In class assignment: Pasque and Plath (2015) review paper

In collaboration with one or two classmates, discuss the following and record your answers to five of the following questions (point form is fine).

What are the main differences between mouse ES cells and human iPS cells in terms of XCI?

Mice ES cells first maintain two X chromosomes and then undergo one round of random X chromosome inactivation, however they can respond to external factors that induce X chromosome reactivation (XCR). By contrast, human IPS cells can never reactivate X chromosomes once they have been inactivated.

What does what we know about XC reactivation (XCR) suggest about the roles of pluripotency factors in maintaining pluripotency and preventing/hindering cell determination?

X chromosome reactivation can occur in an inactivated chromosome, however, it is necessary that multiple factors and signals need to be present to bring the chromosome back in the reactivated state. Similar to this mechanism, it is highly likely that many pluripotency factors must be present and working together in order to maintain the pluripotent state in a cell. The default pathway would be for the cell to undergo detemination, but the pluripotency factors prevent this.

Given what is known about XCR during ES cells reprogramming, what do you think happens to the rest of the chromosomes during this process? Try to give some concrete examples.

Pasque et al. found that a dramatic reorganization of the epigenome occurs during the reprogramming of somatic cells to iPSCs. Changes in Xi-specific, as well as global  chromatin states, noncoding RNA expression, and pluripotency-associated factor expression occur. This could suggest that increases in gene expression on the other non-Xi chromosomes could also occur during XCR.

The authors describe a lot of observations they (and others) made about XCR (see for example the second column on page 77). Some include causal relationships, but many are purely descriptive. Select one “step” of XCR described there and propose an experiment to investigate cause-effect relationships between two factors.

Descriptive Relationship: The enrichment of the PCR2 protein EZH2 on the Xi, not seen in the starting mouse fibroblasts, appears after the mesenchymal to epithelial transition, before pluripotency gene activation, then disappears in fully reprogrammed iPSCs. Pasque et al. speculate that recruitment of EZH2 to the Xi during reprogramming is not required for XCR, but instead represents an intermediate reprogramming stage in which cells are in a de-differentiated state that precedes pluripotency

Investigation of Causality: Complete a knockdown experiment of EZH2 and then observe the Xi. If it undergoes XCR, this shows that EZH2 is not necessary for reprogramming. If the Xi does not undergoe XCR and remains active, EZH2 is necessary for XCR.

What do you think is the key factor to reactivate Xi? Do you think there is a single key factor? If not, what might be the advantage (for a developing cell or organism) of having multiple factors and processes involved? What are the consequences for generating iPS cells?

Although there are many important molecules involved in XCR, there does not seem to be one key factor that is necessary and sufficient to reactivate the Xi. Neither DNA demethylation or Xist repression is sufficient on its own to activate the Xi. In addition, cells lacking the pluripotency gene NANOG, that is involved in XCR, can still undergo XCR in its absence, although the reprogramming reduced in efficiency.

What is the role of Tsix in mouse, and in mouse XCR?

Tsix is required for the repression of Xist in the mouse XCR. However, XCR can still occur in the absence of Tsix. In mice, the inactivated X chromosome is coated with Xist., but Tsix can easily access the activated X chromosome to interact with Xist, by preventing it from binding.

What is one proposed explanation for female ES cells hypomethylation? What suggests that this is connected to there being 2 Xa’s? (Include at least three pieces of evidence)

Explanation: Maintaining the ES cells in a pluripotent state for too long may result in hypomethylation of an X chromosome (it can be both).

Discussio Question: Is hypomethylation of one X chromosome a result of maintaining the cell in a pluripotent state for too long, or a mechanism that allows the cell to be kept in this state? ie. which is cause and which is effect?

Evidence for Explanation:

• Two activated X chromosomes show hypomethylation compared to the XY ESC’s
• When one X chromosome is removed, the female cells return to the male level of DNA methylation (high).
• When cells are grown on serum-free media, DNA methylation is LOW for both male and female.

What is Xi erosion? In what cells does it happen?

Xi is an epigenetic alteration of the inactivated X chromosome. Loss of promoter DNA methylation, and Xist. This phenomenon occurs in human pluripotent stem cells when they are maintained in the pluripotent state for too long) the XC begins to erode.

After carefully reading this review, and discussing with your classmates, would you be worried about receiving human iPS cells “transplants”? How would you check the cells to ensure they are of the highest quality?

If the iPS cell transplant cells were derived from my own cells, I would not be worried about any differences in gene expression or other characteristics that might affect me.

If the iPS cell transplant cells were derived from someone else’s cells, I would be worried that my body may recognize them as “foreign” and reject them by signalling an immune response.

Since iPS cells need to proliferate in order to generate enough tissue for transplantation, certain genes controlling cell growth and division need to be artificially “turned on”. If these genes are not subsequently “turned off” before the transplantation, they can experience uncontrolled growth and result in tumors or cancers.

To check if the cells are of the highest quality, we could ask where or who’s cells they are derived from. It would also be important to understand how the iPS cells have been prepared for transplantation – has growth been adjusted to normal levels? Etc.

People don’t tend to publish negative results. What problems does this lead to?

The problem with researchers not publishing negative results is that other researchers may repeat their experiments or conduct similar ones unknowingly and waste time finding that same negative result. This may happen often when there are many research groups or labs that are studying the same phenomenon or area. Hypotheses and theories in that area of research may be shared between these labs which could lead to many people conducting similar experiments. If one group were to publish their negative results, that would save other people from wasting time conducting those experiments. If people published their negative results more often, research on a large scale would be much more efficient and could move more quickly.

Learning Journal 4

A Technique

One technique that I have learned a lot about in BIOL 463 is Chromatin Immunoprecipitation, or ChIP. ChIP is an experimental technique used to investigate interactions between proteins and DNA in the cell. It allows the user to determine whether specific proteins are associated with specific regions of the genome. For example, ChIP can be used to analyse the binding of proteins such as transcription factors on promoters or other DNA binding sites. Another application of this technique is to determine the specific location of various histone modifications in the genome, therefore identifying the targets of histone modifiers.

Anything Difficult?

Emily, Sina and I had the great opportunity of teaching the ChIP technique to our classmates during the presentations of ‘commonly used’ experimental techniques. When we gave our presentations, there was one aspect of the technique that most students found confusing and it was also the same step that I found confusing when I was learning about ChIP. The experimental method that was quite difficult to understand was the use of a bead in the antibody-binding or immunoprecipitation step. The reason I believe this step was so confusing is because I, and most of my peers have probably only learned about antibodies directly binding antigens. The purpose of having a bead in the immunoprecipitation step was often presented unclearly. The antibodies are commonly coupled to agarose, sepharose or magnetic beads, which are then immunoprecipitated in complexes (i.e., the bead–antibody–protein–target DNA sequence complex). The bead provides a larger component whereby collection of antigen-antibody complexes can be made easier. The complexes are then washed to remove non-specifically bound chromatin, the protein–DNA cross-link is reversed and proteins are removed by digestion with proteinase K.

A Question to Test One’s Understanding

If I were to test whether someone truly understands how the technique above works, I would ask them what the purpose of each step is, and what would happen if this step was left out, or done incorrectly. In my experience, it can be fairly easy to memorize the sequence of steps that make up an experimental protocol. However, knowing the exact purpose of each step can show a thorough understanding of how the technique works. In addition, understanding what would occur if a step was excluded or done incorrectly would demonstrate knowledge of how that step relates to the rest of the experiment. For example, if the protein crosslinking never occurred, the student should say that proteins would never be fixed to the DNA they interact with. As a result, after immunoprecipitating, only naked DNA would be collected and we would not know which proteins interact with that DNA since they were always free to dissociate into the solution.

Learning Journals are a tool commonly used in professional schools (medical school, nursing school, teacher college, etc.) and in the humanities both to help learners engage in metacognition and to help instructors evaluate students’ learning.

Think about the work you did for BIOL463 so far (in and out of class, formally and informally), then try to address each question to the best of your abilities. You can then copy and paste your answers in your wordpress blog. Please do not build a new page, but rather add this LJ above or below your LJ1 entry.

One thing that stood out for you on the midterm or during your midterm preparation

This week you will write (or may already have written) your first midterm for the course. What is one thing that stood out to you either during your preparation for the midterm, or on/during the midterm itself? Is this something that had stood out to you before, or is it something new?

Briefly describe it and explain what made it stand out for you.

One things that stood out for me in preparation for the midterm was the emphasis on answering exactly what the question is asking, and being concise in data interpretations. I’ve found that exam questions are not the type that prompt memorized anwers of facts or details. Instead, I have noticed that understanding figures and experimental data and being able to interpret this information is much more valuable. I’ve found that it’s very important to distinguish between what the data show (exact description of what you see), and what you can conclude from it (what the data prove). In addition, neither of these questions suggest providing a mechanism that COULD possibly describe the data. Its important to never jump to conclusions about mechanisms or cause/effect relationships in data interpretation exam questions. This is something that has been mentioned many times in previous classes, however, I feel as though the testing style for this class emphasizes it even more.

Learning Journals are a tool commonly used in professional schools (medical school, nursing school, teacher college, etc.) and in the humanities both to help learners engage in metacognition and to help instructors evaluate students’ learning.

Think about the work you did for BIOL463 so far (in and out of class, formally and informally), then try to address each question to the best of your abilities. You can then copy and paste your answers in your wordpress blog. Please do not build a new page, but rather add this LJ above or below your LJ1 entry.

One new (or ‘improved’) concept

Now that we have looked a variety of experiments and techniques (e.g. honeybee papers, techniques presentations), some epigenetics mechanisms, the effects and characteristics of boundaries/insulators, etc., identify and briefly describe one concept that is either new for you, or that you understand better or in a new way.

How insulators can regulate gene expression is a brand new concept for me. Before this course, I only knew of transcription factors, enhancers, or inhibitors regulating gene expression but never insulators. How insulators work is they can flank the coding region of a gene, or exist directly adjacent to a gene and block effects of any nearby enhancers on that gene. The result of having an activated insulator is decreased or completely silenced transcription of the gene. How insulators can be activated or deactivated is through their methylation or demethylation. The effect of methyl groups added to an insulator can differ case to case (sometimes activate and sometimes deactivate).

Thinking about your new or ‘improved’ concept

How did you identify your new or ‘improved’ concept? How did you decide that it is a concept (and not, for example, a fact or a skill or a technique)?

What made you realize/decide that you understand it better or in a new way?

Do you think having a good understanding of this concept is important and/or useful? Why/how?

I identified insulators as a new concept because never before have I seen a molecule or component of the genome act to ‘block’ the effects of another transcription regulator. Normally I have seen mechanisms that either directly enhance or directly inhibit transcription. However, this is the first time I have seen interactions between components to produce a result (change in gene expression).

I realized I understood the concept of insulators when I was able to predict the expression of a gene in two different situations: one with an active insulator and one  with an inactive insulator, where both had enhancers for the gene nearby.

Having a good understanding of insulators is useful because it helps us understand another one of the many ways in which gene transcription is regulated.  When assessing data of gene expression levels under varying conditions, having insulators present in the expression mechanism could give rise to particular results. Knowledge of this concept can help us interpret experimental data with much more accuracy.

TECHNIQUES PRESENTATIONS

1.   Names and contributions of group members:

Emily Clarke

Elena Giuchici

Sina Sahebpour

2.   Technique chosen:

ChIP (Chromatin Immunoprecipitation)

3.   What general biological, chemical, and/or physical principles and concepts is this technique based on?

Protein-DNA Interactions

4.   What does this technique ‘do’?

• ChIP is a technique used to identify the sequences where certain proteins bind to and interact with specific DNA sequences in vivo.

5.   What applications is this technique employed for?

• ChIP can be used to determine the specific location of various histone modifications in the genome, therefore identifying the targets of histone modifiers.
• ChIP allows the researcher to determine whether specific proteins are associated with specific regions of the genome.
• Proteins that are not bound directly to DNA or that depend on other proteins for binding activity in vivo can be analysed with ChIP.
• ChIP can be used to analyse the binding of proteins such as transcription factors on promoters or other DNA binding sites.
• Virtually any protein that interacts with DNA can be identified through ChIP experiments.

6.   What questions relating to gene regulation and/or development can be addressed using this technique? Provide two examples (peer-reviewed papers) that use this technique.

One question that can be answered is whether or not a set of genes are regulated by a specific transcription factor. The paper called “ A chromatin immunoprecipitation (ChIP) approach to isolate genes regulated by AGL15, a MADS domain protein that preferentially accumulates in embryos” isolates gene areas associated with the AGL15 transcription factor to analyze and sequence the genes that bind and become regulated by it, through the use of ChIP technology.

Another question that can be investigated is an association of histones with genes. ChIP allows researchers to study how regulation of a specific gene works by looking at how tightly the histones are bound to the gene of interest. The paper called “Direct Examination of Histone Acetylation on Myc Target Genes Using Chromatin Immunoprecipitation” uses ChIP to isolate the DNA of interest and study the level of acetylation on histones, and therefore understand how regulation of the Myc genes works.

7.   What critical reagents are required to use this technique?

Protein of interest

Chromatin from nuclei of your organism’s cells

Formaldehyde to crosslink the protein to the DNA

Antibody specific to the protein of interest

Beads to bind antibody (usually Streptavidin magnetic beads or agarose beads)

PCR Kit

8.   What critical information is required to be able to employ this technique?

• The protein of interest that is believed to interact with a specific area of DNA
• An antibody specific to that protein

9.   At least two resources/information sources that you recommend for learning more about this technique (one or more may be developed by your group):

1. Orlando, V. (2000) Mapping chromosomal proteins in vivo by formaldehyde-crosslinked-chromatin immunoprecipitation. Trends in biochemical sciences, 25 (3): 99-104.
2. R&D SYSTEMS a biotechne bran. N.p., 11 Dec. 2002. Web. 3 Oct. 2016. <https://www.rndsystems.com/resources/protocols/chromatin-immunoprecipitation-chip-protocol>.

10.   List of references consulted:

References:

1. Chromatin Immunoprecipitation (ChIP) Protocol. (2002) [Online]. Available from: https://www.rndsystems.com/resources/protocols/chromatin-immunoprecipitation-chip-protocol [Accessed 10/02 2016].
2. Eberhardy, S., D’Cunha, C. and Farnham, P. (2000) Direct examination of histone acetylation on Myc target genes using chromatin immunoprecipitation. Journal of Biological Chemistry, 275 (43): 33798-33805.
3. Hawkins, R.D., Hon, G.C. and Ren, B. (2010) Next-generation genomics: an integrative approach. Nature Reviews Genetics, 11 (7): 476-486.
4. Kuo, M. and Allis, C. (1999) In vivo cross-linking and immunoprecipitation for studying dynamic protein: DNA associations in chromatin environment. Methods-a Companion to Methods in Enzymology, 19 (3): 425-433.
5. Lee, T.I., Johnstone, S.E. and Young, R.A. (2006) Chromatin immunoprecipitation and microarray-based analysis of protein location. Nature Protocols, 1 (2): 729-748.
6. Mardis, E.R. (2008) The impact of next-generation sequencing technology on genetics. Trends in Genetics, 24 (3): 133-141.
7. Nelson, J.D., Denisenko, O. and Bomsztyk, K. (2006) Protocol for the fast chromatin immunoprecipitation (ChIP) method. Nature Protocols, 1 (1): 179-185.
8. Orlando, V. (2000) Mapping chromosomal proteins in vivo by formaldehyde-crosslinked-chromatin immunoprecipitation. Trends in biochemical sciences, 25 (3): 99-104.
9. Park, P.J. (2009) ChIP-seq: advantages and challenges of a maturing technology. Nature Reviews Genetics, 10 (10): 669-680.
10. Wang, H., Tang, W., Zhu, C., et al. (2002) A chromatin immunoprecipitation (ChIP) approach to isolate genes regulated by AGL15, a MADS domain protein that preferentially accumulates in embryos. Plant Journal, 32 (5): 831-843.

Factual knowledge

Briefly describe one new piece of factual knowledge that you acquired or developed since the start of the course.

I’ve learned that nutrition can play an important role in determining the developmental pathway of an organism, when we learned how drone, worker, and queen bees develop. By feeding bee larvae royal jelly, they grow larger and have higher rates of fertility and longevity which help them with reproduction and expanding the hive. On the contrary, drone and worker bees  are fed “bee bread”, which is a combination of pollen and honey that doesn’t provide the same nutrients as royal jelly.  As a result, they develop better foraging abilities. The bees’ diet controls their development through genetic interactions, where a protein in royal jelly causes methylation of DNA, unwinding it, and allowing replication of specific genes to occur. The genes that end up being expressed are those contribute to the formation of female reproductive organs, and fertility.

Making connections (conceptual knowledge!)

How does this new piece of knowledge fit into what you already knew?

Learning that an organism’s diet can control the methylation of their DNA, and therefore, the expression of certain genes, fit into the concept of how diet and nutrition can alter growth, development, and body shape. However, before learning this, I only thought diet had control over body type through provision of vitamins and other nutrients that act as co-factors in enzymatic biochemical reactions. It was their role in genetics and gene expression that was a brand new idea for me.

What other facts is it connected to, and how?

Proteins from food acting as regulators of gene expression is connected to the whole idea of constitutive and facultative expression of genes through methylation. DNA exists in two forms: euchromatin, when it is loosely wound around DNA, and heterochromatin, when it is tightly packed and genes are not being expressed. Heterochromatin can also be found in constitutive and facultative forms, where facultative heterochromatin is sometimes wound tightly, and sometimes unwound. To achieve this flexibility, the DNA can be methylated in order to interfere with the interactions between its backbone and histone proteins.  This is how DNA methylation allows access of transcription factors to DNA, and increases gene expression.

Does it fit into any concepts? How?

The idea of proteins in diet affecting gene expression, which in turn affects morphology fits into multiple concepts:

1. Gene Regulation: As mentioned previously, proteins can cause methylation of DNA, allowing gene expression to increase.

2. Diet affecting morphology: Proteins in the diet can affect an organisms size, behaviour, fertility, and other characteristics.

Other comments (optional)

Please feel free to include any additional comments that you wish to share.

It would be interesting to know if there are any nutrients, foods, or diets that can cause noticeable changes in humans. For example, could eating a certain food (that contains some protein or vitamin), affect humans behaviour, size, or fertility?

Many attributes of morphology are assumed to be 100% dependent on genetics, such as height, or hair color. However, could humans create a diet that fosters the development of certain morphologies such as taller people, or people with darker hair? The bee example seems to suggest that this may be possible.

I have heard that certain foods act as aphrodisiacs for humans. If true, do these foods act through a protein/gene expression pathway, a hormonal pathway, or by some other mechanism? It would be interesting to understand.

Question: At what stage in human development are the first neurons formed and what mechanism guides their formation?

1. I am interested in investigating this question because I have learned a lot about neuron development and research of the subject through working in a neuroscience lab. Through much experimentation, my PI found that experiments that require observation of neuron growth in its early stages are quite difficult. This is in part because we work with C. elegans and many of their neurons are formed while it is in an egg stage, and the egg shell makes it difficult to view neuron formation through a microscope. This difficulty is what first brought the questions about human neuron development to mind.

2. If we could find out when certain neurons are first formed in the human brain, we could better investigate the mechanisms that guide their initial growth and extension. We may also be able to pinpoint the initial growth of specific neurons. Knowing more about their development may allow us to discover new cures for neurological diseases, especially since most growth and alteration of neurons occurs during brain development (the first couple years of life).