I chose this assignment because I enjoyed discussing the aspects of this paper after studying for the quiz. It showed how much I actually know about the paper and also this assignment helped fill the holes in my understanding of the paper. This was a very difficult paper for me because of the clinical side of things.

Monday March 10^th in-class “assignment”:

studying SRS and BWS patients as a way to elucidate regulatory mechanisms of imprinted loci

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

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?

Comparisons

1. Both congenital disease, growth disorders

2. Contrast
   1. SRS:
      1. Associated with growth restriction
      2. Facial characteristics altered
         • Shape, etc.
      3. BWS:
         1. Associated with overgrowth
         2. Minor details
            1. Lip size, nasal shape, ear crease

• Internal defects

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.
   1. Looked up using the accession number: 130650
   2. States: Beckwith-Wiedemann Syndrome Chromosome Region, Included; BWCR included

3. Look at the pedigree in Figure 1.
4. What can immediately be concluded about BWS (even without knowing who inherits the mutant allele from whom)?
   1. Not sex linked; autosomal
   2. Roughly 50% of offspring inherit
      1. Makes sense because only on one of the inherited chromosomes

1. 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.
   1. It depends on which maternally imprinted chromosome the offspring inherits
      1. For individual I-4, only one of her chromosomes has the microduplication. Both of the chromosomes are maternally imprinted.  This means that offspring have a 50% change of inheriting the microduplicated maternally imprinted chromosome.  If the offspring happen to inherit the microduplicated chromosome, they will have the BWS 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.

1. BWS:
   15. 160 kb inverted microduplication (in cis) consisting of ICR2 sequence and exons 12-15 of the KCNQ1 and 5’ 20kbp of the long ncRNA (KCNQ1OT1) in locus 11p15.5
   16. Methylation of duplicated ICR2: hypomethylated, which is inconsistent with the usual methylated status.
   17. This mutation results in the expression of a truncated long ncRNA and a distinct down regulation of the protein coding genes within the adjacent cluster (CDKN1C)
2. SRS
   1. 2 Mbp inverted duplication (in cis) within the locus 11p15.5
   2. Methylation: is (mainly) consistent with the control group
      1. However, there is a double dosage of the protein coding genes from the maternal chromosome
      2. This results in twice the expression of these genes.

5. Explain what the data in Figure 7 show, and how you interpret them.
6. Figure 7: Looking at the interaction of the long ncRNA with the chromatin using qPCR analysis of ChRIP-purified RNA
   1. From the co-precipitated RNA, they differentiate between maternal and paternal alleles using specific SNPs.
   2. Figure 7a: Overall, compared to the controls, there is a significantly enriched interaction between the long ncRNA and the chromatin.
   3. Figure 7b: The paternal allele (full length RNA) is not significantly different from the control but the maternal copy is significantly more enriched for the patient showing the BWS phenotype. Most interestingly, this is consistent for a patient with no duplication but hypomethylation of ICR2 (BWS P4).  Majority of this difference is coming from the maternal allele as.  This hints at a common mechanism of action between the duplicated patients (P2 and P1) and patient P4.
   4. Figure 7c: They conduct sequence analysis at SNPs of cDNA obtained from ChRIP. Results are consistent with that seen in Figure 7a and 7b.  In the control, only see 1 full length copy, but in all BWS patients, you see the truncated as well as the full length copy of the long ncRNA.
7. How to interpret:
   1. Need to compare the histograms (control vs. two different patient groups)
   2. Figure A and B: Is measuring fold enrichment of KCNQ1OT1 over IgG- quantitative
   3. Figure C: Is using sequencing to analysis expression- qualitative

6. What valuable fundamental information was gained about imprinting control regions through the study of these patients?
   1. Imprinting is not only regulatory sequence dependent (eg. ICR2) but also requires information from the adjacent sequences.
   2. Imprinting mechanism in sperm and oocyte are distinctly different.

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.
   1. Although the authors concluded that the ICR2 sequence in the microduplication is insufficient for proper imprinting, what other factors are required?
   2. Pertaining to Figure 7: with the histograms. If Patient 4 had similar expressions of ICR2 since ICR2 is hypomethylated in both and with the microduplication, one copy of ICR2 is normally methylated, so the genetics seems like it should be of the same results (since they sort of equal to each other); why is there such a huge difference in chromatin enrichment in P4?
   3. How is imprinting erased and re-established during gametogenesis?

8. Bonus Question

• No: Because this is one specific case of this duplication. Other patients with BWS can inherit the disorder differently or have different epigenetic and genetic mutations in both coding and non-coding that cause this phenotype.  For example, Patient 4 in the paper has no duplication and instead just hypomethylation that causes the same sort of symptoms.

This assignment was my first exposure to imprinting. I found this paper a little difficult to tackle but after my group discussion and finishing this assignment, I was able to understand what was going on.

Monday March 2^nd in-class “assignment”: the mystery of the lacZ transgene

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

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

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:
2. What transgenic lines did the author use (do these lines look somewhat familiar)?
   1. HoxD^rel5
   2. HoxD^{Inv(rel5-Itga6)}

These lines were created by the insertion of the transgene Hoxd9lacZ upstream of the Evx2 HoxD locus.

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

• Used a stain-and-look approach to observe lacZ
• The data shows that the 2.7Mb inverted genomic fragment containing the reporter resulted in the absence of lacZ expression in the offspring possessing the maternally inherited allele while lacZ expression in the offspring possessing the paternally inherited allele was increased relative to the uninverted HoxD^rel5 line.

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

• Upon inversion, there is a substantial change in lacZ expression between the offspring possessing the maternally inherited allele and the offspring possessing the paternally inherited allele while this substantial change in the HoxD^rel5 line is not observed.

1. What conclusions do you make from the data?

• Positioning Hoxd9lacZ closer to Itga6 ex1-24 results in parental-specific expression of the reporter lacZ where expression from the paternal allele is observed, but not from the maternal allele.

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?

• lacZ-specific
  • They relocated the native Hoxd11 gene not including the lacZ transgene into Itga6 Upon staining for Hoxd11 expression, they found no significant difference between the alleles inherited from either parent.  This result implies that the imprinting to repress the expression of the maternal allele requires the presence of the lacZ sequence.
• Position/site-specific?
  • Inserted the Hoxd9lacZ into various positions within the HoxD centromeric landscape, none of which resulted in parental-specific expression. This result showed that this is position specific.
  • When the Hoxd9lacZ reporter was placed before the inversion, the transgene did not show parental-specific expression, illustrating that the imprinting was site-specific.

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).

• White circles: unmethylated C’s
• Black circles: methylated C’s
• Each row indicates an individual biological replicate.

1. What do the data in Figure 2 show?

• In the inverted line, it is only the maternal alleles that show methylation of the cytosine’s and this result is also seen at the level of oocytes and sperm.
• Inconsistent intermediate methylation was observed in escaper genes from both the maternal and paternal alleles.

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

• Mothers don’t have sperms.
• Fathers don’t have oocytes.

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

• Despite inversion of the genomic sequence, escaper embryos did not have maternal-inherited imprinting.
• This was shown by looking at lacZ activity and the researchers determined that the escapers had clonal patches of cells and streaks with 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 ChIP experiments were done to look at the accessibility of the chromatin in these specific locations by analyzing H3K27me3, H3K9me3, and H4K20me3.
• The repressive marks (H3K9me3 and H4K20me3) were more prominent in the maternal allele.

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).

• The figure shows how the change in the local chromatin conformation affects the expression of neighbouring genes, such as Dlx1/2.
• Proposed a hypothesis:
  • In the maternal case for the inversion, because it can no longer associate with the Itga6 locus, the digit enhancers are now closer to the Dlx1/2 promoter, thus increasing expression of these genes. There was a slight up-regulation in the paternal case, but it was much more significant in the maternal case.  The result was observed because the digit enhancer in the paternal case is still able to interact with the transgene, thus reducing the interaction with the Dlx1/2 locus.

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

• Why is this maternal imprinting specific to the lacZ sequence?
• There was a high incidence (33%) of escapers of the maternal imprinting.

In the inversion line, there was an observed increase in lacZ expression in the paternally inherited allele

I chose to highlight this assignment because I really enjoy dissecting and answering questions as a group. It really helps me learn and understand the paper a lot better. This assignment also opened my eyes in the importance of studying limb development.

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

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

• Coordinated determination of three axes:

1. Anteroposterrio (AP)- earliest axis to be defined
2. Proximodistal (PD)- controlled by apical ectodermal ridge (AER)
3. Dorsoventral (DV)-

• ZPA = important signaling centre = expresses SHH
  • Defines AP axis
• AER produces growth factor and signals needed for PD growth of limb
• Need feedback loop between ZPA and AER that maintains both of them and for coordinated growth along axes and DV polarity maintenance

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?

• Helps in categorizing types of malformations, subsequently guides the therapies that may be used to treat the malformation
• Each finger looks different which means each digit had to develop differently to result in these morphologies. Spatial and temporal order is important and can help you understand why specific malformations result.
• Easily observable

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: mutations in the cis-regulatory regions of a gene (this means that it only affects that location of genes)
• Syndromic: other phenotypes, if there’s an error in the coding region of transcription factor it will affect the regulation of many genes, resulting in the 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?

• PAX6: locus on Ch11
  • Haploinsufficiency is cause of congenital eye malformation aniridia
  • ELP4: Multiple enhancers for PAX6 have been identified within ELP4 introns (synteny)
  • SIMO: conserved ocular enhancer involved in autoregulation of PAX6
    • If you have a mutation in SIMO, it interferes with the binding of PAX6
    • Says: “in a patient” – (Bhatia et al. 2013)
      • Not statistically significant
      • Need a larger sample size (more patients)
    • Design in vitro studies to confirm and further understand the relationship between SIMO and PAX6 binding affinity
      • How does a mutation in this enhancer reduce PAX6 binding affinity?
      • What is different about the mutated PAX6 protein? Does the amino acid sequence change?

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:
  • Order in which gene is located within a chromosome, that can be passed on
  • Heritable pattern of gene position on a chromosome
  • You can trace evolution through this (eg. If a cat and a dog have a gene in the same location, shows evolutionary relationship)
• If you have the CRE in the intron of one gene regulating the transcription of another gene, it shows their evolutionary relationship
  • You need both genes to be maintained in order for the second to be functional

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?

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?

• Patients and Subjects
  • Detection of diseases (diagnostics) and stratification (putting diseases into different categories)
    • Stratification of cancer: can all be in different categories
    • This is important for designing treatment that targets the specific cancer type (ie. Molecular aberrations)
  • Identification of drugs that may be more effective for patients with certain molecular aberrations (ie. Patient specific, personalized medicine)
• Community benefits
  • Understanding the genetic component important for screening and predicting/prevention or at risk (eg. BRCA)

TECHNIQUES SPEED-DATING PRESENTATIONS: WRITE-UP
Names and contributions of group members:
Mandy Feng: in-depth research for mini report, prop preparation, final editing
Jordan Henriksen: group coordination, in-depth research for mini report, prop preparation
Phillip Chau: in-depth research for mini report, final editing
Abhijit Parolia: in-depth research for mini report, final editing
Ryan Yen: in-depth research for mini report, final editing

Video: CRISPR-Cas Technique
https://www.youtube.com/watch?v=MXDwHBMDq8g

Technique chosen:
CRISPR-Cas Technology: Clustered, Regularly Interspaced, Short Palindromic Repeats-CRISPR Associated

What does this technique ‘do’?
Introducing double-stranded breaks in DNA in a sequence-specific manner

What applications is this technique employed for?
The central purpose of employing this technique is to study gene function in physiological and diseased states by one of the following methods:
1. Simultaneous editing of multiple genes mediated by exogenous small guide RNAs (sgRNA) and Cas9 nuclease complex (Pennisi, 2013).

-Wang et al. (2013) verified mutations in all of the 5 targeted gene sequences in a single                             eukaryotic cell using gene-specific sgRNAs.
2. Reversible gene knockdowns, which is a complementary technique to RNA interference (Pennisi, 2013).

-In a prokaryotic cell, Qi et al. (2013) demonstrated ~100-fold downregulation of a reporter gene using ‘dead’ Cas9 (dCas9):sgRNA complex directed to the -35 box, upstream of the coding sequence.
-In an eukaryotic system, which involves more robust regulatory control mechanisms, this approach is enhanced by fusion of dCas9 to a mammalian transcriptional repressor domain. (Gilbert et al., 2013).
3. Activation of specific genes by delivery of synthetic transcriptional activators to promoter sequences (Gilbert et al., 2013).
-Gilbert et al. (2013) demonstrated that fusion of dCas9 (still directed to the gene promoter) with transcriptional activators effectively resulted in target gene up-regulation, in some cases by >25-fold.

Some additional applications of this technology include:
• Generating gRNA libraries
• Labeling specific chromosomal loci (Sander & Joung, 2014).

What questions (give a couple of examples) relating to gene regulation and/or development can be addressed using this technique?
• Loss of Function Experiments
o Is this gene/protein necessary for a particular function?
o Enables targeted genome editing by insertion or deletion of DNA sequences.
§ If you simultaneously disrupt an enhancer in an ELP4 intron and the enhancer SIMO using the CRISPR-Cas technique, how is PAX6 expression affected (Bhatia & Kleinjan, 2014)?
• Gain of Function Experiments
o Is this gene/protein sufficient to cause a change?
o Allows for over-expression analysis through mutation or the delivery of synthetic transcription factors.
§ If you use the CRISPR-Cas technique to induce a mutation in an enhancer that increases the binding affinity of a particular transcription factor for that enhancer, how does this affect development?

What critical reagents are required to use this technique?
Based on the paper by Sander & Joung (2014), the following reagents are required:
• RNA-guided nuclease
o Cas enzyme specific for required function
• Specific sequence guide RNA (gRNA)
o CRISPR RNA (crRNA) and transactivating CRISPR RNA (tracrRNA)
• Non-replicating plasmid
o For expression of Cas enzyme and gRNA
• Transfection reagents
o Such as electroporation, nucleofection, or Lipofectamine.

What critical information is required to be able to employ this technique?
1. DNA sequence of the gene-of-interest – The ‘seed’ region of the sgRNA is restricted by mandatory presence of a protospacer adjacent motif (5’ NGG) for the Cas9 nuclease activity to occur (Pennisi, 2013).
2. Choice of promoter of the transgenic sgRNA and Cas9 genes – for instance, it needs to be compatible with the eukaryotic transcriptional machinery (Sander & Joung, 2014).
3. Empirically established regulatory protein domains that can affect expression of the target gene – for example, it is critical to know if VP16 can act as a transcriptional activation domain for the gene in question (Gilbert et al., 2013).
4. Configuration of the viral capsid to allow specific targeting of cells – more so in the case of in vivo application (Sander & Joung, 2014).

References:
Bhatia, S., Kleinjan, D. A. (2014). Disruption of long-range gene regulation in human genetic disease: a kaleidoscope of general principles, diverse mechanism and unique phenotypic consequences. Hum Genet 133, 815-845.

Gilbert, L. A., Larson, M. H., Morsut, L., Liu, M., Brar, G. A., Torres, S. E., Stern-Ginossar, N… Qi, L. (2013). CRISPR-Mediated Modular RNA-Guided Regulation of Transcription in Eukaryotes. Cell 154, 442-451.

Pennisi, E. (2013). The CRISPR Craze. Science 341, 833-836.

Qi, L. S., Larson, M. H., Gilbert, L. A., Doudna, J. A., Weissman, J. S., Arkin, A. P., Lim, W. A. (2013). Repurposing CRISPR as an RNA-Guided Platform for Sequence-Specific Control of Gene Expression. Cell 152(5), 1173-1183.

Sander, J. D., & Joung, J. K. (2014). CriSPr-Cas systems for editing, regulating and targeting genomes. Nature Biotechnology, 32(4), 347-355.

Wang, H. (2013). One-Step Generation of Mice Carrying Mutations in Multiple Genes by CRISPR/Cas-Mediated Genome Engineering. Cell 153, 910-918.

This post was from a very early assignment from the beginning of the year. I picked to highlight this assignment because I get to practice how to write a layperson’s paragraph for my final project.

Limb malformation is an important model of gene expression control during the development of an embryo. These diseases are generally a result of a mutation in the DNA which causes some genes to be expressed too much or too little. For an example, if there is too much expression of a certain gene, you may end up with more than five fingers on one hand. Limb development is a good model system to study because it is easy to identify what went wrong during development. For instance, each finger on our hand is different from one another. These differences are due to the different genes being expressed at different locations and time when the baby is developing inside their mother. If we compare the DNA of a patient without thumbs to a normal person, we may be able to find the genes responsible for developing thumbs through the differences in their DNA. It is important to study these diseases to understand how normal limb development works. This allows us to understand what mutations are causing these diseases and by looking for these mutations in a person’s DNA, you may be able to inform potential parents about having children with these deformities. Studying limb development can help researchers find what elements in the DNA are responsible for malformations and can improve methods to identify these elements.

Note: It was difficult to identify what is truly important for the general public to know. There needs to be a balance between being detailed enough to be able to explain my points and not going too in depth which you would need a biology background to understand.