Contributors:
Sam Livingston, Elkie Fung, Kattie Sepehri, Grace Lin, Jordan Whittier

Technique Chosen:
Flow cytometry

What does this technique do?

• Cell counting, identification, and sorting based on the analysis of laser light scattering from individual cells.

What applications is this technique employed for?

Cell counting: Detect relative population sizes of different cell types within a sample

• Eg. Analyze composition of cells in blood

Cell sorting: physically separating cells or particles of interest from a heterogeneous mixture

• Biomarker detection – Identification of cells based on a specific molecular characteristic (protein, lipid, DNA/RNA etc.)
• Apoptosis detection – identification of cell state by detecting DNA damage, caspase activation, or other apoptotic characteristics
• Protein engineering – rapid sorting of cells expressing desirable proteins
• Cancer detection – identification of many blood cancers by using antibodies that stick to only one type of cell

Cell identification: Identify cells based on external and internal characteristics using light scatter and/or fluorescence (eg. antibody tagging)

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

• Are the cells of a shrinking tissue/organs the result of cellular ‘shrinkage’, apoptosis, or necrosis?
  • Useful for identifying degenerative conditions and abnormalities in normal organ/tissue homeostasis

• Can the development of cancerous tissue be analyzed through this technique? Useful in identify proteins with improper folding or cells that have passed through stages of the cell cycle which they shouldn’t have.
• Which cells have progressed to a certain cell fate during a specific time period in development?
• How many/which cells of a tissue/organ produce my biomarker of interest?

What critical reagents are required to use this technique?

• A flow cytometer
• fluorescent labels ( eg. specific antibodies or probes)
• solution to suspend cells – usually PBS
• Propidium Iodide for non-specific DNA labelling of apoptotic cells

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

• What particle is being detected and how to tag it (eg. antibody tagging)
• Predicted size of the cell and their surface morphology that can be observed as forward and side scatter by the detectors of the flow cytometer
• Surface properties for tagging/ internal properties for tagging

References

Davies, D. Flow Cytometry. London: Humana Press, 2007. Ebook.

Gasol, Josep M., et al. “Significance of size and nucleic acid content heterogeneity as measured by flow cytometry in natural planktonic bacteria.”Applied and Environmental Microbiology 65.10 (1999): 4475-4483.

Hodge, Sandra J., et al. “Flow cytometric characterization of cell populations in bronchoalveolar lavage and bronchial brushings from patients with chronic obstructive pulmonary disease.” Cytometry Part B: Clinical Cytometry 61.1 (2004): 27-34.

Wittrup, K.D. “Protein engineering by cell surface display.” Elsevier  12.4 (2001): 395-392.

Janossy, George, et al. “Affordable CD4+ T cell counts by flow cytometry: II. The use of fixed whole blood in resource-poor settings.” Journal of immunological methods 257.1 (2001): 145-154.

Macey, Marion G. Flow Cytometry. Humana Press Incorporated, 2007.

Sosik, Heidi M., et al. “Growth rates of coastal phytoplankton from time‐series measurements with a submersible flow cytometer.” Limnology and Oceanography 48.5 (2003): 1756-1765.

Honey bees-  I have always found honey bees and their community structure very fascinating. Since this lecture I have been thinking about what can actually happen to you if you ingest royal jelly …

Limb Malformations –  Before this lecture I had never heard about limb malformations in mammals, so it was really interesting to get more information about human genetic diseases.

SRS and BWS – This article was quiet challenging to understand, however I liked the way that the study was done, based on pedigrees and on phenotypes seen in humans (not animals).

LacZ transgene – The way that LacZ was used to stain the embryos was a really cool/ smart way of looking at gene expression. I did not know that LacZ can be used even in mammals…

Epigenetics and Development

Our Goals:

* To build our own definition of “epigenetics”;

* To classify the major mechanisms of chromatin remodeling;

* To investigate how external environment/behaviour can trigger a completely different developmental fate

* To propose, and interpret some experiments that allow us to shed light on this phenomenon (in a specific case)

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

• Signalling pathway -> Hormones
• on/off genes -> methylation

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

• Factors that influence gene expression and development that is not directly related to the pattern of nucleic acid or DNA of the individual

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

• Heritable modifications of DNA
• inheritance of a phenotype that is not explained by differences in DNA sequence

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

• Methylation/acetylation
• Histone modifications and the mechanisms involved -> turn on/off areas of the genome
• The environment, for example nutrients
• Developmental pathways

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

• Chromatin remodelling factors
• Different histones
• Methylation/acetylation of histones
• Coiling of chromatin
• Methylation of DNA

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

• Worker bees have reproduction related genes shut down

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?

• There is up regulation of foraging genes and down regulation of reproductive genes in worker bees, and queens bees show the opposite pattern

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

• methylation -> can test with ChiP analysis, or inject methyl transferase inhibitors into larva
• Test other suspected  proteins by injection of RNAi into larva

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

• DNA Methyl transferases are important
• Knock down of Dnmt3 mimics royal jelly – induces queen bee development
• Workers have more of their DNA methylated

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

• lipid, 10HDA, Royal Jelly Acid

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

• Activates Fas expression
• Overall increase in acetylation of histones when treatment with Royal Jelly acid

9. ***optional*** Using these pieces of data that we just discussed, construct a model of how consumption of the food in question leads to each of the developmental trajectories.

In-class questions (group)

Discuss these questions with members of your group, come to a consensus answer fro each question (you may incorporate several opinions/ideas into one answer), and hand in your group’s answers via email after class (ONE set of answers per group, please include the first names of all contributors). Please also send a copy to all contributors, and if you feel so inclined, please feel free to include this piece of work in your portfolio.

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

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

• Pattern formation, differential gene expression, timing

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?

• Easy to see and compare at early stages
• Expression mechanism can explain phenotype/genotype
• Developmental problem, possible solution for disorders/illnesses

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 malformation is a defect in a single system and can have many causes, while syndromic malformation is a pattern of anomalies that have a single cause (made some corrections from the group discussion).

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?

• ZRS enhancer controls Shh expression in developing limb bud
• single base pair substitutions or duplications that encompasses the ZRS region
• The point mutation results in a wrong 3D structure that affects level of  Shh expression. Ex. it regulates the Shh because it binds to other regulatory protein at higher affinity. The change in Shh causes the bud to express extera digits (at the phenotype – Cuban and Belgian mutation)

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 is the degree to which the patterns/orders of genes are similar in 2 compared genomes. Some Cis-resulatory regions have been conserved in evolution. Recent common ancestors will have more synteny between their chromosomes.

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 could be recruited via
  • Volunteers
  • Direct request from researchers
  • Paid
  • Anonymous information from hospitals
  • Depending on the researchers doing the study it could be free access on the web, in journals, or sold in a journal. The researchers and medical institutions could all have access to the information. It could be interesting for physicians, other researchers working in the same area and other individuals with the genetic condition. The phenotypes under study are depicted as descriptions, pictures or experimental data.

OR: (please answers only one of Q6 or Q7, not both)

7. Understanding human “pathologies” that have a genetic basis is almost always listed as one of the benefits of uncovering the genetic and molecular mechanisms that cause a given phenotype. How does the knowledge obtained in this field benefit the patients/subjects? How does it benefit the community at large?

Assigned paper: Chiesa et al. (2012) The KCNQ1OT1 imprinting control region and non-coding RNA: new properties derivedfrom the study of Beckwith–Wiedemann syndromeand Silver–Russell syndrome cases

You are welcome to work on this set of questions collaboratively or individually, and to request help as/if needed. You will hand in your answers at the end of class (one set of answers per group if you work collaboratively).

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?

-More facial morphology defects in SRS than BWS, pre-natal growth failure in SRS but not BWS patients. The BWS babies tend to be heavier than normal (except patient 1). SRS patient is below normal weight at pre-natal weight and at present weight.

-Umbillical hernia seem to be associated with BWS but not SRS

-some nervous system problems are observed in SRS, but not BWS

-Even though all the patients in this article have mutations in the same imprinted cluster, the mutation is not the same. That is, the duplication of entire cluster resulted in phenotype of SRS and the partial duplication resulted in phenotype of BWS. This differences in phenotypes may be because the different level of transcription of different genes as a result of the different types of duplication which have different level of methylation of ICR2.

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.

* overlaps with KCNQ1, associated with BWS and multiple cancers, expressed from the paternal allele

* KCNQ1OT1 promoter is a repressor of neighboring genes. However, highly conserved repeats in the 5′ end of the KCNQ1OT1 transcript is not necessary for imprinting

* KCNQ1OT1 is transcribed in all tissues but its role in imprinting is tissue specific

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 inheritance is autosomal inheritance, not sex-linked. Both male and female could have BWS. and around 1/2 of the offspring would get BWS

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.

Since the inheritance in autosomal, the chance of passing the mutant chromosome/alleles from mom to child is 50% for each child. If the mutant maternal copy is passed to child, then child have BWS. If WT maternal copy is passed to the child, then the child is normal.

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.

BWS: The mutation is an inverted duplication of ICR2 and a small portion of 5‘ of KCNQ1OT1 in cis. When this mutation in inherited from mother, only one of the maternal ICR2 allele is methylated while the additional allele is not. Also, the paternal allele is not methylated. The additional partial KCNQ1OT1 is transcribed because of lack of methylation so the total amount of KCNQ1OT1 transcribed is higher than WT. The transcribed KCNQ1OT1 interacts and represses CDK1NC expression so there is lower overall level of growth inhibitor CDK1NC (essentially, the paternal KCNQ1OT1and transcribed truncated maternal KCNQ1OT1 both repress CDK1NC), which could affect growth at molecular level and macroscopic level so that the patient have higher birth weight.

If mutation is inherited from father, then there is no deviation from WT cellularly or macro- phenotype. The methylation pattern is normal: methylated for maternal allele, and non-methylated for the paternal allele (and the duplicated allele). But the expression of protein IGF2 might be higher than normal.

SRS: The inverted duplication of entire 11p15.5 cluster in cis is detected. Both maternal copies are methylated. Methylated ICR2 inhibits the transcription of KCNQ1OT1 and this result in the higher transcription of KCNQ1 and expression of this protein. Also there is higher level transcription of CDK1NC, and low relative level of IGF2, a growth factor. This high level of CDK1NC protein and low level of IGF2 protein (relatively) affects downstream genes/ protein expression which ultimately may result in the slow growth of the patient at organismal level.

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

Fig 7 a): Experimental results after performing qPCR analysis of ChRIP-purified RNA from lymphoblast cell lines. Using primers located 500bp downstream of transcription start site, the researcher showed that KCNQ1OT1RNAS interacts with chromatin in all four cell lines. In BWS patients, KCNQ1OT1 RNA was detected to be much higher than in control. This is true for BWS patients that had maternal duplication of ICR2, or patients with ICR2 hypomethylation & no microduplication. This high level of KCNQ1OT1 RNA transcript detected in BWS patients likely due to the lack of ICR2 methylation.

Fig 7b): The researchers uses SNPs to detect whether the KCNQ1OT1 RNA from (a) is the paternal allele or maternal allele. They showed that similar level of paternal allele is detected for control and all BWS patients. But while no maternal allele is detected for control, the BWS patients have significantly higher maternal allele KCN1OT1 RNA in the cell lines. This part of figure showed that BWS patients’ high KCNQ1OT1 RNA detected in (a) is likely due to the high transcription level of maternal allele, not the paternal allele.

Fig 7c): The ChRIP RNAs from the control and BWS patients were retrotranscribed and sequenced. The sequence clearly shows that the BWS patients had a dual RNA type expression while control only have one allele transcribed. They also sequenced the control’s DNA to demonstrate that the control have heterozygosity genetically, so the single allele detection at ChRIP RNA is not because of having two copies of same allele but because only one allele was transcribed.

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

It’s possible that the sequence of the ICR itself is not sufficient for establishing imprinted methylation. Orientation of the ICR sequence and it’s methylation pattern may play a significant role in imprinting.

7. Please list any questions and points of confusions relative to imprinting and the regulation of Hox genes (if nothing much comes to mind, please think about it and post on FB or on the Connect Discussion board). We will take the time to clear up your questions before starting on X inactivation.

8. Bonus: do you think the phenotypes of BWS patients 1-3 are representative of BWS? justify.

* BWS patients 1-3 do not represent BWS phenotype as whole because of the wide range genetic variations that result in BWS and it’s correct diagnosis is still under study. In this paper, it is only a 160kb duplication that seems to result in BWS in the three patients, it’s possible the duplication can have different sizes and occur in different locations of the 11p15.5 locus.

Assigned paper:Lonfat et al. (2013). Transgene- and locus-dependent imprinting reveals

allele-specific chromosome conformations. PNAS 110(29): 11947-51

You are welcome to work on this set of questions collaboratively or individually, and to request help as/if needed. You will hand in your answers at the end of class (one set of answers per group if you work collaboratively).

Before you start: spend about 4-5 minutes comparing your answers to the “background questions” within your group.

Specific questions about the article

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

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

Looking at the paper again – They used Hoxd9LacZ and Hoxd11LacZ , positioned into the integrin- alpha- 6 gene.

b) What do the data show (Panel C)?

-whole-mount staining of beta-gal activities in embryos heterozygous for the transgene with (HoxDrel5) and without the inversion (HoxDInv(rel5-Itga6).

– when the transgene is not inverted, the beta-gal activities staining is similar regardless of whether the transgene is inherited from mother or father.

– Embryos with inverted transgene inherited from father also show betal-gal expression as the “non-inverted” embryos. But the inverted- transgene inherited from mother did not show beta-gal activities

c) What is striking/unexpected about the results, and why?

* Paternal inherited inverted-transgene embryos expressed heavier betal-gal expression because it is higher expression level than without inversion.

* Maternal inherited inverted-gransgene embryos expressed little to no beta-gal expression, this is suprsing because the paternal inherited inverted transgene embryos have much higher expression.

d) What conclusions do you make from the data?

Depending on which parent donates the inverted-transgene, the beta-gal expression will be different. For maternal inhierted: there is no beta-gal expression ad for paternal inherited: there is high beta-gal expression.

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?

-insert inverted transgene in many places that are upstream or down stream of Hga6. There was no imprinting, only found imprinting at Itga6 locus

* they remove lacZ from Hoxd/lacZ found through RT-QPCR insitu hybridization that there was no imprinting when there is no lacZ. This showed lacZ is required for imprinting at Itga6 site

* Replaced Hoxd9/lacZ insert with Hoxd11/lacZ and found there is imprinting in inverted transgene embryos.

* Yes these experiments showed that the imprinting is specific to lacZ, site specific, and imprinting is not due to Hoxd9

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).

The black dots represent methylated bases and the white circles represent The more black, the more methylation of the given sequence.

b) What do the data in Figure 2 show?

A. Shows the degree of methylation in parental methylatable cytosine nucleotides in the hoxd9/lacz reporter gene. The paternal data shows a lack of methylation and the maternal data shows high methylation of the transgene.

B. This figure shows the degree of methylation of “escapers”(which are from individuals with lesser degrees of methylation of the transgene) The higher degree of methylation implies a higher degree of imprinting. A ‘strong’ escaper has less methylation.

C. This figure shows that sperm (paternal) has less methylation on their transgene than oocytes (maternal)

D. This figure shows the degree of methylation with noninverted transgenic individuals. There is low methylation in both maternal and paternal transgenes

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

Sperm is always paternal, oocytes are always maternal

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

Escapers are referring to individuals that had a lesser degree of methylation than would be expected based of which parents donates the transgene. (EG maternally donated transgenes are expected to be high in methylation. A low degree of methylation would be an escaper.)

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)

They were comparing the H3K9 trimethylation in paternally and maternally inherited transgenes, because H3K9me3 is a marker for transcriptional repression. Tri-methylation marker was highly present in maternal/+embryos but not in paternal/+embryos, which shows that maternal imprinting likely occurred in all embryonic cells.

5. 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. You don’t need to answer this question in writing.

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

A: The degree of interactions between the transgenes of maternal and paternal donated copies. Paternal copies of the transgene showed higher interaction between transgene with Dlxx1 and Dlx2 genes. Maternal transgene have lowers interaction between transgene and dlx& dlx2

B: the RT-qPCR on mRNA Maternal transgene have a significantly higher dix1/2 expression than WT. The paternal transgene have also a higher dix 1/2 expression than WT but not as high as maternal transgene.

C & D. diagrams show that possible models of the interactions between the inverted transgenes and the digit enhancer regions and the digit enhancers to dlx 1/2 locus. For maternal inherited inverted transgene, the highly methylated region chromatin compaction prevents interactions between the digits enhancers and invervted transgene.

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

o They concluded that presence of LacZ reporter was necessary for maternal derived expression.

o The inversion of the lacZ transgene has a different expression depending on which parent it is donated from.