Research proposal
Priyanka Pahan
Background/survey
Chromatin modifications can continue through generations and this can happen due to transcriptional memory (Zachaioudakis 2007). Transcriptional memory is turning on gene expression when exposed to a stimulus that was presented before. The chromatin in the nucleus (which exists in eukaryotic cells) can remember what happened before so there is a quicker response to the signal the next time. Ways that this can happen is the location of gene in nucleus, the gene’s ability to interact with nuclear pore complex, the ability to activate RNA pol2 with protein factors, and chromatin packaging can change. The advantage of transcriptional memory is that the next time the response to the signal will be faster. (DUrso 2016) Chromatin packaging allows access to gal1 promoter via other transcriptional regulators and plays a role in control of gene expression through histone and chromatin modification. Histones are in eukaryotic cell nuclei that make nucleosomes by packaging DNA and chromatin is the packaged DNA found in nucleus.
Examples of transcriptional memory are in Drosphilia and Arabidopsis. In Drosophilia if the mother experienced transcription then there is a 4X higher chance of the daughters receiving the gene as well. This allows better development during embryogenesis (Ferraro 2015). Also Arabidopsis uses stress-induced transcriptional memory, Arabidopsis is an organism in plants that uses stress-induced transcriptional memory where if you treat them with heat, the plants respond to the heat. If the heat is presented again after a gap, the gene responds faster. (Feng 2016)
For yeast, glucose is the main source. When the media is switched to galactose, the gal1 promoter is involved. When the yeast is switched back to galactose again, the advantage is that the response is faster so there is more yeast growth compared to the yeast that was only exposed to galactose once. Making gal1 involves transcription of Gal1 and that depends on the TATA box. Gal1 is an important memory determinant (Zachaioudakis 2007). The genes responsible for reactivation interact with the nuclear pore complex and the gene’s ability to interact with this complex affects transcriptional memory (Sood 2017). But the nuclear pore complex is needed for faster Gal1 reactivation cuz there are other transcriptional factors downstream that affect memory (Sood 2017). access to gal 1 promoter via other transcriptional regulators and how its controlled by DNA packaging via histones.
Specific aims/ hypothesis
The goal of this experiment is to see what mutations in the histone would affect chromatin packing. Chromatin packing would affect transcriptional memory of the gal1 promoter which is needed for galactose catabolism. The purpose of this experiment is to see if mutations in the histone affect the Gal1 transcriptional memory in yeast. Our histone modifications are S22A and H3delta24-111. The serine is mutated to an alanine at the 22nd position on the tail. The second mutation is also serine to alanine but is in the H3 core. The mutation in the tail would negatively affect gal1 transcriptional memory because the mutation of serine to alanine is a non-conserved substitution. Since it is becoming less polar, it would affect the interaction with the adaptor proteins that are needed for chromatin remodeling. which causes the chromatin to pack tighter which would most probably affect transcriptional memory negatively. The amino acid mutation in the histone core is also a non conserved substation and would affect transcriptional memory because the charge or polarity would cause DNA to pack tighter which would also affect transcriptional memory negatively.
Experimental design
In Experiment 1 we had 6 mini cultures of wild type, S22A, and H3delta24-111 yeast grown in glucose, glucose, galactose or galactose, glucose, galactose. We added a yeast colony into the cultures with glucose or galactose. We incubated the samples in either glucose, then glucose, then galactose or galactose, then glucose, then galactose. We want to maintain the yeast cultures in the log phase so there is a higher rate of growth. After the incubation, we checked the absorbance of the yeast using a spectrophotometer to determine the density of growth and so we can add the right amount into the culture tube. To collect the final growth data, we use a 96 well plate reader. We want to see which type of yeast has the most growth. The controls are the wild type yeast grown in glucose and galactose. This is needed so we can compare the 2 mutants.
In experiment 2 we want to check that the effects we see on transcriptional memory are gal1 dependent. We need to make the yeast more competent for transformation so it can express neomycin which inactivates G418. G418 blocks protein synthesis. We do colony PCR to confirm the presence and location of the cherry gene insert. The cherry gene is needed to track the presence of gal1 and has neomycin resistance so protein synthesis can continue. We can tell that we amplified the sequence by running the PCR product on an agarose gel. If it is at 3.5K base pairs then it is the correct size and at the correct location. By this process we can see the changing amounts of gal1 protein over time. This experiment is to ensure that the change in growth is happening due to Gal1 and not some other factor that the histone mutation is causing. From this experimental design we would know what happens but we wouldn’t be able to tell what the mechanism is. From this experimental procedure we can’t tell what is happening to the chromatin packaging to affect transcriptional memory.
Data analysis
We ran a PCR of the product from week 1 through an agarose gel. We wanted to compare the product to the standard. Both A and B were 3.5K base pairs which showed that the correct product was made and was at the right location since it is known that the mutant gal1 is 2.5K base pairs. The S22A mutant grown in galactose grew slower compared to the wild type which could mean that the amino acid mutation does affect transcriptional memory negatively. The S22A mutant grown in glucose grew faster than the wild type. It is possible that this was caused without any transcriptional memory by the mutation causing Gal1 expression. The H3delta24-111 mutant grown in galactose grew at a lesser rate compared to the wild type which could mean that the mutation does affect transcriptional memory. The H3delta24-111 mutant grown in galactose showed no growth when it was returned to galactose. That sample had a very low absorbance indicating that there wasn’t as much yeast to begin with. We can’t tell how the mutation would have affected growth and would need to repeat this part of the experiment. The next week no yeast colonies grew in the H3delta24-111 mutant so only one agar plate with the S22A mutant was used. The H3delta24-111 mutant didn’t have growth which would imply that PCR product wasn’t taken up by the mutant. For the S22A mutant, the PCR product was taken up by the yeast to integrate into their genome since there were 3 colonies. The second set of overnight culture data showed the same results with wild type galactose having the most growth, then S22A mutant grown in galactose, then S22A mutant grown in glucose, and wild type glucose with the least growth. The second set of data for the H3delta24-111 had the same results as the S22A mutant. This shows that the H3delta24-111 mutant has a similar effect as the S22A mutant. These results matched our hypothesis that the mutated amino acid would affect transcriptional memory negatively. It is not certain why the mutant in glucose grew more than the wild type in glucose.
Conclusions/ future directions
It can be said that the S22A mutation affects transcriptional memory negatively since the mutant in galactose grew at a lesser rate than the wild type. The mutant grown in glucose grew faster than the wild type. This could have been some other effect that the mutation caused. This part of the result wasn’t clear. The H3delta mutation showed change similar to the S22A mutation which cold indicate that the mutation affects transcriptional memory negatively. The first set of the H3delta mutation was inconclusive because there was no growth for the H3delta mutant in galactose. There was very little yeast to begin with according to the absorbance value so there was most probably no growth. The results from experiment 2 should show a band at 3500 base pairs as that would indicate that the cherry was inserted after the gal1 gene. We want to check the absorbance and fluorescence over time. If there is more growth in galactose by the mutant, then it would be due to more gal1expression. The experiment should be repeated again so we can confirm the effect of H3delta mutant. We also want to check why the mutant grown in glucose is growing at a greater rate than the wild type. We should repeat it to confirm the result that the amino acid mutation affects transcriptional memory negatively. We would then be able to conclude that this was caused by the mutation causing Gal 1 expression even without any transcriptional memory.
References
D'Urso, A, and J H Brickner. “Epigenetic Transcriptional Memory.” Current Genetics., U.S. National Library of Medicine, June 2017, www.ncbi.nlm.nih.gov/pubmed/27807647.
Fengab, Xuan Jun, et al. “Xuan Jun Feng.” Proceedings of the National Academy of Sciences, National Acad Sciences, www.pnas.org/content/113/51/E8335.abstract.
Ferraro, Teresa, et al. “Transcriptional Memory in the Drosophila Embryo.” Current Biology : CB, U.S. National Library of Medicine, 25 Jan. 2016, www.ncbi.nlm.nih.gov/pmc/articles/PMC4970865/
Sood, V, et al. “Epigenetic Transcriptional Memory of GAL Genes Depends on Growth in Glucose and the Tup1 Transcription Factor in Saccharomyces Cerevisiae.” Genetics., U.S. National Library of Medicine, Aug. 2017, www.ncbi.nlm.nih.gov/pubmed/28607146.
Zacharioudakis, I, et al. “A Yeast Catabolic Enzyme Controls Transcriptional Memory.” Current Biology : CB., U.S. National Library of Medicine, 4 Dec. 2007, www.ncbi.nlm.nih.gov/pubmed/17997309.