1. What could be the underlying mechanism driving the different developmental trajectory of the organisms studied in today’s class?

Since worker and queen bees have practically identical genotypes, some sort of environmental cue must be affecting the phenotypes arrived at from the same genotype. These environmental factors most likely affect epigenetic factors like DNA methylation, histone methylation and histone variants to change the dosage of certain gene products produced that results in different physical features. (APPAREANTLY DIET IS THE KEY TO THE DIFFERENT DEVELOPMENTAL TRAJECTORIES).

2. a) With one or two partners, take two minutes to come up with a definition of “epigenetics”:

Heritable modifications to gene expression that are also unrelated to the DNA sequence.

b) After listening to other classmates’ ideas, provide a more complete (if necessary) definition of “epigenetics”:

After hearing Pam’s description of the unicellular protist that can be physically manipulated such that their offspring inherit the physical manipulation, it might be the case the epigenetics isn’t specific to modifications to gene expression but also more gross anatomy manipulations.

3. a) What are different mechanisms that can affect developmental trajectory, and that could be affected/directed by an “outside factor”?

Signaling pathways that affect that production of hormones that have downstream affects on gene regulation.

On/Off gene switches that result in methylation patterns amongst other manipulations to chromatin structure.

b) What are the mechanisms that can affect chromatin structure?

Supercoiling

Histone variants

Histone modifications

Locations in the nucleus

DNA modifications

Chromatin remodeling factors (slide nucleosome along the DNA)

4. What would you predict about gene expression patterns in the two distinct developmental trajectories if epigenetics is driving the phenotype?

Each distinct developmental trajectory will have differential gene expression of particular genes related to development of size, life span and reproductive organs since queen bees are larger, live longer and can reproduce whereas the worker bees are smaller, live shorter lives and cannot reproduce.

5. Researchers (Grozinger et al., 2007) actually checked… what do you notice about the gene expression patterns in individuals following each of the two developmental trajectories?

Genes involved in reproduction and longevity are upregulated in Queen bees and genes involved in foraging behavior are upregulated in worker bees. Furthermore, genes involved in foraging behavior are down regulated in Queen bees and genes involved in reproduction and longevity are down regulated in worker bees.

6. What kind of protein/factor could be a key component of the epigenetic control of developmental trajectories? How would you test your hypothesis?

Since honey bees have a fully functional methylation system, the differential gene expression might be the result of differential methylation patterns. Therefore, the key component of epigenetic control might be a DNA methyltransferase.

7. What did Kucharski and colleagues find, and what does their experiment suggest?

Kucharski et al found that knocking down the DNA methyltransferase Dnmt3 by injection of siRNA results in an increased number of ovarioles per ovary that is a distinguishing feature of the queen phenotype. Knockdown of Dnmt3 mimics royal jelly in producing the queen phenotype. Therefore, there might be a compound in royal jelly that has similar effects as knocking down Dnmt3 has.

8. a) What component of the food in question is most likely to affect gene regulation?

Protein since enzymes are proteins and enzymes are the most likely compound to have activity that can regulate DNA methylation and other epigenetic markers.

b) How does the food in question activate a transcriptionally silenced gene?

Royal jelly acid or 10HDA activates transcriptionally silenced genes by removing methyl groups from the DNA of a gene and, also, by inhibiting the deactylation of histones of the nucleosomes of the genes of interest.

 

TECHNIQUES SPEED-DATING: Quantitative Polymerase Chain Reaction

Names and contributions of group members:

Kenrick Ocampo-Tan, Natasha Tripp, Lorenzo Luis Casal, Melissa Chen, Kimmy Wong

Technique chosen: Quantitative PCR and its derivatives

What does this technique ‘do’?

• Quantitative polymerase chain reaction estimates the starting copy number of a DNA/mRNA segment
  • Can be estimated using the final product concentration after ‘X’ cycles (end-point qPCR) or by detecting the amount of products generated after each cycle (real-time qPCR)1

What applications are this technique employed for?

• Estimating DNA copy number or detecting DNA from a sample2
• Gene expression analysis by quantifying mRNA (RT-qPCR)2,4

What questions (give a couple of examples) relating to gene regulation and/or development can be addressed using this technique?

• How does the copy number of a certain gene compare with others? (qPCR)
• What housekeeping genes are active during specific time in development of fetal animals? (RT-qPCR)
• What genes are used in development but rarely expressed in adulthood? (RT-qPCR)
• Is there differential expression of genes under different stress conditions? (RT-qPCR)

What critical reagents are required to use this technique?

• Both qPCR and RT-qPCR require standard PCR reagents (Forward and reverse primers, dNTPs, thermophilic polymerase, Mg2+, appropriate buffers)
  • qPCR also requires DNA template and either a dye or probe that fluoresces when hydrolyzed or hybridized4
  • RT-qPCR requires reverse-transcriptase (and other standard RT-PCR reagents)  in addition to all of the above
• real-time PCR requires a PCR machine capable of detecting the amount of product after each cycle

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

• Sequence data to be able to create primers
• Proper annealing temperature for reaction specificity

References:

1. Heid, Christian A., Junco Stevens, Kenneth J. Livak, and P. Mickey Williams. “Real time quantitative PCR.” Genome research 6.10 (1996): 986-994. Web. 01 Feb 2015.
2. Karlen, Yann, Alan McNair, Sébastien Perseguers, Christian Mazza, and Nicolas Mermod. “Statistical significance of quantitative PCR.” BMC bioinformatics 8.1 (2007): 131. Web. 01 Feb 2015.
3. Lederman, Lynne. “QPCR.” BioTechniques 47.4 (2009): 817-19. Web. 01 Feb 2015.
4. VanGuilder, Heather, Kent Vrana, and Willard Freeman. “Twenty-five Years Of Quantitative PCR For Gene Expression Analysis.” Biotechniques 44.5 (2008): 619-26. Web. 01 Feb 2015.

 

 

1. Compare and contrast the phenotypes of the SRS vs. BWS patients (you will need to look at Table 1). Do you notice any trends? Knowing that all the patients studied in this article have mutations in the same imprinted cluster, what could explain the differences in phenotypes?

The SRS patient had low birth weight, low birth length, had post and pre natal growth failure, facial defects, digit deformities, and had nervous muscle deficiencies. The BWS patients were generally large at birth, maintain large stature, and do not have facial or digit deformities. The SWS patient has a duplication of all of the genes between ICR1 and 2, but the methylation pattern is the same meaning there is no inhibition by the lnRNA in ICR2. This means that there would be an increased amount of CDKN1C which is a cell growth inhibition, leading to growth deficiencies. The BWS patients have a mircoduplication that results in a truncated lnRNA that inhibits expression of the growth inhibitor, leading to an increase in growth and large size.

2. The authors report using OMIM to obtain some information for their research. Take a few minutes to look up Kcnq1ot1 on OMIM and see what information you get.

Maps to chrom 11p.15.5, lnRNA, overlaps KCN1 in antisense

3. Look at the pedigree in Figure 1.

a) What can immediately be concluded about BWS (even without knowing who inherits the mutant allele from whom)?

The syndrome is probably not recessive autosomal and probably not X linked. Individuals with the syndrome are fertile, and the syndrome is not extremely lethal.

b) II-2 and II-4 both have BWS, and both have one child with BWS and one child without BWS. Briefly explain how this is possible.

If the affected mother passes on the allele that she inherited from her father (with the correct maternal methylation) then the allele will be normal resulting in a normal phenotype.

4. Briefly describe the mutation detected in the BWS patients and the mutation detected in the SRS patient, and their respective effects at the molecular, cellular, and organismal levels (use figures 2, 3, and 8, as well as your answer to Question 1).

NOTE: molecular level = DNA sequence, DNA methylation, gene expression; cellular = proteins present in the cell, potential effects on the cell; organismal = effects on the entire organism.

SRS – 1.2 Mb duplication and inversion of the entire 11p15.5 region, methylation is consistent with maternal methylation (ICR1 is not methylated and ICR2 is methylated), the lnRNA is not expressed on the maternal chromosome, there is a double dosage effect of the genes in the 11p15.5 region on the maternal copy, increased cell growth inhibitor decreases cell growth, has the syndrome phenotype (low birth weight, low birth length, had post and pre natal growth failure, facial defects, digit deformities, and had nervous muscle deficiencies)

BWS – 160 kb duplication and inversion of the ICR2 and part of the KCNQ10TI sequence, methylation on the original copy is normally methylated but the partial RNA sequence is un-methylated, there is expression of the partial lnRNA leading to the down regulation of the cell growth inhibitor, cells grow larger, the individual is larger

5. Explain what the data in Figure 7 show, and how you interpret them.

BWS patients have increased expression of the lnRNA and this is due to increased expression on the maternal allele. Paternal lnRNA expression is unchanged among individuals. The control individual only shows expression of one allele, while the DNA sequence shows that there are 2 different alleles present. The BWS patients show Chirp sequences from both alleles, meaning that there is both maternal and paternal expression of the lnRNA.

6. What valuable fundamental information was gained about imprinting control regions through the study of these patients?

Having a sequence in cis to the ICRs does not necessarily mean that it will be properly imprinted. This is shown by the improper imprinting of the duplicated region in the BWS patients.

 

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

1a)  What transgenic lines did the author use (do these lines look somewhat familiar)? Hoxd9/lacz (HoxD^rel5) and HoxD^{Inv(rel5-Itga6)}. These lines look similar to the line we discussed on Friday where there was also a large inversion.

b) What do the data show (Panel C)? There is normal expression of both parental copies without the inversion. Inversion causes loss of expression when inherited from the mother.

c) What is striking/unexpected about the results, and why? F1 progeny either had strong or almost no expression of lacZ. This indicates that the inversion places the lacZ sequence into a region where it is then maternally imprinted. This is striking because this is very site-specific.

d) What conclusions do you make from the data? Maternal imprinting is position dependent and occurred near the Itga6 locus.

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 researchers used a different mouse line that has HoxD11 replacing the HoxD/lacZ transgene in the inverted sequence. Then they looked at the expression of HoxD11. There was no change when the inverted allele was maternally or paternally passed down, so when lacZ is not present there is no imprinting of the allele. The position specific aspect is shown in Figure 1. Their data interpretation and conclusion sound plausible.

3. Refer to Figure 2.

a) 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). Circles represent potential methylation sites at C residues. White circles are un-methylated and black circles are methylated.

b) What do the data in Figure 2 show? Methylation in escapers is much more variable. Maternal inverted alleles are heavily methylated and paternal inverted alleles are mostly un-methylated. This pattern is also seen in eggs and sperm. D shows the normal amount of methylation in the non-inverted line.

c) Why aren’t there a “paternal/+” and a “maternal/+” groups for sperm and oocytes? Sperm and eggs are haploid, so only have one allele. This only shows the methylation that is sex specific.

d) What are “escaper” embryos, and how were they identified prior to bisulphite sequencing? These embryos had some lacZ expression when the inverted reporter as maternally transmitted. Other maternal/- embryos had little to no expression of lacZ.

4. What was the purpose of the authors’ ChIP experiments, and why did they choose to look for specific histone modifications? What did they find? (Expected answer: max two sentences)

The purpose of ChIP was to look at which regions were associated with H3K27me3. They found that the histones associated with the HoxD9 promoter and lacZ were heavily methylated compared to wild type.

6. What does Figure 4 show? (Please describe/summarize its content including specific information).

A shows a strong interaction between Dlx1/Dlx2 and the transgenes in paternal/+. B shows that Dlx1/Dlx2 expression is much higher in Maternal/+, less in Paternal/+, and even less in WT when the transgene was inverted. C and D show chromosome compaction models for how the digit enhancers can interact with Dlx1/Dlx2.

 

Part I – The study of limb phenotypes (10 min)

1.       What big processes of development are involved in the formation of a human limb?

The formation of three axes (AP, PD, and DV) is very important for limb development. The anterioposterior (AP) axis is formed first. These axes are caused by gradient differences in proteins like SHH and other molecules.

2.       Think about human limb development (wild-type or mutant) as a phenotype of interest. From a fundamental research perspective, why is it a useful phenotype to study? Why is it a good model system for the study of development? What are the advantages?

It is a good phenotype to study because it is very visual, caused by non-lethal mutations and changes in regulation, and it is a relatively complex model for even more complex systems. Limb malformation studies have been happening for a very long time even without genetic techniques.

3.       What is the difference between an isolated and a syndromic malformation, and what kinds of mutations are they postulated to be associated with?

isolated -> change in regulation of a gene in only one aspect of development. ie. SHH production is only effected in limb development but not everywhere else.

syndromic -> either a change in regulation in multiple areas or a mutation in the gene itself that causes phenotypic effects in many areas, leading to a group of symptoms that result in a syndrome

Part II – The study of cis-regulatory elements (20 min)

4.       Select one of the loci discussed in the review by Bhatia and Kleinjan. As a group, prepare a model of its regulation (can be in words, diagrams, a mixture thereof, etc). Then:

•   list the evidence that the authors use as a basis for each part of the model;

•   evaluate the evidence (decide if it is sufficient to support the various parts of the model);

•   if applicable, select a part of the model for which we do not (yet) have much supporting evidence. What additional piece(s) of evidence would help strengthen the model? What experiment(s) could you do to obtain them?

HoxD is surrounded by intergenic space and many regulatory islands that include enhancers. This includes the global control region (GCR) that has enhancing activity over many genes in a tissue specific manner; this is only one example of the many regulatory element groups surrounding HoxD. This information was found using histone markers that signify the presence of enhancers in the intergenic space surrounding HoxD. We think that finding histone makers that are often found near enhancers is a correlation rather than truly a signifier of enhancer sequences. Further investigation using enhancer traps and other techniques is necessary for verifying the presence of these regulatory islands.

5.       What is synteny? How does progress in our identification of cis-regulatory elements help explain some cases of synteny? (And thus making the connection between genome structure, function and evolution relevant?)

synteny -> physical co-localization of genes on the same chromosome

An understanding of the identity and location of cis-regulatory elements shows which aspects of regulation are essential for specific structure development. The conversation of these structures may lead to conservation of the regulation and groups of genes that are regulated by the same elements are more likely to remain together throughout speciation, resulting in syntenic regions.  

 Part III – Where do the cases are from, and who is the information for? (10 min)

6.       Think about all the research conducted on human limb malformation. How do you think the subjects for the study were recruited? How do you think the information gained from these studies was disseminated? Who had access to it? Who could it be useful or interesting for? How are the phenotypes under study depicted?

Subjects were likely recruited through doctors’ offices, medical histories, or speaking with people who have limb malformations. There may have also been some study of dead embryos (most likely in mice). The information gained from these studies was likely distributed through medical case studies, medical journals, and potentially in some scientific journals. Medical professionals, medical researchers and students, and other scientists would have had access to this information. This information would be interesting to medical students, science students, doctors, genetic counselors, researchers in the field, and people who are affected or know someone affected. The phenotypes are depicted in pictures, x-rays, and medical terms.