desired mutation(s) without leftover exogenous DNA sequences in the genome. This technique takes advantage of the efficient homologous recombination system in yeast and has several features in common with other similar techniques developed by other groups - most notably the Delitto Perfetto, site-specific genomic (SSG) mutagenesis, and cloning-free PCR-based allele replacement methods5,6,7. However, the technique we describe has an aspect that makes it particularly well-suited for mutagenesis of histone genes. In haploid yeast cells, each of the four core histones is encoded by two non-allelic and highly homologous genes: for example, histone H3 is encoded by the HHT1 and HHT2 genes, and the open reading frames (ORFs) of the two genes are over 90% identical in sequence. This high degree of homology can complicate experiments designed to specifically target one of the two histone-encoding genes for mutagenesis. Whereas the aforementioned methods often require the use of at least some sequences within the ORF of the target gene to drive homologous recombination, the technique we describe here makes use of sequences flanking the ORFs of the histone genes (which share much less sequence homology) for the recombination step, thus increasing the likelihood of successful targeting of mutagenesis to the desired locus. Moreover, the homologous regions that drive recombination can be very extensive, further contributing to efficient targeted homologous recombination.

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</table>

targeted in situ mutagenesis of histone genes in budding yeast

We describe a PCR- and homologous recombination-based system for generating targeted mutations in histone genes in budding yeast cells. The resulting mutant alleles reside at their endogenous genomic sites and no exogenous DNA sequences are left in the genome following the procedure. Since in haploid yeast cells each of the four core histone proteins is encoded by two non-allelic genes with highly homologous open reading frames (ORFs), targeting mutagenesis specifically to one of two genes encoding a particular histone protein can be problematic. The strategy we describe here bypasses this problem by utilizing sequences outside, rather than within, the ORF of the target genes for the homologous recombination step. Another feature of this system is that the regions of DNA driving the homologous recombination steps can be made to be very extensive, thus increasing the likelihood of successful integration events. These features make this strategy particularly well-suited for histone gene mutagenesis, but can also be adapted for mutagenesis of other genes in the yeast genome.

We describe the generation of an hht2 allele expressing a histone H3 mutant protein harboring a substitution at position 53 from an arginine to a glutamic acid (H3-R53E mutant) as a representative example of the targeted in situ mutagenesis strategy.
We generated a strain in which the entire ORF of HHT2 is replaced by the URA3 gene (see step 1 of the protocol). This strain, yAAD156, also harbo...

Watch this Scientific Journal Video about Targeted in Situ Mutagenesis of Histone Genes in Budding Yeast at JoVE.com

Targeted in Situ Mutagenesis of Histone Genes in Budding Yeast

A strategy for generating mutations in histone genes at their endogenous location in Saccharomyces cerevisiae is presented.

Targeted in Situ Mutagenesis of Histone Genes in Budding Yeast

We describe a PCR- and homologous recombination-based system for generating targeted mutations in histone genes in budding yeast cells. The resulting ...

The overall goal of this procedure is to generate specific mutations in histone genes at their endogenous chromosomal locations in budding yeast cells. This method can be used to determine the contribution of specific histone residues in a variety of processes that use chromatin as the substrate, such as DNA transcription, every combination. The main advantage of this technique is that specific histone genes can be targeted for mutagenesis despite the fact that in yeast, each histone protein is encoded by two highly homologous, non-allelic genes.After generating a yeast strain with the target histone gene knocked out and replaced by URA3, generate PCR products for two partially overlapping fragments of the target histone gene by setting up the following reaction for the first half of the gene. For the second half of the gene, set up a similar PCR reaction using different primers. Place the reactions in a thermocycler and set up the following program.Run 20 to 50 microliters of the PCR reactions on a 0.9%low melting point agarose gel in TBE buffer. Then use a clean razor blade to cut the PCR products out of the gel and transfer each section to a 1.5 milliliter tube. Store the agarose pieces at 20 degrees Celsius until ready to use.To prepare template DNA for the PCR reactions to generate full size PCR products, melt the agarose sections in a heat block at 65 degrees Celsius for five minutes. Vortexing the tubes every one to two minutes to help with melting. Transfer a set amount of melted agarose from each sample into a single Microcentrifuge tube and mix the tube by vortexing.Use this as the template for fusion PCR and store at 20 degrees Celsius until use. To amplify a large quantity of full sized PCR product, set up six PCR reactions each with the following reagents. Place the tubes in the thermocycler and run the reactions with the following settings.Concentrate the PCR products through precipitation and use them for cotransformation with the backbone plasmid according to the text protocol. To screen for the loss of the URA3 gene as a result of PCR product integration, replica plate cells from plates 1 to 20 by removing the plate lid and pressing the plate containing colonies onto sterile velvet. Transfer the cells from the velvet to a 5-FOA plate by pressing the plate on the velvet.Incubate the plates at 30 degrees Celsius for two days. Then carefully inspect the plates for growth. After streaking and incubating candidate colonies on YPD plates according to the text protocol, replica plate each YPD purification plate to a fresh YPD plate, to a dropout plate lacking uracil to check for the loss of the URA3 gene, and a second dropout plate to monitor for the presence or absence of the backbone plasmid.Following incubation, identify a colony from each candidate sample that is growing on the YPD plate but not growing on either dropout plate. Restreak these colonies onto fresh YPD plates. As say for proper integration of the mutant allele using PCR according to the text protocol.This figure shows the PCR results for generating two partially overlapping HHT2 fragments with the desired mutations that ultimately result in an AGA to GAA codon change producing an R53E mutation. The result from a fusion PCR reaction that generated the full sized PCR products is illustrated here. The full sized PCR products contained 195 and 220 base pair regions homologous to the regions upstream and downstream of the HHT2 open reading frame respectively to drive homologous recombination.In this example transformation, the full sized PCR products were cotransformed with the HIS-3 marked plasmid and transformants were selected on plates lacking histidine. HIS positive colonies were then screened for 5-FOA resistance. Examples of candidates as well as samples unlikely to represent the desired integration events are shown.12 candidate samples were identified and subjected to PCR analysis for replacement of the URA3 gene with mutant PCR products. Four candidates showed integration of a PCR product at the correct location. One of these candidates in shown in lane four.Since the AG2GA mutation generates a new EcoR1 restriction site, digestion with EcoR1 was carried out on one of the successful integration PCR samples to demonstrate that the mutant sequence had indeed been incorporated into the genome. Once all reagents are available, it should be possible to obtain yeast cells harboring the desired histone mutations in the time frame or 15 to 20 days. Once the desired mutation has been generated in the host strain, cells expressing the target histone protein solely from the mutagenized gene can be obtained through appropriate genetic crosses.While attempting this procedure, it's important to remember that the perimeters for the PCR reactions may need to be adjusted based on the nature of the specific primers used in the experiments and the expected sizes of the PCR products. Although this procedure is well suited for histone gene mutagenesis as it allows for targeting a specific histone gene despite the presence of a second highly homologous, non allelic gene, it can also be used to generate mutations at other genomic locations. After watching this video, you should have a good understanding of how to generate targeted NC2 histone mutations in budding yeast using a combination of PCR approaches and yeast genetics techniques.Don't forget that exposure to UV light can be extremely hazardous, and protective eyewear, shields, and clothing should always be used while working with a UV transilluminator. Also wear gloves, goggles, and protective clothing throughout most steps of the procedure.

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Preparation of rAAV9 to Overexpress or Knockdown Genes in Mouse Hearts

Controlling the expression or activity of specific genes through the myocardial delivery of genetic materials in murine models permits the investigation of gene functions. Their therapeutic potential in the heart can also be determined. There are limited approaches for in vivo molecular intervention in the mouse heart. Recombinant adeno-associated virus (rAAV)-based genome engineering has been utilized as an essential tool for in vivo cardiac gene manipulation. The specific advantages of this technology include high efficiency, high specificity, low genomic integration rate, minimal immunogenicity, and minimal pathogenicity. Here, a detailed procedure to construct, package, and purify the rAAV9 vectors is described. Subcutaneous injection of rAAV9 into neonatal pups results in robust expression or efficient knockdown of the gene(s) of interest in the mouse heart, but not in the liver and other tissues. Using the cardiac-specific TnnT2 promoter, high expression of GFP gene in the heart was obtained. Additionally, target mRNA was inhibited in the heart when a rAAV9-U6-shRNA was utilized. Working knowledge of rAAV9 technology may be useful for cardiovascular investigations.

In this manuscript, a method to prepare recombinant adeno-associated virus 9 (rAAV9) vectors to manipulate gene expression in the mouse heart is described.

Controlling the expression or activity of specific genes through the myocardial delivery of genetic materials in murine models permits the investigation ...

Optogenetic Random Mutagenesis Using Histone-miniSOG in C. elegans

Forward genetic screening in model organisms is the workhorse to discover functionally important genes and pathways in many biological processes. In most mutagenesis-based screens, researchers have relied on the use of toxic chemicals, carcinogens, or irradiation, which requires designated equipment, safety setup, and/or disposal of hazardous materials. We have developed a simple approach to induce heritable mutations in C. elegans using germline-expressed histone-miniSOG, a light-inducible potent generator of reactive oxygen species. This mutagenesis method is free of toxic chemicals and requires minimal laboratory safety and waste management. The induced DNA modifications include single-nucleotide changes and small deletions, and complement those caused by classical chemical mutagenesis. This methodology can also be used to induce integration of extrachromosomal transgenes. Here, we provide the details of the LED setup and protocols for standard mutagenesis and transgene integration.

Optogenetic Random Mutagenesis Using Histone-miniSOG in C. elegans

Genetically-encoded histone-miniSOG induces genome-wide heritable mutations in a blue&#160;light-dependent manner. This mutagenesis method is simple, fast, free of toxic chemicals, and well-suited for forward genetic screening and transgene integration.

Forward genetic screening in model organisms is the workhorse to discover functionally important genes and pathways in many biological processes. In most ...

CRISPR/Cas9-mediated Targeted Integration In Vivo Using a Homology-mediated End Joining-based Strategy

As a promising genome editing platform, the CRISPR/Cas9 system has great potential for efficient genetic manipulation, especially for targeted integration of transgenes. However, due to the low efficiency of homologous recombination (HR) and various indel mutations of non-homologous end joining (NHEJ)-based strategies in non-dividing cells, in vivo genome editing remains a great challenge. Here, we describe a homology-mediated end joining (HMEJ)-based CRISPR/Cas9 system for efficient in vivo precise targeted integration. In this system, the targeted genome and the donor vector containing homology arms (~800 bp) flanked by single guide RNA (sgRNA) target sequences are cleaved by CRISPR/Cas9. This HMEJ-based strategy achieves efficient transgene integration in mouse zygotes, as well as in hepatocytes in vivo. Moreover, a HMEJ-based strategy offers an efficient approach for correction of fumarylacetoacetate hydrolase (Fah) mutation in the hepatocytes and rescues Fah-deficiency induced liver failure mice. Taken together, focusing on targeted integration, this HMEJ-based strategy provides a promising tool for a variety of applications, including generation of genetically modified animal models and targeted gene therapies.

CRISPR/Cas9-mediated Targeted Integration In Vivo Using a Homology-mediated End Joining-based Strategy

The clustered regularly interspaced short palindromic repeats/CRISPR associated protein 9 (CRISPR/Cas9) system provides a promising tool for genetic engineering, and opens up the possibility of targeted integration of transgenes. We describe a homology-mediated end joining (HMEJ)-based strategy for efficient DNA targeted integration in vivo and targeted gene therapies using CRISPR/Cas9.

As a promising genome editing platform, the CRISPR/Cas9 system has great potential for efficient genetic manipulation, especially for targeted integration ...

Gene-targeted Random Mutagenesis to Select Heterochromatin-destabilizing Proteasome Mutants in Fission Yeast

Random mutagenesis of a target gene is commonly used to identify mutations that yield the desired phenotype. Of the methods that may be used to achieve random mutagenesis, error-prone PCR is a convenient and efficient strategy for generating a diverse pool of mutants (i.e., a mutant library). Error-prone PCR is the method of choice when a researcher seeks to mutate a pre-defined region, such as the coding region of a gene while leaving other genomic regions unaffected. After the mutant library is amplified by error-prone PCR, it must be cloned into a suitable plasmid. The size of the library generated by error-prone PCR is constrained by the efficiency of the cloning step. However, in the fission yeast, Schizosaccharomyces pombe, the cloning step can be replaced by the use of a highly efficient one-step fusion PCR to generate constructs for transformation. Mutants of desired phenotypes may then be selected using appropriate reporters. Here, we describe this strategy in detail, taking as an example, a reporter inserted at centromeric heterochromatin.

This article describes a detailed methodology for random mutagenesis of a target gene in fission yeast. As an example, we target rpt4+, which encodes a subunit of the 19S proteasome, and screen for mutations that destabilize heterochromatin.

Random mutagenesis of a target gene is commonly used to identify mutations that yield the desired phenotype. Of the methods that may be used to achieve ...

Visualization of Microbiota in Tick Guts by Whole-mount In Situ Hybridization

Infectious diseases transmitted by arthropod vectors continue to pose a significant threat to human health worldwide. The pathogens causing these diseases, do not exist in isolation when they colonize the vector; rather, they likely engage in interactions with resident microorganisms in the gut lumen. The vector microbiota has been demonstrated to play an important role in pathogen transmission for several vector-borne diseases. Whether resident bacteria in the gut of the Ixodes scapularis tick, the vector of several human pathogens including Borrelia burgdorferi, influence tick transmission of pathogens is not determined. We require methods for characterizing the composition of the bacteria associated with the tick gut to facilitate a better understanding of potential interspecies interactions in the tick gut. Using whole-mount in situ hybridization to visualize RNA transcripts associated with particular bacterial species allows for the collection of qualitative data regarding the abundance and distribution of the microbiota in intact tissue. This technique can be used to examine changes in the gut microbiota milieu over the course of tick feeding and can also be applied to analyze expression of tick genes. Staining of whole tick guts yield information about the gross spatial distribution of target RNA in the tissue without the need for three-dimensional reconstruction and is less affected by environmental contamination, which often confounds the sequencing-based methods frequently used to study complex microbial communities. Overall, this technique is a valuable tool that can be used to better understand vector-pathogen-microbiota interactions and their role in disease transmission.

Visualization of Microbiota in Tick Guts by Whole-mount In Situ Hybridization

Here, we present a protocol to spatially and temporally assess the presence of viable microbiota in tick guts using a modified whole-mount in situ hybridization approach.

Infectious diseases transmitted by arthropod vectors continue to pose a significant threat to human health worldwide. The pathogens causing these ...

Single Molecule Fluorescence In Situ Hybridization (smFISH) Analysis in Budding Yeast Vegetative Growth and Meiosis

Single molecule fluorescence in situ hybridization (smFISH) is a powerful technique to study gene expression in single cells due to its ability to detect and count individual RNA molecules. Complementary to deep sequencing-based methods, smFISH provides information about the cell-to-cell variation in transcript abundance and the subcellular localization of a given RNA. Recently, we have used smFISH to study the expression of the gene&#160;NDC80&#160;during meiosis in budding yeast, in which two transcript isoforms exist and the short transcript isoform has its entire sequence shared with the long isoform. To confidently identify each transcript isoform, we optimized known smFISH protocols and obtained high consistency and quality of smFISH data for the samples acquired during budding yeast meiosis. Here, we describe this optimized protocol, the criteria that we use to determine whether high quality of smFISH data is obtained, and some tips for implementing this protocol in&#160;other yeast strains and growth conditions.

Single Molecule Fluorescence In Situ Hybridization smFISH Analysis in Budding Yeast Vegetative Growth and Meiosis

This single molecule fluorescence in situ hybridization protocol is optimized to quantify the number of RNA molecules in budding yeast during vegetative growth and meiosis.

Single molecule fluorescence in situ hybridization (smFISH) is a powerful technique to study gene expression in single cells due to its ability to detect ...

Mating-based Overexpression Library Screening in Yeast

Budding yeast has been widely used as a model in studying proteins associated with human diseases. Genome-wide genetic screening is a powerful tool commonly used in yeast studies. The expression of a number of neurodegenerative disease-associated proteins in yeast causes cytotoxicity and aggregate formation, recapitulating findings seen in patients with these disorders. Here, we describe a method for screening a yeast model of the Amyotrophic Lateral Sclerosis-associated protein FUS for modifiers of its toxicity. Instead of using transformation, this new screening platform relies on the mating of yeast to introduce an arrayed library of plasmids into the yeast model. The mating method has two clear advantages: first, it is highly efficient; second, the pre-transformed arrayed library of plasmids can be stored for long-term as a glycerol stock, and quickly applied to other screens without the labor-intensive step of transformation into the yeast model each time. We demonstrate how this method can successfully be used to screen for genes that modify the toxicity of FUS.

This article presents a mating-based method to facilitate overexpression screening in budding yeast using an arrayed plasmid library.

Budding yeast has been widely used as a model in studying proteins associated with human diseases. Genome-wide genetic screening is a powerful tool ...

Characterizing Histone Post-translational Modification Alterations in Yeast Neurodegenerative Proteinopathy Models

Neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS) and Parkinson&#39;s disease (PD), cause the loss of hundreds of thousands of lives each year. Effective treatment options able to halt disease progression are lacking. Despite the extensive sequencing efforts in large patient populations, the majority of ALS and PD cases remain unexplained by genetic mutations alone. Epigenetics mechanisms, such as the post-translational modification of histone proteins, may be involved in neurodegenerative disease etiology and progression and lead to new targets for pharmaceutical intervention. Mammalian in vivo and in vitro models of ALS and PD are costly and often require prolonged and laborious experimental protocols. Here, we outline a practical, fast, and cost-effective approach to determining genome-wide alterations in histone modification levels using Saccharomyces cerevisiae as a model system. This protocol allows for comprehensive investigations into epigenetic changes connected to neurodegenerative proteinopathies that corroborate previous findings in different model systems while significantly expanding our knowledge of the neurodegenerative disease epigenome.

This protocol outlines experimental procedures to characterize genome-wide changes in the levels of histone post-translational modifications (PTM) occurring in connection with the overexpression of proteins associated with ALS and Parkinson&#39;s disease in Saccharomyces cerevisiae models. After SDS-PAGE separation, individual histone PTM levels are detected with modification-specific antibodies via Western blotting.

Neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS) and Parkinson&#39;s disease (PD), cause the loss of hundreds of thousands of lives ...

In Vivo Forward Genetic Screen to Identify Novel Neuroprotective Genes in Drosophila melanogaster

There is much to understand about the onset and progression of neurodegenerative diseases, including the underlying genes responsible. Forward genetic screening using chemical mutagens is a useful strategy for mapping mutant phenotypes to genes among Drosophila and other model organisms that share conserved cellular pathways with humans. If the mutated gene of interest is not lethal in early developmental stages of flies, a climbing assay can be conducted to screen for phenotypic indicators of decreased brain functioning, such as low climbing rates. Subsequently, secondary histological analysis of brain tissue can be performed in order to verify the neuroprotective function of the gene by scoring neurodegeneration phenotypes. Gene mapping strategies include meiotic and deficiency mapping that rely on these same assays can be followed by DNA sequencing to identify possible nucleotide changes in the gene of interest.

In Vivo Forward Genetic Screen to Identify Novel Neuroprotective Genes in Drosophila melanogaster

We present a protocol using a forward genetic approach to screen for mutants exhibiting neurodegeneration in Drosophila melanogaster. It incorporates a climbing assay, histology analysis, gene mapping and DNA sequencing to ultimately identify novel genes related to the process of neuroprotection.

There is much to understand about the onset and progression of neurodegenerative diseases, including the underlying genes responsible. Forward genetic ...

Expression, Purification, and Liposome Binding of Budding Yeast SNX-BAR Heterodimers

SNX-BAR proteins are an evolutionarily conserved class of membrane remodeling proteins that play key roles in sorting and trafficking of protein and lipids during endocytosis, sorting within the endosomal system, and autophagy. Central to SNX-BAR protein function is the ability to form homodimers or heterodimers that bind membranes using highly conserved phox-homology (PX) and BAR (Bin/Amphiphysin/Rvs) domains. In addition, oligomerization of SNX-BAR dimers on membranes can elicit the formation of membrane tubules and vesicles and this activity is thought to reflect their functions as coat proteins for endosome-derived transport carriers. Researchers have long utilized in vitro binding studies using recombinant SNX-BAR proteins on synthetic liposomes or giant unilamellar vesicles (GUVs) to reveal the precise makeup of lipids needed to drive membrane remodeling, thus revealing their mechanism of action. However, due to technical challenges with dual expression systems, toxicity of SNX-BAR protein expression in bacteria, and poor solubility of individual SNX-BAR proteins, most studies to date have examined SNX-BAR homodimers, including non-physiological dimers that form during expression in bacteria. Recently, we have optimized a protocol to overcome the major shortcomings of a typical bacterial expression system. Using this workflow, we demonstrate how to successfully express and purify large amounts of SNX-BAR heterodimers and how to reconstitute them on synthetic liposomes for binding and tubulation assays.

Here, we present a workflow for the expression, purification and liposome binding of SNX-BAR heterodimers in yeast.

SNX-BAR proteins are an evolutionarily conserved class of membrane remodeling proteins that play key roles in sorting and trafficking of protein and ...