If I were a Developmental Biologist….

1. What are the specific molecular mechanisms involved in regulating cellular apoptosis during development and how are they activated in some cells and not in others?
2. Which of these aforementioned pathways remain active after the organism has fully developed?
3. (Q1) Mechanisms regulating cellular apoptosis are of interest to me as they play such a critical role in how an organism develops. As was mentioned in class, without this sort of altruistic cell death we would all have webbed-fingers and all sorts of other unnecessary and potentially maladaptive tissues. I think it is interesting that during development an embryo has cells that are superfluous and need to be deleted after they have served their purpose.  I would like to know how they come about and how they know when they are supposed to die off.

Findings would have an immediate benefit to cancer research as proteins that play a role in inhibiting the apoptosis pathway could become potential targets for anti-cancer drugs.  In this way, this research could also benefit the larger community by potentially providing new cancer treatments.  As programmed cell death is an essential part of the development of multicellular organisms I think that it could definitely improve our understanding of the molecular underpinnings that determine how an organism develops.

Reflection/Comment:

I selected this item partly because I feel like it was thought out a little better than some of the in class assignments that we worked on. I had more time to think about it and it is much better written then the answers that we quickly typed out for the in-class assignments.  The reason I decided to discuss apoptosis was because during the summer I worked as a co-op student in a lab where my project involved studying some of the non-canonical roles of the pro-cell survival protein Bcl-2.  So when I was writing this I already had some idea of the sort of roles these sorts of proteins might play in development.  So in spite of my lack of experience with developmental biology I still felt that I could say something useful about these proteins and their role in development.  I feel that at that early stage in the course I really couldn’t have said much else about development with much confidence.  Now with what I have learned I feel that I have the knowledge necessary to make these questions a little more specific.  For example I could assess the roles of certain cis-regulatory elements in modulating the expression of these apoptosis genes during development.

Assigned Readings Questions: Plank and Dean (2014)

1. The majority of the studies sited in the article use some variation of the 3C (chromatin conformation capture) technique. The 3C technique is built on the principle that proteins and DNA can be crosslinked together using formaldehyde or other compounds. If these proteins form a chromatin loop, then they too will be bound together, additionally, if these proteins happened to be bound to DNA at that time and that DNA will also become fixed in its looped position. The DNA can then be sheared, after that the DNA is treated with restriction endonucleases and ligated back together, if there is a loop present in your DNA of interest, then the two separate strands should be ligated together, this DNA can then be sequenced.  In that way the 3C technique can be used to verify the presence of loop forming enhancer elements in a specific sequence of chromosomal DNA.  There are other variations of this technique, which can be used to different ends, ultimately, however all of the 3C related techniques function using the method described above.  The main advantage of this technique is that it managed to provide the first solid evidence that the enhancer-gene loop was the mechanism by which enhancers interact with their target gene.  Prior to this, the interaction between enhancers and the genes that they regulate was clear.  This technique provided the mechanistic data needed to determine a method by which genes and enhancers interact and in that way, this technique is good for proving which specific proteins are needed for the formation of enhancer-gene loops.  This is shown in the Plank and Dean article in the first section, in which they indicate the importance of 3C studies in verifying which protein are involved in loop formation.  For example they indicate that commonly expressed proteins such as cohesion and CTCF are also involved in loop formation.
2. The existence of roughly 90000 tissue and stage-specific enhancers in mice is interesting, considering that mice have approximately 23000 genes. At first glance, this suggests that multiple enhancers exist for the purpose of amplifying one specific gene. If one considers the tissue specificity of these enhancers, the existence of 90000 of them begins to make sense.  It is well-established that many of the same genes are active in different cell types.  It is possible that different tissue specific enhancers are used to activate these genes in different cell types.  The article itself suggests this by citing that genes like Myc and Pim1 make use of different enhancers as development moves forward in certain cell types.  The existence of tissue and stage specific enhancer regions may act to inactivate genes that are no longer necessary in the cell by inactivating certain enhancers, while still allowing for the expression of genes that are necessary for the cell to function by having them switch to other enhancers.
3. A latent enhancer is an enhancer that is lying dormant waiting for a signal to activate it. This doesn’t necessarily mean that the enhancer is permanently inactive; just that it is only active under a certain set of conditions.  The review article by Plank and Dean provides an example of latent enhancers action in macrophages.  Upon treatment with LPS, these enhancers undergo epigenetic changes that allow them to activate.  The article also suggests that under standard conditions these genes appear to be packed away as heterochromatin as in their inactive state they are not sensitive to digestion by nucleases.  This raises the interesting question of as to how a cell may select this DNA to be activated over other inactivated enhancers.  In this regard, the article states that it is not yet known how this occurs.
4. A number of different proteins seem to be required for chromatin loop formation. The article illustrates a number of methods by which this can be accomplished in its first section. One example was that of the lineage specific transcription factors, which were determined to be necessary for the formation of loops involved in the transcription of genes that are specific to certain cell types.  Looping also seems to be driven by proteins such a cohesin, which is a protein generally known for holding sister chromatids together before the cell undergoes mitosis.  Finally, enhancer loops seem to also be driven by proteins such as mediator, which acts as a kind of go between the transcription complex and the transcription factors on the enhancer site.  In addition to this, the article states that enhancer loops are more likely to form within topologically associated domains (TADs).  The article suggests that TADs are regions of DNA that contain clusters of enhancers and genes that interact with each other.  The article suggests that this clustering effect limits the amount of searching the enhancer proteins have to do along the chromosome before they encounter their target site.  This is important to loop formation as it could potentially act to limit the sizes of the loops that may form.  It would be more favourable for a cell to form smaller loops within a cluster than to interact with genes that could be very far from the enhancer site.
5. I found that I had to read this article through a couple of times in order to retain the information that I needed to answer the questions. One of the difficulties I had with it was dealing with a lot of the protein names. Many of the proteins that were involved in regulating very specific transcription events, although important within those specific interactions from a mechanistic standpoint, had very little bearing on the overall purpose of the article.  I felt like I had to wade through all of the information to get through to the main point.  In terms of the content, I felt that I had the most difficulty with the epigenetics segment.  I am a bit fuzzy on histone modifications and how they tie into gene expression and activation.  I am not sure that I ever properly understood DNA methylation and acetylation and how they specifically activate or inactivate segments of the genome.  I am definitely interested in learning more about this aspect of the biology though.

Reflection/Comment:

I selected the above assignment for essentially the same reason that I selected the first assignment.  I feel like I had more time to write it out properly.  In terms of my learning I think it played a big role in solidifying my understanding of the 3C technique.  Additionally, working on this assignment ahead of time helped me to perform better on the in-class assignment on the same article.  I had already considered an answer for the  latent enhancer question when it came time to answer it on the in-class assignment.  In terms of the course I feel like I got the most out of the enhancer section and it was probably because I put the most effort into doing the reading and assignments during this section.

Plank and Dean (2014) In-class Assignment

Kate-Marie Neufeld, Fareeha Salahuddin, Mitchell Beattie, Christy Kwok, Garry Bains

Questions:

1. We had troubles understanding some of the jargon in the paper due to the fact that it takes a very mechanistic approach. Understanding how all of the proteins affect each other was difficult. In addition, we had difficult understanding how eRNA gets methylated, and the affect and changes that methylation has on the overall structural integrity.

 

2. It is important to study different model systems, cell types and developmental stages because most enhancers have specific characteristics and properties. For example, there are:

• Tissue-specific enhancers, which only activate when present in certain genes. For example, the proteins and genes expressed in mouse cells result in over 90,000 enhancers (Nord, et al. 2013).
• latent enhancers that activate only when certain elements are present like lipopolysaccharides.
• enhancers that only activate during certain developmental stages.

If studies were only limited to one specific model system, cell type or developmental stage, scientists would potentially miss a lot of information regarding when specific enhancers are activated.

 

3. In general, we agree that chromatin structure/ packaging at the combinations of histone modification and at the proteins associated with an enhancer element depicts whether an enhancer is active, poised or inactive. The binding of transcription factors and the histone modifications show when an enhancer is active.

 

However, in special circumstances, like with latent enhances for example, it is unclear when latent enhancers are activated. This is because latent enhancers lack transcription factors and histone markers.

 

4. Based on the review article, we hypothesize that some of the genes are localized whereas others are not. Enhancer looping that is key to development is generally localized and show a specific a pattern; however, with most cases, there are also numerous exceptions. Some transcription factors, like OTX2, pioneer new enhancer sites, which allow relocalization. This allows new sets of active genes to activate.

Reflection/Comment:

I picked this assignment because I felt that although the answers were hastily put together I feel that at least for the most part they effectively answered the question.  With the exception of question 3 which seems a bit cluttered and hard to interpret.  I think what we were trying to articulate was that for the most part epigenetic markers can be used to identify gene activity.  But as Phoebe Lu stated in her guest lecture, the so-called “epigenetic code” is not quite as clear cut as was initially thought.  For example, now we have discovered latent enhancers which on the surface resemble inactive genes put can be activated under certain conditions, unlike “actual’ inactive genes.   Other than that, I think that it provides a good demonstration of the effort that I put into trying to understand that particular paper.  Additionally, as this was the first group assignment it provided an indication of what other group assignments would be like.  Working in a group provided a different dynamic and it involves considering others opinions, as such it took much longer to answer questions.  But I think in the long run it was more valuable to have everyone’s collective knowledge rather than just working by myself.

Assignment-Nov.2 Lonfat et. al.

Questions:

1. Recall the general rule, “Figure 1 is often the most important figure in the paper”. Referring to Figure 1:

1. What transgenic lines did the author use (do these lines look somewhat familiar)?

A Hoxd9lacZ reporter transgene was inserted into several sites upstream of HoxD. An inverted Hoxd11lacZ transgene was also inserted upstream from Hoxd13.

1. What do the data show (Panel C)?

In noninverted Hoxd9lacZ transgene insertions, LacZ staining is identical in both maternal and paternal propagated embryos. On the other hand, with inverted Hoxd9lacZ transgene insertions, LacZ staining is expressed in significant quantities throughout the entire paternally propagated embryo whereas very minimal staining is observed in maternally propagated embryos.

1. What is striking/unexpected about the results, and why?

The site-specific expression of the transgene was striking and unexpected. This is because prior research only shows that whether the transgenes are inverted or not influence expression, so the phenotype of the escape embryos are unexpected.

1. What conclusions do you make from the data?

From the data, we can conclude that imprinting of Hoxd9LacZ transgene is site-specific, as there is significant LacZ staining in paternally propagated inverted genes in comparison to maternally propagated inverted genes. In addition, in non-inverted genes, LacZ staining is the same in both maternally and paternally propagated embryos.

2. How did the authors show that the observed imprinting is lacZ-specific and position/site-specific? Do you agree with their data interpretation and with their conclusion?

The authors shows that the observed imprinting is lacZ-specific and position/site-specific by comparing the expression of Hoxd11 after paternal and maternal inheritance at the same position as the imprinted Hoxd11lacZ transgene without any lacZ sequence. Using in situ hybridization and quantitative RT-PCR, the quantification showed that there was no significant variation in Hoxd11 expression levels in either of the parental alleles. This shows that the LacZ sequence is necessary to elicit maternal imprinting. Through LacZ staining, the authors observed a position/site-specific response.

We agree with their data interpretation and their conclusion.

3. Refer to Figure 2.

1. Briefly explain how to “read” the diagrams shown (i.e. what do the rows of circles represent, what do the white vs. black circles represent).

The diagrams show the allelic DNA methylation of different gene sequences in either paternally or maternally propagated embryos. Each circle is an allelic DNA. It also shows differences in allelic DNA methylation between the same sequences at different times (aka sperm vs. oocyte). The blacked out dots represent methylated allelic DNA whereas the blank dots represent unmethylated allelic DNA.

1. What do the data in Figure 2 show?

In the inverted insertions of the transgene, the allelic DNA of the maternally propagated embryos are mostly methylated whereas the allelic DNA of the paternally propagated embryos are mostly unmethylated. The oocytes similarly possess mostly methylated allelic DNA whereas the sperm possess mostly unmethylated allelic DNA. There is not much of a difference in allelic DNA for rel5 in both maternally and paternally propagated embryos. For the escapers, whether the allelic DNA is methylated or unmethylated will differ based on whether the escapers are strong or intermediate.

1. Why aren’t there a “paternal/+” and a “maternal/+” groups for sperm and oocytes?

The sperm and the oocytes are haploids.  So the sperm contains only the paternal chromosomes whereas the oocyte contains only maternal chromosomes.

1. What are “escaper” embryos, and how were they identified prior to bisulphite sequencing?

Escaper embryos are embryos that exhibit phenotypes that differ from the expected genotype expression. The escaper embryos were identified through LacZ staining prior to bisulphite sequencing.

1. What can we conclude from the data?

Methylation of the allelic DNA might be correlated to the loss of expression of the maternal allele.

1. How do the data in Figure 2 support the claim in the title?

This data shows that only the maternal allele showed signs of DNA

methylation, which is generally considered to be an indication of gene suppression.  In this way this data supports the title’s claim that suppression was allele specific. Figure D shows that transgene methylation was specific to the inverted gene which suggests that the modifications are locus-dependent as the title claims.

4. Figure 3 depicts the results of a series of 3D chromatin conformation capture (aka “4C”) experiments. Try to “read” the figure and see if you can identify the information described in the text.

1. What was the experiment, and what are the results?

4C was used to find distant sequences of DNA that are associated with the LacZ transgene.  The results show that in the maternal inverted gene shows less interaction with distant sequence it is more focused around the area immediately surrounding the gene, whereas the paternal allele shows interaction with a number of sequences downstream of the LacZ gene.

1. What can be directly concluded from the data?

Parental lacZ inversion interacts with a number of enhancer sequences downstream of the gene, whereas the maternal gene does not.

1. How do the data in this figure support the claim in the title?

This data shows that there is an allele specific chromosome conformation that may play a role in regulating the position dependent imprinting of the maternal LacZ transgene.

5. List any findings that you and your group found surprising.

-Genetic imprinting was a new idea and I didn’t know anything about it prior to reading this study and the background material.

-It seemed strange that a transgene which is foreign to the organism could be imprinted upon in the first place.  It was even more surprising to see that it was position dependent.

Reflection/Comment:

I selected this assignment as I felt it was answered better than some of the other group assignments that I had done.  For example, with the Chiesa et al paper.  I had two exams in that week and as such I basically skimmed over that paper instead of taking the time to read it thoroughly.  However with this one I felt that I had read it a little more closely and so overall I think it was the better assignment.  In terms of learning that I gained from doing the assignment I feel like that was well expressed in the assignment itself.  In addition to solidifying my knowledge of imprinting, I hadn’t even heard of gene imprinting prior to learning about it in class.  In addition to that it seemed like an odd discovery to find that a transgene, an ectopic gene that is not normally expressed alongside mouse Hox genes could be specifically imprinted in such a manner that it depended on the location of the transgene within the chromosome.  I also found it interesting that the researchers themselves did not seem to expect the result themselves and decided to pursue the position dependent imprinting of the LacZ gene further.

 

Annotated Bibliography:

1. Kraushaar, DC., Jin, W., Maunakea, A., Abraham, B., Ha, M., and Zhao K. (2013). Genome-wide incorporation dynamics reveal distinct categories of turnover for the histone variant H3.3. Genome Biology, 14(10):R121

This paper provided a model system that could be used to effectively study the formation of H2A.Z/H3.3 nucleosomes in mammalian cells.  The paper itself used the system to study the turnnover of H3.3 in different regions of the genome.  It found that H3.3 is turned over in active gene bodies and that the turnover is linked to transcription.  This provided a link to the study by Jeronimo et. al . from which I based my experiment.

2.  Jin, C., and Felsenfeld, G. (2007). Nucleosome stability mediated by histone variants H3.3 and H2A.Z. Genes & Dev. 21, 1519-1529.

This paper describes the unstable nature of H2A.Z/H3.3 nucleosomes and also functions to characterize the nature of the bond between H2A.Z and H3.3 within nucleosomes and why specifically, it is less stable then that of other nucleosomes.  They also characterized the localization of these nucleosomes to the promoters and transcription start sites of a number of genes using chIP-qPCR.  I used this paper for that information and also for its uses of the double chIP technique that it used to isolation H2A.Z/H3.3 nucleosomes.  That seemed like the best way to test my hypothesis.

3.  Jeronimo, C., Watanabe, S., Kaplan, C.D., Peterson, C.L., and Robert, F. The histone chaperones FACT and Spt6 restrict H2A.Z from intragenic locations.  Mol. Cell, 58, 1113-1123.

This paper provided a basis for my experiments.  Here the authors describe how mutations in yeast Spt16 and Spt6 genes result in in the localization of H2A.Z into the gene bodies in yeast.  They also indicate that H2A.Z is normally evicted from active gene by transcription.  This made me wonder if this mechanism could occur in mammalian cells and how it might occur.

4. Hardy, S., Jacques, P.-E.,  Gevry, N., Forest, A., Fortin, M.-E., Laflamme, L., Gaudreau, L., and Robert, F. (2009).  The euchromatic and heterochromatic landscapes are shaped by antagonizing effects of transcription on H2A.Z deposition. PLoS Genet. 5, e1000687.

This paper indicates that H2A.Z accumulates in inactive gene bodies in both yeast and human cells.  It also indicates that the removal of H2A.Z is dependent on transcription.  This provided evidence that the model described in Jeronimo et. al. could potentially have a basis in a mammalian system.

5.  Ray-Gallet, D., Woolfe, A., Vassias, I., Pellentz, C., Lacoste, N., Puri, A., Schultz, D.C., Pchelintsev, N.A., Adams, P.A., Jansen, L.E.T., and Almouzni, G. Dynamics of histone H3 deposition in vivo reveal a nucleosome gap-filling mechanism for H3.3 to maintain chromatin integrity. Mol. Cell, 2011: 44(6): 928-941.

This paper describes a model by which H3.3 is deposited into actively transcribed gene bodies.  This mechanism is transcription dependent and thus provides a correlation between the deposition of H3.3 and the removal of H2A.Z.  In terms of the project it was used as evidence for the transcription based deposition of H3.3 into active gene bodies.  This provided more of a basis for the study described in my project.

6. Conerly, M.L., Teves, S.S., Diolaiti, D., Ulrich, M., Eisenman, R.N., and Henikoff, S. (2010). Changes in H2A.Z occupancy and DNA methylation during B-cell lymphomagenesis. Genome Res. 20, 1383–1390.

This describes the deposition of H2A.Z into active gene bodies in a mouse B-cell lymphoma model.  This implies that their may be a link between improper H2A.Z deposition, nucleosomes composition and cancer, which helped to provide relevance to my study.

7. Garcia, H., Miecznikowski, J.C., Safina, A., Commane, M., Ruusulehto, A., Kilpinen, S., Leach, R.W., Atwood, K., Li, Y., Degan, S., Omilan, A.R., Guryanov, O., Papantonopoulou, O., Wang, J., Buck, M., Liu, S., Morrison, C. and Gurova, K.V. (2013). Facilitates Chromatin Transcription Complex Is an “Accelerator” of Tumor Transformation and Potential Marker and Target of Aggressive Cancers Cell Reports 4(1): 159-173.

This paper describes the role of FACT as a driver of mutations in cancer cells.  This is done by describing its activation is cells that normally do not express it.  However for the purposes of the final project, it was used as evidence that FACT is expressed in my model system this providing a basis for the method of study that I intended to use.

8. Melters, D.P., Nye J., Zhao, H., and Dalal, Y. (2015). Chromatin dynamics in vivo: A game of musical chairs. Genes (Basel), 2015; 6(3): 751-776.

This review describes the differential deposition of a variety of histone proteins.  They provide information about both H3.3 and H2A.Z and their deposition and function.  This was primarily useful as a reference in my introduction and as a source of ideas when I was initially researching the project.

9. Huang, C., and Zhu B. (2014).  H3.3 Turnover: A mechanism to poise chromatin for transcription, or a response to open chromatin? Bioessays, 36(6): 579-584.

This paper describes the turnover of H3.3 in various genomic regions.  This describes aspects of H3.3 turnover and suggests potential mechanisms for its deposition and removal from the genome.  This was useful in providing me the initial information that helped me to formulate my question and my hypothesis as it suggested that H3.3 deposition in active genes was mediated by transcription.

10.  Sigma-Aldrich Protocol: file:///C:/Users/Owner/Downloads/shclngbul_2.pdf, (Accessed Dec 7^th, 2015.)

This pamphlet describes Sigma’s wide array of shRNA products that can be used to perform knockdowns.  This provided me with ideas for controls that I could perform in my model system to optimize and validate my knockdowns.

 

Reflection/Comment:

Preparing an annotated bibliography forced me to think more critically about why I was using a particular reference.  As you can see some of these are more critical to the project than others.  For example Kraushaar et al. was of critical importance to the development of my experiment.  It provided the ideal model by which the hypothesis could be tested in mammalian cells. Where as some of the others such as the review article by Huang and Zhu was more useful in the initial research stage as it fed ideas for questions that I could test.  It also proved to be of use in the project’s introduction as a source of background information.  The Sigma pamphlet is not a scientific paper, but I feel it was important enough to merit a mention on this list over some of the other material that I cited in my project, as it provided information regarding what is available in terms of products that can be used to study molecular biology.  Overall, I think the annotated bibliography was a useful tool for me to use to assess the importance of each of my sources.