Monthly Archives: March 2015

Learning Journal 4 Week 11

A. Factual knowledge

Please describe, briefly, two new, distinct pieces of factual knowledge that you acquired or developed since the last learning journal

I learned that XIST and Xist is responsible for X chromosome inactivation (XCI) in humans and mice, respectively. This long noncoding RNA coats the X chromosome to inactivate it.

I learned that there are certain histone modifications that are involved with activating genes (H4ac) and inactivating genes (H3K9me3).

B. Conceptual knowledge

Since connections and models make for conceptual knowledge… please describe any connections (direct or indirect) that you can see between the two pieces of knowledge described in A.

Histone modifications involved in inactivating genes are important for XCI. H3K9me3 is found on the promoter of Xist in mice in order to prevent the expression of Xist, which will inactivate an X chromosome. The modifications are lost during development in order to allow XCI.

C. Metacognitive knowledge (no skills this time!)

If you are like most students in the class, you probably spent a significant amount of time reading, studying, and dissecting the article assigned for MT2.

Please describe, briefly, the strategy that you employed to complete the task.

First, I quickly read through the paper once to get a feel of the paper. The second time I look at each figure and try to annotate what they observed and concluded. I also underline the results and the conclusion

Thinking about your experience with reading and dissecting this article, what was the hardest part?

There was a lot to keep track of since there were so many experiments and figures. It’s sometimes hard to see the overall picture of the entire paper.

Thinking about your experience with reading and dissecting this article this article, what did you feel most comfortable with/confident about? Why do you think that is?

I was most comfortable with understanding the difference between inner cell mass and the trophectoderm cells because it was something discussed it in class and I actually understood the difference in XCI between these two types of cells.

Final Project Draft for Peer Reviewing

Effects of Paternal Alcohol Consumption on the Epigenome of Sperm

Introduction

Background

Consumption of alcohol is a common aspect of today’s society; however, it is known to have detrimental effects on humans when a significant amount is consumed. Although children may not consume alcohol, they may come into contact with alcohol’s teratogenic effects during development through their parents’ habits. This may lead to fetal alcohol spectrum disorders (FASD); fetuses have decreased pre- and postnatal growth, distinct facial abnormalities, and improper development of the central nervous system, which may cause mental and cognitive disabilities (Lee et al. 2013). These defects are associated with excessive maternal alcohol consumption during pregnancy (Burd et al. 2003). Prenatal alcohol exposure via the placenta allows ethanol and toxic metabolites to come into contact with the fetus (Zelner and Koren, 2013). However, studies have shown that preconception paternal consumption of alcohol have transgenerational effects on the fetus (Lee et al., 2013, Knezovich and Ramsay, 2012).

Lee et al. (2013) speculated that these effects were passed down as changes in the epigenome of the sperm. The epigenome consists of inheritable chemical groups independent of the DNA sequence that can regulate gene expression (Holliday, 2006). These changes include methylation of cytosine bases, which is usually associated with gene silencing, and chemical groups that are added to histones. Epigenetic modifications are used to silence and active genes in a parent-of-origin specific manner called imprinting. Imprinting control regions (ICRs) are areas of DNA which are differentially methylated when inherited maternally compared to paternally. Alcohol is shown to lower the levels of DNA methyltransferase transcript and/or activity in sperm (Bielawski et al., 2002). Ouka et al. (2009) found a correlation between chronic alcohol use and demethylation of normally hypermethylated imprinted regions in human sperm DNA. All these studies agree that sperm DNA could be a potential medium to transmit alcohol induced epigenetic mutations; however, there is not a study that shows a direct link.

Lee et al. (2013) treated male mice with alcohol and mated them with untreated females. They observed skull malformations in some fetuses and speculated the cause was due to changes in the methylation signatures in the sperm. Laufer et al. (2013) identified three loci that are differentially methylated when the fetus is exposed to alcohol prenatally. There is currently no link between these observations from these two studies. These cranial abnormality may be caused by a change in methylation in these three ICRs that are related to neuron development of the fetus. My specific question I would like to answer is are Stfmb2, Snrpn-Ube3a, and Dlk-Dio3 differentially methylated in the sperm DNA and fetal brain tissue DNA when CD1 male mice are treated with ethanol?

By answering this question, we would be able to finally find a direct link in how a father pass on negative transgenerational effects onto his offspring. There is already public awareness in which pregnant women should not consume alcohol as that would have a direct effect on their babies. If we can find an explanation that alcoholic fathers can still pose a risk on birth defects, then we can raise awareness for paternal alcohol related defects. Fetal alcohol spectrum disorders are easily preventable if the parents are responsible. Raising awareness and education is an excellent preventative measure for FASD.

Hypothesis

I hypothesize that Stfmb2, Snrpn-Ube3a, and Dlk-Dio3 are differentially methylated in the sperm DNA and fetal brain tissue DNA when CD1 male mice are treated with ethanol. Lee et al. (2013) observed preconception paternal consumption of ethanol resulted in skull malformation of fetuses. In another study, Knezowich and Ramsay (2012) found that preconception paternal alcohol exposure showed reduction in methylation of two ICRs (H19 and Rasgrf1) in sperm of exposed males and somatic DNA of the offspring. Although they did not observe an overall change in methylation in the sperm, they speculated that this was due to their method of identifying methylation statuses (more of this will be explained in the experimental design. Laufer et al. (2013) identified three murine ICRs that are differentially methylated after prenatal alcohol exposure: Stfbm2, Snrpn-Ube3a, and Dlk-Dio3. The Stfbm2 region contains neuron-specific transcripts expressed during development (Kagami et al., 2008). The Snrpn-Ube3a region contains a neuron-specific polycistronic transcript (de los Santos et al., 2000). The Dlk-Dio3 region contains over 40 miRNA in two clusters that are expressed in the embryo, placenta, and in adult brains (Seitz et al., 2004). These three ICRs are relevant because they affect endophenotypes that are observed in FASD including impaired growth, craniofacial abnormalities, and behaviourial and cognitive disabilities (May and Gossage, 2001). These ICRs are associated to neuron development in fetus, which may explain the skull malformation observed by Lee et al. (2013).

Prediction

If these ICRs are found to be differentially methylated in sperm and fetal brain tissue DNA, we may be able to explain how the negative effects of alcohol could be passed on through changes in the epigenome of sperm DNA. Finding epigenetic changes in these three ICRs may be able to explain the skull malformations observed in the fetuses. There is a correlation that alcohol consumption increases the demethylation sperm DNA (Ouko et al., 2009). I predict that these changes will in methylation in sperm DNA will be passed to the offspring. This will cause an abnormal expression of some genes controlled by the three loci during development and cause skull malformations.

Experimental Design

To test the hypothesis, five CD1 mice (postnatal day 49) are treated with a 4 g/kg EtOH with 0.9% saline orally once in the morning and once in the evening, for a seven week period. Five control mice would be fed a saline solution. After the seven weeks of alcohol treatment, the mice are allowed to relax for a week then they will be mated with non-treated females. A female will be housed with a male in a cage overnight. When sperm plugs are found, that would be considered as gestation day (GD) 0. After mating, mature sperm will be harvested from all the male mice and DNA is extracted. Dams are sacrificed on GD 16.5. Brain tissues from all the fetuses will be harvested and DNA will be extracted.

A portion of each DNA sample (sperm and fetal brain tissue DNA) will be treated with potassium perruthenate (KRuO₄) and sodium bisulfite for oxidative bisulfite sequencing (OxBS-seq). The other portion will be sequenced without any treatment for reference. All samples will be amplified by PCR using 3 sets of primers that each flank Sfmbt2, Snrpn-Ube3a, Dlk-Dio3 in triplicate. The samples will be then prepared and sent for sequencing. After receiving the sequences, look for 5-hydroxymethylcytosine (5-mC) by looking for cytosines in the OxBS-seq data. Using the reference, each methylated CpG will be mapped and counted for each loci. The triplicates for each sample will be averaged for the number of 5-mC for each loci. Assess for changes in methylation between the experimental and control groups for sperm DNA and fetal brain tissue DNA using Mann-Whittney U test.

Knezovich and Ramsay (2012) used a bisulfite sequencing which could not differentiate between 5-methylcytosine (5-mC) and 5-methylhydroxymethylcytosine (5-hmC) and proposed that this led to the observations of very little demethylation of the sperm DNA after preconception alcohol consumption. Tet dioxygenases changes 5-mC to 5-hmC through hydroxylation and is implicated in active DNA demethylation (Wossido et al., 2011). Additionally, alcohol metabolism has been proposed to cause oxidation of 5-mC to 5-hmC (Wright et al., 1999). Knezovich and Ramsay speculated that the undetected 5-hmC in sperm DNA would manifest itself as demethylated DNA in the offspring. In this study, OxBS-seq is used to differentiate between 5-mC and 5-hmC through selectively oxidizing 5-hmC, which will register as T when sequenced, whereas 5-mC would register as a C (Booth et al., 2012). This results in a better representation of the methylation status of alcohol-exposed sperm DNA. The Mann-Whittney U test is used in this study for determination of statistical significance of difference in methylation in the loci. This test is more suitable when a particular population tends to have larger values than the other, the control is expected to have more 5-mC compared to the experimental group since alcohol induces demethylation (Bielawski et al., 2002). As a precaution, a synthetic oligonucleotide containing a known number of 5-mC and 5-mhC will be used as a control to see if OxBS-sequencing is able to distinguish between the two nucleotides.

Discussion of Possible Results

A possible outcome of this experiment is that the hypothesis is correct and alcohol does cause demethylation in the three loci in both sperm DNA and in the fetal brain tissue DNA. This will provide evidence for a link that epigenetic mutations caused by alcohol can be passed down to the fetus through sperm DNA. Another outcome can could be that the only a change in methylation is seen in the fetus, not in sperm DNA, as observed by Knezowich and Ramsay (2012). This means that alcohol does not necessarily affect the methylation in sperm but rather affect other mechanism such as RNA mediated effects and histone modifications.

Another possible outcome would be that these three loci are not differentially methylated in sperm DNA and/or the fetal brain tissue DNA. This means that the three loci are only affected through prenatal alcohol exposure and not by preconception paternal alcohol exposure. Perhaps there are other effect loci that are involved with toxic transgenerational effects of alcohol.

References (not complete yet)

Laufer et al. 2013 – found three differentially methylated ICRs when the fetus were treated with alcohol that are related to development of the brain

Laufer & Singh 2012 – more in depth review of Sfmbt2, Snrpn-Ube3a, and Dlk-Dio3

Knezovich & Ramsay 2012 – found epigenetic mutations in sperm DNA, will be using an adapted experimental from this paper

Lee et al. 2013 – found skull malformations in mice fetuses when the fathers are fed alcohol

Liu et al. 2009 – found alcohol to changes the DNA methylation in mouse embryos during early development of neurons

Layperson Paragraph

Fetal Alcohol Spectrum Disorders (FASD) is a full range of disabilities, including growth deficiency, craniofacial abnormalities, and mental disabilities, caused by excessive maternal alcohol consumption during pregnancy. Alcohol and its toxic metabolites can reach the developing fetus through the placenta. However, recent studies have showed that the father may be responsible too. The researchers speculate that alcohol induces changes to the DNA in sperm (epigenetic mutations); thus, can be passed onto his offspring. Epigenetics does not change the code of DNA but changes the way how DNA is expressed through inheritable chemical groups added to DNA. There have been a few studies which showed that alcohol leads to the removal of these groups and change the expression of some genes during the development of the fetus. There is currently no experiment that proves a father can transmit transgenerational toxic effects of alcohol through changes in sperm DNA. I would like to perform an experiment that answers this question and see if alcohol induce epigenetic mutations to sperm that can be found in the offspring’s DNA in mice. If I can find evidence that these toxic effects can be passed down paternally, there will be greater awareness of the risks of birth defects caused by alcohol. FASD is an easily preventable disorder that can be countered by responsible parenting through awareness and education.

More on CRISPR

The biggest biotech discovery of the century is about to change medicine forever

This article talks about CRISPR, its origin and mechanism, and also how it may potentially be used. I enjoy seeing articles like this that is published for the general audience to hype up what science has been doing recently. Most advances that are often in the media are technological advances. However, it is rare to see some Biotechnology talked about in the media. Usually, people have a bad connotation with Biotechnology due to genetically modified foods. However, I believe with enough education, we can show the world that genetically modified foods can be controlled and used safely. It is just another tool that humans can use to improve their lives.

Application of CRISPR!

http://rt.com/news/243097-dna-mammoth-cloning-progress/

This article talks about using CRISPR to insert mammoth DNA into elephant cells! It is interesting to see how CRISPR is used in science now after learning about how it works! I hope to see more application of CRISPR in the future.

Learning Journal 3 Week 8

	*Three things that stood out*	*Type of knowledge*	*What makes these things stand out for you*	*Evidence/how you would test someone on this (select one “thing” only!)*
1	GOF experiment shows sufficiency while LOF experiment shows necessity	Conceptual	This is the biggest take home message of this course! This is the basis of all the examples we look at in class. Most experiments in developmental biology are either GOF or LOF and it is important to know what can be concluded from these experiments.	Give a list of a short description of different experiments and ask the students what can be concluded from each experiment.
2	Homeotic genes are usually expressed collinearly in terms of location and time that it is expressed	Conceptual	I found this to be very interesting how homeotic genes can be expressed according to their position on the chromosome. There are many interesting and complicated regulation in Hox genes which also makes this “thing” stand out.
3	Transcription of a particular gene can affect the expression of nearby genes; the gene product may not necessarily be responsible for the effect.	Conceptual	I never thought of this concept before but after learning about it in class, it blew my mind. At first, I was confused how this could work but when I realized that the transcription complex for one gene may block the binding of another transcription complex for a nearby gene.

Animals in Research

Animals in research has always been very important in the advancements in science. There is a large amount of research conducted in embryo development; however, there are major ethical issues with studying using human fetuses. Using animals is the closest model to humans that scientists can study without raising major ethical issues. There is a continual drive for scientists to understand more about the world in order to better our society. With our intellect, it is a way of evolution. We have a competitive advantage over other living organisms because our intellect allows us to improve our living conditions to foster a better society. The use of animals in developmental biology allows us to study a similar system to human embryonic development. Allowing us to understand the mechanism of development, scientists would be able to predict malformations and perhaps find ways to prevent these malformations from happening. In the end, scientist strive to pass on this information to the next generation to aid in their survival. This is almost the basis of any cell in the world. Changes in the genetic material of the cell get passed on to the next generation. If this change helps the next generation survive better, the information is then passed on. In the end, you would get information that is highly optimize to survive in that particular environment.

With the growing of technology, we may perhaps be able to reduce the number of animals sacrificed for our progression in science. There is a starting trend in using computers to model these animal systems. We may be able to use computer simulations to conduct experiments instead. However, we will only be able to model what we have already observed from the real animal model systems. This method may reduce the number of animals sacrificed; however, the continual use of animals in research will be important to explore more of the unknowns of the world in developmental biology.

Musings of a BIOL463 Student