Biology Research Projects 2022

Ahado Ali

Advisor: Tamara Davis

The Maintenance of DNA Methylation at Primary and Secondary DMRs

The overall research topic of our lab is genomic imprinting. Genomic imprinting is the process in which a gene's expression is dependent on its parental origin; for instance, though mammals inherit two copies of a gene, one from each parent, only one copy of that gene, either maternally or paternally inherited, will be expressed while the other remains silenced. DNA methylation helps regulate genomic imprinting by distinguishing the paternally inherited copy from the maternal copy and determining which copy gets expressed. This process is achieved by attaching methyl groups to the cytosines present in cytosine:guanine dinucleotides; this often leads to the silencing of a gene. A DMR refers to a differentially methylated region. Primary DMRs are acquired during gametogenesis and remain methylated throughout development, while secondary DMRs acquire methylation after implantation. The regulation and maintenance of methylation in these regions is crucial to the expression of imprinted genes and the proper growth and development of mammals. The improper maintenance of these regions can result in developmental disorders such as Beckwith-Wiedemann and Angelman syndrome.

A goal of the lab is to determine how methylation in these regions is maintained and whether methylation is maintained differently at primary vs. secondary DMRs. To analyze this, we assessed methylation patterns across various developmental stages. My current project consists of analyzing methylation patterns at both non-imprinted loci and primary and secondary DMRs associated with imprinted loci in 18.5 day-old mouse embryos. I am comparing methylation patterns in wild-type mice to homozygous P allele mice. The P allele mice have a mutation in the Dnmt1 gene responsible for maintaining DNA methylation. This mutation makes it difficult for them to maintain global DNA methylation during embryogenesis and leads to early death. Despite the loss of global methylation and methylation at secondary DMRs, methylation at primary DMRs is well maintained, which leads us to believe that the maintenance of methylation at primary vs. secondary DMRs requires separate mechanisms. By further analyzing DNA methylation patterns in wild-type and homozygous P allele mice, we will better understand the mechanisms responsible for acquiring and maintaining DNA methylation at secondary DMRs. The techniques we use in our analysis are bisulfite mutagenesis, which helps us track methylated sites by converting unmethylated cytosine into uracil; PCR then amplifies primary and secondary DMRs associated with imprinted genes as well as sequences associated with non-imprinted genes; Gel electrophoresis then helps determine if the DNA we amplified is the correct size and whether they're suitable for extraction. Future techniques will consist of submitting the samples for next-generation sequencing so that we may analyze the sequence data and compare the methylation levels at each site to understand better how methylation is regulated at both primary and secondary DMRs.

Adelma Argueta-Roman

Advisor: Thomas Mozdzer

Intraspecific variation in photosynthetic rates of Phragmites australis in a common garden study

Salt marshes are one of the most productive ecosystems worldwide, allowing scientists to determine how global change affects ecosystem productivity. Phragmites australis is an invasive species found globally in marshes, and a newly proposed model organism to investigate the role of heritable traits that are demonstrated to influence ecosystem carbon cycling. There is limited data on how global change is affecting heritability in genetic traits. Working with P. australis will help increase understanding of how global change factors like elevated CO2 and nitrogen, influence the way salt marshes may respond due to the widely spread genetic variability of salt marsh plants, and whether plants can evolutionarily keep up. The study will be focusing on photosynthetic factors like ETR, stomatal conductance (gsw), and water usage from 66 genetically unique P. australis. I hypothesize that within the intraspecific variation there will be genotypes that are consistently performing better in terms of higher photosynthetic rates, independent of changing atmospheric conditions. To investigate the role of heritable trait variation, 66 genetically unique plants from two populations were studied in a common garden located at Arnecliff in ÷ÈÓ°Ö±²¥ in order to standardize the conditions the plants are exposed to. Under standard conditions of light, water, temperature, and nutrients, genetic variation can be attributed to significant finds. Plant traits known to influence carbon cycling in salt marshes, specifically the light-saturated photosynthesis (ASAT), stomatal conductance (gs), and water use efficiency are tracked using a fluorometer, among other conditions. All 66 plants will be tracked in a single day over the course of the summer, and ten plants (five of each population) will be tracked overly to determine dinural photosynthetic responses.

Sachi Bower

Advisor: Thomas Mozdzer

Heritability of functional traits and elemental composition in P. australis

Due to their ability to sequester carbon (C), foundation species in coastal wetlands are vitally important to study to understand the effects of climate change. Little is known about how changing functional traits will influence the ability of these foundation species to store carbon.

An ongoing global change experiment at the Smithsonian Global Change Research Wetland on the common reed, Phragmites australis, has been exposing plant communities to elevated concentrations of carbon dioxide (CO2) and nitrogen (N2) since 2011. Previous research has revealed 64 unique genotypes growing in the field experiment, which have also demonstrated tradeoffs to elevated CO2 and N2 exposure. Our driving question is, are plants traits including elemental content of C, N, P, and/or lignin content genetically inherited.

To test this, we grew genotypes from two populations in a common garden at ÷ÈÓ°Ö±²¥ in 2021 and collected the biomass in fall 2021, which will be used to assess for differences in C, N, P, and lignin between genotypes. First, I separated stems and leaves from each genotype to be analyzed in three distinct procedures. C and N content will be measured on an elemental analyzer, phosphorus will be measured spectrophotometrically after combustion and acid digestion; and lignin will be measured using the acetyl bromide method.

This study will be among the first to evaluation the variation of these traits both within, and among populations. The findings will contribute to a parallel decomposition experiment and inform models of the influence of plant traits on wetland carbon sequestration.

Clementine Chen

Advisor: Bárbara Bitarello

Quantifying the evidence for adaptive evolution of the TAS2R38 bitter receptor gene in primates

Taste is a sense that allows the perception and distinction of different flavors via specialized cells in the tongue. Taste buds are in charge of forming taste perception, and type II taste receptors (TAS2R) are the gene family that codes for bitter taste receptors, which are considered G-protein coupled receptors. Combined, these receptors can bind to a large number of molecules, and each receptor has a specific affinity to different molecules. Since bitter taste is important for animals to avoid swallowing potentially harmful chemicals, we believe that the TAS2R genes that code for bitter taste receptors may have evolutionary significance to humans and other primates. Inside the protein family, there are 25 protein-coding genes and 11 pseudogenes that are found in humans.

Specifically, TASR38 is the gene that codes for taste receptor type 2 number 38, which binds to 23 known ligands. The G-protein coupled receptor is associated with the perception of taste phenylthiocarbamide (PTC), which is tasted differently by different people. The TASR38 gene is the most studied protein-coding bitter receptor gene from an evolutionary perspective. In humans, two divergent TAS2R38 haplotypes exist at intermediate frequencies, which has been interpreted as evidence that they have been maintained by balancing selection. However, a more recent study argues that the recent evolution of this gene can be explained fully by ​â¶Ä‹demographic events in human evolution. Does the TAS2R38 gene play a role in the adaptive evolution (positive selection and/or balancing selection) of humans and other primates? We hypothesize that specific binding sites in this gene may have been subjected to adaptive evolution in humans. If the hypothesis is correct, there will be an increased dN/dS value (ratio of nonsynonymous substitution rate to synonymous substitution rate) at certain binding sites of the gene, corresponding to those that determine ligand specificity. Although this gene can be found in most mammals sequenced to date, we will focus on primates, since this is the taxonomic group including humans and our closest relatives. We will also test whether evidence for adaptive evolution is different for different subgroups of primates, such as the great apes (chimps, bonobos, gorillas, orangutans, humans), New World, and Old World Monkeys. Another test will also be done to detect if a specialized diet of humans and other primates affected the evolution of their TAS2R38 gene. If so, feeding habits might be a factor that determines the genetic variations of the TAS2R38 gene.

In the Bitarello lab, we will be focusing on using computational methods to conduct experiments at the level of comparative genomics. The data used will be publicly available. To find out the evolutionary relationship between species, the obtained protein-coding sequences will be aligned to create phylogenetic trees. Codon substitution models will be implemented in software PAML and HyPhy to measure the evidence for natural selection at the protein level for different groups (sites in the gene or branches in the phylogeny). 

The phylogenetic analysis results may bring new insights to other scientific fields. Since taste affects food intake, knowing the ligand-binding mechanism helps drug developers understand how bitter taste forms so that they are able to create drugs with higher acceptance by patients. Also, based on a recent study, homozygous carriers of a certain variant of the TAS2R38 gene may be related to pine nut syndrome which suggests that dietary habits may be a potential factor to determine the evolution of bitter taste genes. Besides that, the evolutionary significance of the TAS2R38 bitter receptor gene promotes people’s understanding of cultural evolution in human history and how dietary preferences shape human evolution.  

Kuankuan Hu

Advisor: Thomas Mozdzer

Identification of heritable trait variation in the common reed, Phragmites australis

Phragmites australis, the common reed is a newly identified model organism for ecological and evolutionary studies given its global distribution, and its relatively conservative genetic variation.  In a long-term field experiment at the Smithsonian Global Change Research Wetland (GCREW) where Phragmites was exposed to elevated CO2 concentrations, elevated nitrogen, or both for nearly a decade, previous research identified changes in genetic diversity and genotypic tradeoffs in the response to these two global change factors. To evaluate the heritability of plant traits, 63 unique Phragmites genotypes were collected from GCREW and a second population at Parker’s Creek, MD. These genotypes were grown in a common garden at ÷ÈÓ°Ö±²¥ established in 2021. This research aims to identify traits that reflect genetic differences, and future work on the offspring of these genotypes (F1s) will determine the heritability of these traits in the future. Relative growth rate is a known heritable plant trait in previous research and is an excellent candidate to examine trait variation in Phragmites. Each week, I measure the height of five tagged shoots from each unique genotype, and these data will be modelled in R to evaluate differences among genotypes and the two populations.  In addition, I will measure specific leaf area and root functional traits to evaluate the breadth of heritable traits in this species. This study is the first to evaluate heritable trait variation within populations of Phragmites and will challenge results of previous research. Through study of inheritable traits, we also expect to gain more insights in development of Phragmites populations, or even of the ecosystem, under current global change factors.

Lana Hwang

Advisor: Monica Chander

Investigating the function of SoxR and Act in Streptomyces coelicolor         

Chander Lab investigates what mechanisms a soil bacterium Streptomyces coelicolor, a producer of four antibiotics including pigmented Red and Act, employs to protect itself from redox-active compounds it produces. Data from previous experiments done in the lab suggest that different mutants have different pellet morphology. It has been observed that when SoxR, a transcription factor that gets activated by Act for a regulon of genes associated with antibiotic resistance, is deleted, pellets tend to be small. A similar trend was observed for the pellet size when Act is not produced. 

The primary goal of my summer research is to confirm that these data are reproducible. We will first look at SoxR complementation clones under a microscope. If the size of ΔsoxR pellets becomes similar to that of the wildtype when SoxR is reintroduced, it is likely that the small pellet size is due to the absence of SoxR. Furthermore, in regards to the function of SoxR, we would like to investigate if the protective role SoxR plays is specific to Act or not. We will look at M510 and M510ΔsoxR which produce a substantial amount of Act and at M1141 and M1141ΔsoxR in which the entire Act cluster is deleted. If SoxR regulates Act only, strains that are not producing Act should look similar to each other and also smaller since SoxR is supposedly inactive when Act is not produced. This leads us to hypothesize that pellets of M1141, M1141ΔsoxR and 510ΔsoxR would be small whereas M510 is huge.

In addition to the microscopy projects described above, we plan to examine microscopic phenotypes of SoxR-regulated genes: ecaA, ecaB, ecaAB and ecaD. We are particularly interested in ecaB since it is an ortholog of a putative monooxygenase PumA found in Pseudomonas aeruginosa that has been contributed to normal biofilm development and phenazine resistance. Via microscopy, I hope to collect data that can provide clues to answering the question of what the functions of SoxR and Act are in S. coelicolor.

Julia Kesack

Advisor: Tamara Davis

Asymmetric Methylation Patterns in Secondary Differentially Methylated Regions of Imprinted Genes

Our lab studies genomic imprinting, which is the differential expression of genes based on parental origin achieved through epigenetic modifications. Many organisms inherit a set of alleles, one from each parent. At imprinted genes, only the maternal or paternal allele is expressed. Differential expression of these genes is achieved via epigenetic modifications, which are chemical modifications to the chromatin that regulate gene expression. Our lab is focused on DNA methylation, or the addition of a methyl (CH3) group to the DNA. Methylation occurs at cytosines followed immediately by guanine, or a CpG, creating a 5-methylcytosine. Imprinted genes contain differentially methylated regions (DMRs) that influence their expression, with the methylated allele often being silenced.

Since methylation controls gene expression, it needs to be stably maintained and this is usually achieved by DNA methyltransferase. DNA methyltransferase faithfully copies the methylation patterns of genes following replication. Previous research completed in our lab has found a higher occurrence of asymmetric methylation at secondary DMRs, which are acquired during gestation, than at primary DMRs, which are inherited at fertilization. Asymmetric methylation is when one DNA strand is methylated at a certain nucleotide, while the complementary strand lacks complementary methylation. Asymmetry does not promote stability, creating questions regarding the potential cause for this asymmetry.

We hypothesize that asymmetric methylation may be caused by the enrichment of 5-methylcytosine to 5-hydroxymethylcytosine. To test this hypothesis, we will analyze 5-methylcytosine and 5-hydroxymethylcytosine levels at primary and secondary DMRs associated with imprinted genes, as well as non-imprinted genes. In bisulfite mutagenesis, both 5-methylcytosine and 5-hydroxymethylcytosine (5hmC) will remain cytosine while unmethylated cytosine will be converted to uracil, showing up as thymine when sequenced. This mutagenesis allows us to differentiate between methylated and unmethylated cytosines. The oxidative bisulfite mutagenesis will then allow for differentiation of 5-methylcytosine from 5hmC. After performing these reactions, we will amplify and sequence the sites of interest to determine if there is 5hmC enrichment specifically at secondary DMRs.

Gillyoung Koh

Advisor: Bárbara Bitarello

Investigation of Long-Term Diversifying Evolution In TAS2R14, A Promiscuous Bitter Taste Receptor In Primates

T2R or TAS2R are Type II taste receptors that are G protein-coupled receptors. When a bitter agonist binds to the receptor, the organism perceives the bitter taste. Bitter-tasting genes code for the proteins which are the bitter-tasting receptors. It is important to perceive bitterness to protect organisms from eating potentially harmful substances, such as cyanide. The receptor encoded from TAS2R14 is very promiscuous. It can bind to more than 150 known, diverse bitter agonists, roughly two-fold more than the second-highest number of known ligands a receptor encoded from a T2R gene can bind to. Possibly, TAS2R14 could have been subjected to long-term balancing selection. In the Bitarello lab, we are exploring whether there is evidence for long-term diversifying evolution in TAS2R14 in the primate lineage. If we can find evidence supporting this, is the long-term diversifying evolution targeting codons in TAS2R14 corresponding to portions of the receptor interacting with the bitter agonists? We hypothesize that this may be true given that certain vertebrate genes have great diversity and that major histocompatibility (MHC) complex genes show balancing selection in certain codons encoding for the binding of antigens. Finally, we will also test whether different subgroups of primates have experienced different selective pressures. 

To answer the research question, we are exploring whether we can find evidence on a phylogenetic scale. We will use publicly available protein-coding data for humans and other primates. Phylogenetic approaches will be used to study the evolution of TASR14 in primates. A phylogenetic tree will be created after performing a multiple sequence alignment. Then, codon substitution models implemented in HyPhy and PAML will be used. Diversifying selection, or adaptive evolution, includes positive and balancing selection but not purifying selection. We are testing whether there has been adaptive evolution in TAS2R14 and if so, whether certain codons have been the target of selection, i.e, those more linked to binding specificities of the receptor. If this is true, we would observe elevated dN/dS (rate of nonsynonymous mutations to the rate of synonymous mutations) in codons that correspond to amino acids that recognize and/or bind the agonists compared to other codons in the gene. We are also testing whether different taxonomic groups (Great Apes, New World Monkeys, Old World Monkeys) or groupings based on dietary habits (herbivore, carnivore, omnivore) have experienced different selective pressures.

Bitter taste receptors are not just found in taste buds, but they can also be found in some areas of human airways. These receptors bind to bitter compounds generated by bacteria and can lead to narrowing of the airways to avoid inhaling the potentially toxic substances. This suggests that bitter taste receptors may fulfill roles that we have not discovered yet and learning more about these bitter taste receptors may aid the healthcare and pharmaceutical industries. Along with protecting airways from bacterial infection, bitter taste receptors may have influenced primates’ diet to avoid eating certain foods which may have been potentially toxic, affecting primate evolution.

Yeipyeng Kwa

Advisor: Gregory Davis

The Role of JH in Specifying Reproductive Fate in the Pea Aphid

Aphids exhibit reproductive polyphenism, a case of phenotypic plasticity in which different environmental conditions determine the mode of reproduction. Aphids will reproduce either viviparously and parthenogenically (asexual females) or oviparously and sexually (sexual females) depending on environmental cues. During the summer months, when nights are shorter (and days are longer), female asexual aphids will produce offspring that are also asexual and female. However, in the late fall, when nights are longer (and days are shorter), these asexual females will produce asexual females, known as sexuparae, that are different in that they will produce offspring comprising sexual females and males. Evidence suggests that the reproductive fate of embryos is dependent on a maternal signal whose transmission is dependent on the environment the mother is exposed to. Results from tests of sufficiency have implicated Juvenile Hormone as a candidate for this maternal signal: topical application of JH in asexual females who will normally produce sexual offspring (i.e., sexuparae) induces them to instead produce asexual females. While this result indicates the sufficiency of JH to induce asexual fate, evidence indicating that JH is required to specify asexual fate is so far lacking. One approach to test the role played by JH is to establish a means to measure JH signaling in embryos specified as either sexual or asexual.  My goal this summer will be to validate a qPCR assay I have been developing as a measure of JH signaling and then apply this assay to embryos specified as either sexual or asexual. If successful, the resulting data should either support or challenge the role of JH in specifying reproductive fate in the pea aphid.

Sunny Li

Advisor: Gregory Davis

When the environmental condition of the terrestrial ecosystem drastically changed, it forces the insect to develop survival strategies, or they will face the problem of extinction. One of the most influential abiotic factors in the environment is cold temperature. Insects in the terrestrial region need a way to survive the cold temperature of winter. One of the strategy strategies, photoperiodism, brings many advantages for multiple species to adjust their adaptation during different seasons. This mechanism works by detecting variation in daytime length and sending the signal to the inner part of the insect to make changes for a better survival rate. The mechanism of Photoperiodism was developed in pea aphids. During the summer months, there are all-female asexual populations, and sexual females and males in the fall. In asexual populations, the oocytes of females are viviparous, which means they complete embryogenesis within the mother (Imagine embryos were ‘stored’ in the mother). On the other hand, the sexual females with fertilized oocytes will begin embryogenesis after the mother lays the eggs. 
During winter fall, the asexual female will interpret the shorter days and send a signal to the embryos developing inside of her to grow as sexual females (and some males). The mother aphid uses the sexual reproductive mode and produces external eggs (with embryos inside) that are frost resistant. In contrast, when female aphids detect longer days, they will switch to the asexual reproductive mode, which is a faster and more efficient model. It is cool to know that the female aphids can adjust to season changes, but what are the factors capable of receiving the long/short days signal and how does the signal interact with which part of the female aphids? Those questions remained unknown.

Based on the result of preliminary research that has been done, insulin influences the switching of the asexual into the sexual reproductive mode in aphids. However, to state that insulin influences aphids, more controlled experiments are needed because there were some uncertain results from the preliminary research. Therefore, the first part of this summer research aimed for a solid experimental result to confirm the effect of insulin on aphids.  Furthermore, a designed method is being created to test where the signal of switching reproductive mode takes place in the aphid. To do that, we will apply two types of insulin (Juvenile hormone (JH) and wortmannin (WT)) together to the virginoparin mother aphids and then analyze the progenies and the reproductive mode of the mother aphids.

Anna Miller

Advisor: Monica Chander

Role of SoxR In Protecting Streptomyces coelicolor From Foreign Phenazine Antibiotics.  

Streptomyces is a versatile and populous genus of bacteria that grows predominantly in soil and decaying vegetation. Characterized by a specialized metabolism that allows for the creation of spores, Streptomyces also produces secondary metabolites including clinically useful antibiotics and immunosuppressives. Historically, Streptomyces griseus was used to mass produce the antibiotic streptomycin, and other Streptomyces species produce an array of other antibiotics. In the soil, Streptomyces encounters foreign antibiotics produced by other bacteria that share the environment. Among these foreign antibiotics are phenazines, which are used for intracellular signaling and electron transport by the bacteria that produce them. I am studying how the model species Streptomyces coelicolor responds to a variety of phenazine antibiotics produced by neighboring bacteria in their shared soil, and how a particular regulatory protein, SoxR, modulates this response. Previous work in the Chander lab suggests that SoxR regulates the metabolism of endogenous antibiotics produced by Streptomyces coelicolor. In order to investigate this hypothesis, we will expose wild type and SoxR mutant Streptomyces coelicolor strains to various redox drugs. We will then perform a complementation to restore SoxR in the mutant strain, or repeat our original experiment to establish if the observed results are due to SoxR’s influence alone.

Tatiana Perez

Advisor: Sydne Record

Seedlings of Prospect Hill

          My name is Tatiana Perez, and I am an Environmental Studies student whose summer research is being mentored by Sydne Record. My project this summer is called "Seedlings of Prospect Hill," in reference to the tract of land on which I am working. Here at Prospect Hill, I am working in the Harvard Forest with a small field team to examine the confounding factors that affect the survial of tree seedlings of different species. Specifically, I am focused on how and if patterns of newer seedling survival are facing a 'decoupling' when it comes to previous land use. That is to say, older tree species here at Harvard forest have trended towards seedling recruitment following a pattern consistent with past land use disturbances - whether that be burning, hurricanes, farming, etc. Now, the pattern is less consistent, and seedling species are beginning to recruit in places whose land use  histories in the past may not have been as favorable towards that type of tree. My goal is to understand why this is happening - non-native insects? pathogens? - as well as what type of seedlings are recruiting where around the forest. Using land use history and bug data provided by Harvard forest, as well as seedling census data and field observations collected by myself and my team, I intend to use r and GIS modeling techniques to map how these seedlings are moving and how this change may continue in the forest. By answering this question, we may be able to scale up my research to apply to larger swaths of New England land, gaining a stronger understanding of how different types of land can affect the tree species which can grow there.

Tillie Ripin

Advisor: Monica Chander

In the Chander lab we are studying the transcription factor SoxR and the genes it regulates in Streptomyces coelicolor. SoxR is activated by the antibiotic actinorhodin (Act) that is produced by S. coelicolor, so we are also looking at the Act biosynthetic gene cluster. Because SoxR is activated by Act, it is hypothesized that SoxR and SoxR regulated genes provide self-resistance mechanisms against Act. This is supported by an increase in Act production in ΔsoxR mutants. However, ΔsoxR mutants are still viable, demonstrating that there are other SoxR-independent self-resistance mechanisms. ActAB, located within the Act biosynthetic cluster, provides one of these self-resistance mechanisms by exporting Act. Even ΔactABΔsoxR mutants are viable, leading us to question if there is yet another self-resistance mechanism. This has been the focus of my project, continued from this past spring semester, which explores the gene ActVA-orf1 that is within the Act biosynthetic gene cluster. ActVA-orf1 is a putative Act exporter, so in order to determine if this is an additional self-resistance mechanism, my project this summer, continued from the spring semester, is to create ΔactVA-orf1 mutants. Once these mutants have been made, the ActVA-orf11 gene will be complemented in order to see if any phenotypic differences compared to wild type ActVA-orf1 are reversed. This should demonstrate what role ActVA-orf1 plays in self-resistance mechanisms of S. coelicolor.

Jacqueline Saulnier

Advisor: Tamara Davis

Defining the cause of asymmetric methylation at genomic imprinting-associated secondary DMRs

Genomic imprinting refers to the differential expression of alleles on a parent of origin-specific basis. Deviating from the simplified view of gene expression whereby each allele is equally receptive to transcription and translation into protein, imprinted genes are imbalanced in the expression of the maternal copy compared to its paternal counterpart. This difference is mediated by the addition of methyl groups to cytosines followed by guanines (CpGs) on one of the two parental alleles. The importance of accurately regulating the expression of maternal versus paternal imprinted genes is demonstrated by the various developmental disorders that can result when imprinting goes awry, such as Beckwith-Wiedemann syndrome.

Despite notable strides in the scientific community’s understanding of imprinting since the 1990’s, much remains unknown about why and how this phenomenon occurs. The research I am doing investigates why secondary differentially methylated regions (DMRs) display distinctive asymmetric methylation. Generally, DMRs result when methylation is present on one of the two parental chromosomes, and are further defined as primary or secondary: Primary DMRs are acquired in gametogenesis, mark the chromosome’s parental origin, and control expression of associated imprinted genes. In contrast, secondary DMRs are established in the implanted embryo and are thought to be involved in maintaining differential gene expression. These two regions are further distinguished by variation in the stability of their methylation patterns. While primary DMRs are 90-100% and symmetrically marked, with both complementary strands displaying methylation, methylation at secondary DMRs ranges from 30-90%, with greater instances of asymmetric methylation.

My search to identify the cause of this asymmetry at secondary DMRs is driven by the hypothesis that asymmetry results from 5-hydroxymethylation (5-hmC) enrichment followed by active and/or passive demethylation. The abnormal structure of 5-hmC, the oxidized form of methylcytosine (5-mC), can provoke its replacement with unmethylated cytosine, eliminating 5-mC entirely. Alternatively, failure to recognize 5-hmC can prevent 5-mC from being incorporated into the replicated daughter DNA strand. While preliminary results offer some support for this hypothesis, I am testing it against a greater number of imprinted and non-imprinted genes, and via a more comprehensive oxidative bisulfite sequencing approach. In this technique, a sample of DNA undergoes bisulfite mutagenesis and oxidation (oxBS), converting unmethylated cytosines and oxidized 5-hmC’s to uracil, while another sample is only bisulfite mutagenized (BS), converting unmethylated cytosines alone to uracil. Polymerase Chain Reaction (PCR) amplifies the DMRs, or a methylated region of a non-imprinted gene. This enables quantification of the region’s remaining CpG cytosines, representing 5-mC in the oxBS sample and 5-mC + 5-hmC in the BS. Subtracting these values gives 5-hmC levels at the DMRs. Ultimately, this approach promises further elucidation of the relationship between asymmetric methylation and 5-hmC levels at secondary DMRs, progressing toward a better understanding of their unique property and role in the wider picture of genomic imprinting.

Chloe Tang

Advisor: Tamara Davis

Methylation Analysis of 9.5 dpc Embryo DNA Cmtm4

In embryonic development, both paternal and maternal gene copies are expressed and

contribute to fetal growth. However, only one of the parental copies will be expressed if the gene is imprinted. Imprinted genes are regulated epigenetically through DNA methylation, where a methyl group (-CH3) is added to the gene and silences one of the copies. Consequently, differential gene expression patterns that are essential for fetal development are obtained through differential methylation of the parental alleles, referred to as differentially methylated regions (DMRs). Methylation at primary DMRs, which are inherited at fertilization, has been shown to be stable across development whereas methylation at secondary DMRs, which are acquired during early embryogenesis, is less stable and is asymmetric. If methylation is not established properly, then imprinted genes are not expressed properly and diseases and disorders can arise.

Previous research done in the lab has indicated that imprinted genes have more variable and more asymmetric methylation at secondary DMRs. The observed asymmetry raised the question of why DNA methyltransferase is working inconsistently resulting in asymmetric methylation when its role is to maintain methylation and promote stability. In order to study what is causing this unusual methylation pattern, it is important to analyze methylation patterns on both imprinted and non-imprinted genes.

The goal of this project is to perform methylation analysis on Cmtm4, a non-imprinted gene, to understand its methylation pattern and compare it with the results of imprinted genes. By covalently attaching the complementary strands of DNA to each other using a hairpin linker, we can analyze both strands in order to quantify symmetric versus asymmetric methylation levels. My goal is to compare the methylation patterns between imprinted and non-imprinted genes to better understand how methylation is regulated and maintained. Methods required for this analysis include bisulfite mutagenesis, Polymerase Chain Reaction (PCR), gel electrophoresis, molecular cloning, and sequencing.