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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