Final Project

My final project is a proposal for a research project investigating the connection between DNA methyltransferase 3A (DNMT3A), HOX9A, and the development of acute myeloid leukemia (AML).

• Full Research Proposal: BIOL 463 Final Project
• Annotated Bibliography

Lay Person Summary

Small molecules called enzymes carry out a wide variety of important processes in the body. One such molecule is DNMT3A, which adds modifications to DNA regulatory regions to prevent genes from being expressed as proteins. This process is called DNA methylation, and it is used to regulate the expression of different genes in the cells of the body.

Studies have shown that DNA methylation patterns are altered in many types of cancer, including acute myeloid leukemia (AML). This is a type of blood cancer in which blood stem cells fail to grow into mature blood cells, leaving immature cells called “blasts” crowding the bone marrow and impairing its function. It is a fast-developing cancer with a five-year survival rate of less than 1 in 4. Mutations in the DNMT3A gene that prevent it from functioning properly has been observed in up to 22% of AML cases, as well as in other types of cancer. DNMT3A is one of the earliest mutations that occurs in AML, which suggests that its dysfunction might predispose cells to leukemia development.

But how does this happen? Few studies have investigated the mechanisms through which defective DNMT3A can affect AML. However, a recent study showed that there may be a link between the DNMT3A mutation and decreased methylation of a group of genes called the homeobox-containing (HOX) genes. One member of this family is HOX9A, . Since decreased methylation of a gene can cause increased expression of the gene’s protein product, it is possible that the dysfunctional DNMT3A protein is unable to methylate the HOXA9 gene, leading to overexpression and AML. This study will investigate this possibility by destoying the DNMT3A gene in blood cells, assessing the methylation levels of the HOXA9 gene, and transplanting the cells into mice to determine their ability to cause leukemia. By developing a better understanding of DNMT3A’s role in leukemia development, it will be possible to identify new targets for therapies to help treat those with AML.

Reflection

The learning objectives that I believe I demonstrated through this assignment are:

• Approach questions, concepts, and facts with curiosity
• Deal positively with frustration, setbacks, and failure – there was a lot of frustration in the process of developing a research question!
• Search the literature and effectively extract relevant information from it
• Integrate new factual and conceptual information into your models/thoughts/ideas
• Communicate information, ideas, and findings clearly and succinctly at a variety of levels – demonstrated through the write-up and the lay person summary
• Develop new questions that can be investigated experimentally
• Construct testable hypotheses based on evidence
• Predict the outcomes of observations, experiments, and correlational studies based on a given hypothesis or model
• Select the most suitable techniques to test a hypothesis
• Evaluate what can and what can’t be concluded from given or predicted data/results

Paper: Shi et al. (2011). Diet and cell size affect queen-worker differentiation through DNA methylation in honeybees (Apis mellifera, Apidae). PLoS One.

What are some other “types” of honeybees, other than queens and workers?

Drones are male bees that result from a haploid unfertilized egg. They do not have stingers, nor do they gather nectar or pollen. Intercaste is another type of bee that has an intermediate phenotype between that of a queen or worker.

What prompted the authors to investigate the effects of DNA methylation on honey bee development?

Recent studies had suggested that DNA methylation was a factor involved in the caste differentiation of honeybees. Honey bees had been found to have DNA methyltransferases that are orthologs of the vertebrate Dnmt1 and Dnmt3 enzymes, and silencing of the Dnmt3 gene was found to result in a higher proportion of queens. The authors were interested in examining whether royal jelly exerts its action through changes in methylation, mediated by Dnmt3. The authors were also interested in examining whether different cell sizes could also contribute to the caste differentiation process, as there had not been much attention previously devoted to this factor.

Considering Table 1, what are the main question(s), experiment, results, and possible conclusions? How do conclusions support the article’s title claim?

Question: What effect(s) do(es) food and cell type have on the queen-worker differentiation process?

Experiment: Examined larval development when they were fed royal jelly versus worker jelly. Also examined development when larvae were grown in different sizes of cells.

Results: There were changes in methylation at 4 sites as the duration of royal jelly feeding increased, indicating that royal jelly duration is correlated with methylation at CpG locations.

Conclusion: This experiment demonstrates a correlation between the duration of royal jelly consumption or growth within a queen cell and development into queens, which provides some support for the article’s title claim. However, another experiment must be done in order to demonstrate convincingly that the methylation pattern changes are what cause the larvae to develop into either queens or workers. Until then, the effects of diet and cell size cannot be said to function “through DNA methylation” in order to affect differentiation.

Consider Figure 2. What is/are the experiment, results, conclusions, and possible inferences? How do the conclusions support the title claim?

Experiment: Larvae were fed with royal jelly for different durations (3, 4, or 5 days). The levels of Dnmt3 enzyme activity, Dnmt3 expression, and methylation of the dynactin p62 gene were measured, and the differentiated state of the honeybees were observed at day 6.

Results: A longer duration of royal jelly feeding was found to be correlated with an increase in the percentage of adults that differentiated into queens. Dnmt3 activity and expression, as well as methylation of dynactin p62, were shown to decrease as the duration of royal jelly feeding increased.

Conclusions: While this experiment does not explicitly establish causation between methylation activity and differentiation into queens, the fact that 5 days of royal jelly feeding was sufficient to make all of the larvae differentiate into queens provides strong support that an element of the royal jelly is responsible for the differentiation into a queen phenotype. 

Consider Figure 3. What does cell size refer to? What was the experimental question, the experiment, results, and conclusions? Do the conclusions support the title?

Cell size refers to the type of cell in which the larva develops – queen cell or worker cell.

Question: What is the effect of cell size on the methylation patterns of honeybees? Does a change in methylation patterns correlate with a change in the percentage of adults that develop into queens?

Experiment: Reared larvae in either worker or queen cells, and assayed them at 3 and 5 days of age. Measured the levels of Dnmt3 activity, Dnmt3 RNA expression levels, and percent methylation of the dynactin p62 gene.

Results: Larvae that were reared in the queen cells had lower levels of Dnmt3 activity, mRNA expression, and overall methylation in the dynactin p62 gene relative to larvae raised in worker cells. However, no queens resulted from larvae reared in queen cells, with 19% differentiating into intercastes.

Conclusions: Rearing larvae in queen cells is not sufficient to differentiate honeybees into queens. However, queen cell size is correlated with decreased levels of Dnmt3 activity, expression, and methylation. These data may suggest that the cell size may be another component of the differentiation process, as rearing larvae in non-worker cells (i.e. queen cells) resulted in a lower proportion of workers overall.

Propose a model, incorporating the conclusions made from the data presented in the article and the information presented in class, that explains how the difference in diet between queen and worker larvae could result in their phenotypic differences.

1. Based on the article, what are the known causes of SRS and BWS? Which of these causes are genetic, and which are epigenetic?

The known causes of SRS and BWS all involve defects in the 11p15.5 imprinted gene cluster, which contains two independent domains that are each under control of a different imprinting control region (ICR):

The genetic causes:

• Mutation of the DNA sequence of the cell growth inhibitor CDKN1C (5% of BWS cases)
• Loss-of-function mutation in a trans-acting factor
• Uniparental disomy
• Chromosomal abnormalities – maternal deletions, paternal duplications, balanced maternal translocations (BWS), maternal duplications (SRS)

The epigenetic causes are:

• Loss of DNA methylation on the maternal allele of ICR2
• DNA methylation defects at ICR1, associated with changes in IGF2-H19 expression

2. What do the data in Figure 5B show, and what do they tell us about the methylation state of the ICR2 region in individuals I-4, II-4 and III-6?

Figure 5B shows the methylation status of the ICR2 region, which is located within the imprinted gene cluster at 11p15.5, in three individuals in the BWS family. Two of these individuals carry the BWS phenotype (II-4 and III-6), while I-4 is normal phenotype. Individuals II-4 and III-6 have the maternal 160 kb duplication that was identified in the paper. The data show that I-4, II-4, and III-6 have hypomethylated ICR2 alleles relative to an unrelated control. The overall methylation pattern is quite similar across the three cases, but I-4 has the maternal allele almost entirely methylated, while the duplicated maternal allele is only partially methylated in II-4 and III-6.

3. Notice how I-4, II-4 and III-6 all have the same number and methylation pattern of ICR2 ‘loci’. How can their difference in terms of having vs. not having BWS be explained?

Individuals II-4 and III-6 have the BWS phenotype, while I-4 does not. This difference can be explained based on the origin of the 160 kb duplication in ICR2. I-4 has a duplicated paternal allele, while II-4 and III-6 have duplicated maternal alleles. In both of the BWS patients, the maternal allele is only partially methylated, which indicates disruption of the imprinted methylation process in these individuals. In I-4, however, the maternal allele is still almost entirely methylated and the paternal allele is unmethylated. This difference suggests that the 160 kb duplication alters the imprinted methylation of ICR2 only when maternally transmitted, and that this is associated with the presence of the BWS phenotype.

4. Explain what Figure 7B shows and how you interpret the data.

This figure shows the interaction between the chromatin and the KCNQ1OT1 RNA transcript in four different cases: an unaffected control, two individuals with BWS from the analyzed pedigree (II-4 and III-6), and an individual with BWS that has ICR2 hypomethylation but no 160 kb microduplication. The data comes from chromatin RNA immunoprecipitation (ChRIP), a technique that precipitates RNA that is bound to specific regions of the chromatin. By synthesizing cDNA from the precipitated RNA transcripts and subsequently performing PCR, the authors were able to determine whether KCN1OT1 transcripts interact directly with the chromatin or exert their silencing through some other mechanism.

The data shows that the KCNQ1OT1 transcript is significantly enriched in the BWS patients relative to control (Fig 7A). The individual alleles were then analysed in Figure 7B, and the data show that the BWS maternal alleles’ interaction with chromatin is significantly higher relative to the control. There does not appear to be a significant difference in chromatin interaction with the paternal allele. Finally, in panel C the authors analyzed the sequence of the cDNA obtained from the ChRIP experiments. In the BWS samples, both parental alleles (A/G) were present, while only G was present in the control.

I interpret these data as indicating that the KCNQ1OT1 transcript interacts with chromatin, and that the maternal duplication of this region observed in the BWS cases correlates with increased interaction. This suggests that the duplicated region of the KCNQ1OT1 transcript is at least partly responsible for the interaction with the chromatin. Taken together with the other data presented in the paper, it also suggests that this chromatin interaction with the duplicated region may be the mechanism of action through which the transcript silences CDKN1C.

5. Propose two possible mechanisms that would explain how the duplication of ICR2 in these patients causes a reduction in the expression of CDKN1C. Based on what you know about Airn, Igf2r, and slc22a3, which of the two hypotheses is most likely and why?

Two possible mechanisms of action:

The mechanism that I think is more likely

Airn is also a cis-acting silencer – it controls the imprinting of the genes in Igf2r locus, which contains Igf2r, slc22a3, and Airn.

6. After reading this paper, how do you think clinical papers describing just a few patients can contribute to our understanding of the regulation of developmentally relevant genes?

Studies like this one are very useful when studying genetic conditions that are fairly rare, such as BWS and SRS. Silver-Russell syndrome’s exact incidence is unknown, but is estimated to affect between 1 in 30,000 to 1 in 100,000 people worldwide [1]. Beckwith-Wiedemann syndrome affects approximately 1 in 13,700 newborn infants worldwide, although this estimate is likely conservative since some individuals with mild phenotypes are never formally diagnosed [2]. Both of these conditions are therefore quite rare, and it can be challenging to find a sufficiently large population to perform large clinical studies. Furthermore, both of these conditions are quite heterogeneous in their etiology – see the causes listed under question 1. The authors also note in the paper that microduplications are rarer occurrences in both of these conditions, which further complicates the issue.

Using a few patients as case studies for specific molecular defects can provide interesting information that may be overlooked or unavailable from larger studies. Additionally, the fact that this data is clinical provides “real-world” examples of the disease’s molecular pathogenesis, which can sometimes be a limitation of animal model studies and cell culture experiments. Nevertheless, there are still limitations to this approach. The findings may not be applicable to . Additionally, since specific family pedigrees are usually studied, it is possible that typical familial resemblances in the genetic sequence could be misinterpreted as being correlated with the disease phenotype. Therefore, it is critical to couple these case studies with data from other studies. The authors of this paper attempted to do this, bringing in findings from mouse models regarding the function of the Kcnq1ot1 transcript to help support their own data. This paper demonstrates that there are many different ways to approach a scientific problem, and that examining specific biological questions from a variety of perspectives can be key to obtaining novel and important insights.

Reflection

I chose to showcase this assignment in particular since I enjoyed working through this paper. I do not typically work with rare genetic conditions and case studies of this nature, so it was interesting to see a different way of approaching the study of a molecular biology question. This paper also taught me quite a bit about the ChRIP technique, which I was not previously familiar with. I needed to do a bit of extra research on the technique in order to understand the results that were presented in Figure 7, which was helpful for me. I also looked into the prevalence of the two syndromes discussed in the paper, since I was curious to know how common it was in the general population. Once I realized how rare these conditions are, I understood more clearly how valuable familial case studies like this can be.

CRISPR_Cas9 Writeup – Prepared by Campbell Drohan, Denny Gombalová, Leon Lai, Carl-Johan Claudi Risom, and Ida Vinggaard Kjeldsen

Naturally-ocurring and engineered CRISPR/Cas9 systems. via Sander & Joung (2014). Nature Biotechnology. doi:10.1038/nbt.2842

Reflection

The questions I investigated in this assignment were:

1. What general biological, chemical, and/or physical principles and concepts is this technique based on?
2. What does this technique “do”?”

I had previously been somewhat familiar with CRISPR, having studied it briefly in MEDG 420 last year. In that course, we learned about gene therapy and gene editing technologies, gaining a surface-level understanding of the technology and its capabilities. This assignment, however, required me to know the in-depth mechanisms through which CRISPR functions, and allowed me to delve much deeper into the technique than I had done previously. The fact that I would be presenting this technique to my peers and would be expected to answer questions made me reflect on the components of the technique that I was a little confused about.

Over the course of this assignment I was able to learn about some new applications of the CRISPR-Cas9 technology. For instance, I was not previously aware that one could use an inactive Cas9 enzyme to target specific genes and add a marker, such as GFP or a radioactive marker. I always find it amazing how the scientific community can take a technique and tweak it so tat it can serve a with variety of purposes. This project has made me think about CRISPR-Cas9 beyond the classic “gene-editing” or knockout applications. I chose to showcase this assignment because it demonstrates my ability to work collaboratively with my peers, to share information in an engaging and accessible way, and to delve deeper into my own existing knowledge of scientific concepts.

This assignment demonstrates that I was able to achieve the following learning objectives:

• Assess your own level of knowledge in a field/area of interest and identify “gaps” in your knowledge or skills
• Integrate new factual and conceptual information into your models/thoughts/ideas
• Search the literature and effectively extract relevant information from it
• Communicate information, ideas, and findings clearly