Jeevika Goyal 1209306

Tutorial Assignment 2

December 2nd, 2013

1. What is the title of the research paper that the news article is referring to? Provide a complete reference indicating authors, date of publication, and journal. Use the AMA referencing style. ( 1 mark) The title of the research paper that the new article is referring to is translating dosage compensation to trisomy 21. Reference: Jiang J, Jing Y, Cost GJ, Chiang JC, Kolpa HJ, Cotton AM., Carone DM, Carone BR, Shivak DA, Guschin D Y, Pearl JR, Rebar E J, Byron M, Gregory PD, Brown CJ, Urnov F D, Hall L L, Lawrence JB. Translating dosage compensation to trisomy 21. Nature. July 17th,2013; 500(7462):296-300. 2. Why did the authors study this topic? ( 2 marks) Down syndrome is one of the leading genetic causes of various intellectual disabilities and affects 1 in 1000 newborns. It is caused by an extra chromosome 21, normally referred to as trisomy 21. Authors study was motivated by the idea to silence the extra chromosome with a gene. This would assist the author in understanding any disruptions in cellular or gene pathways due to Down syndrome. Xist gene in nature silences the extra X chromosome in females, to compensate for only 1 X chromosome present in males. Using its RNA, it induces numerous heterochromatin modifications and architectural changes to silence the transcription in the inactivated Xchromosome. Authors took this novel approach presented by our body to establish natural equilibrium, to find a way to treat Trisomy.2 Additionally, Dr. Jennifer Lawerence, the senior author of this experiment, had previously shown that, Xist RNA paints the inactivated X chromosome, and is further investigating as to how this non - coding RNA, Xist RNA, induces heterochromatin modifications to restructure the chromosome.3 Therefore, this study would be an extension to her project, as the author uses the same Xist gene, to inactivate the additional X chromosome, by inducing the similar heterochromatin modifications. 3. What are the causes of different types of Down syndrome? Briefly explain them. (3 marks) Down syndrome is a genetic disorder, caused by an extra copy of chromosome 21. This additional copy of the chromosome could be a result of nondisjunction or translocation; giving rise to three types of Down syndrome, trisomy 21, mosaicism and translocation. Trisomy 21 is caused by nondisjuction in meiosis I or II, in the egg or sperm cell that becomes zygote, in which a pair of chromosome 21 fails to separate; leading to an extra copy of chromosome 21. Trisomy 21 is the leading type of Down syndrome that affects people as it accounts for about 95% of the cases. Mosaicism, another type of Down syndrome, is similar to trisomy 21, as it is also caused by nondysjunction. However, in Mosaicism, not all of an individuals cell carry an additional copy; and as result a mixture of both types of cells is present in the patient. Mosaicism is the result of Page 1 of 4

Jeevika Goyal 1209306

Tutorial Assignment 2

December 2nd, 2013

nondysjuction after the fertilization, probably during early stage of cell division, giving rise to two types of cell line; one with trisomy and one with normal number of chromosome. Contrarily, nondysjunction in Trisomy occurs prior to fertilization; which is why, unlike Trisomy 21, not every cell is affected in Mosaicism.4 Mosaicism rarely affects individuals, as it accounts for only 1% of all cases. Additionally, individuals with Mosaicism exhibit fewer characteristics of Down syndrome as oppose to individuals with Trisomy 21. Lastly, translocation, another type of Down syndrome, which accounts for 4% of all cases, is caused when a piece of chromosome 21 breaks off and attaches itself to another chromosome. Despite the regular number, presence of extra piece of copy of chromosome 21 causes Down syndrome. Unlike, trisomy 21 and mosaicism, Translocation is hereditary. Translocation in chromosomes, like Trisomy 21, also occurs prior to fertilization in sperm and egg cells that become zygote.5 4. How was Xist gene inserted on chromosome 21 and how was it expressed? Briefly describe the technique. ( 6 marks) The root of this experiment is to utilize the natural function of Xist gene, which is to silence the extra chromosome in females. Doxycycline controlled Xist gene was inserted on chromosome 21 using the zinc finger nuclease (ZFN). The complication in inserting this Xist gene is caused by its large size. In the experiment, Xist transgene was inserted into DYRK1A locus of chromosome 21 and a doxycycline control component was inserted into AAVS1 of chromosome 19 simultaneously. This insertion was done in the pluripotent stem (iPS) cell line obtained from a male Down syndrome patient. The purpose of doxycycline in the experiment is to induce Xist gene expression in the cells.1 Zince Finger Nucleases are engineered protein class that used to create a double strand break with the genome. It is used to insert a target gene into the genome. Upon expression, ZFN binds to their target site to create a specific double stranded break using the Fokl nuclease that acts as its catalytic dimer. The cell tries to repair this break via non homologous end joining or homologous recombination. When the cell is provided with an external donor plasmid; whose end sequence is homologous to the target region, it will use this donor piece of DNA, to initiate homologous recombination repair. ZFN is inserted into the nucleus via a plasmid vector and is expressed using an inducible promoter called TetO2.6 Fluorescence in situ hybridization (FISH) and Southern Blotting were used to confirm whether the gene was accurately added to the chromosome. 5. What is OCT4 and why was used in this study? ( 2 marks) Octamer binding transcription factor 4 (OCT4), also known as POU5F1, is a transcriptional factor that is used to maintain an undifferentiated state of the cells. This is a protein that is encoded by POU5F2 gene in humans. Oct4 controls the transcription of certain genes. This transcriptional factor is required for early embrygenesis, as it is required to maintain undifferentiated cells in human embryonic cells.7 In this study it is for Oct4 immunostaining, in which Oct4 is used as a marker to check for pluripotent cells. This was done before insertion of Xist gene. 1

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Jeevika Goyal 1209306

Tutorial Assignment 2

December 2nd, 2013

6. How was determined that Xist transgene product behave the same as the endogenous Xist ? ( 2 marks) The Xist gene silences the chromosome 21 by coating it with its non coding RNA (Xist RNA), which then triggers stable heterochromatin modifications, silences transcription throughout the chromosome and triggers DNA methylation to form a chromosome 21 Barr Body. Using FISH technique, it was determined that Xist transgene product behaves the same as the endogenous Xist. This is because, just like endogenous Xist RNA, Xist transgene RNA coats the chromosome 21, in order to silence it. Additionally, just like endogenous Xist forms Barr Body to silence the chromosomes, a similar expression of Xist transgene was confirmed by RNA FISH, as five days post the induction of Xist gene, heterochromatin marks were observed in the edited chromosome 21 and in various nuclei chromosome 21 DNA condensed; thereby, indicating the formation of chromosome 21 barr body. Other methods used to confirm expression of Xist gene were: RNA microarray, DNA methylation array, heterochromatin hallmarks, qRT PCR, gene SNP analysis, Barr body formation and RNA FISH to hnRNA.1 7. Briefly describe the importance of this study. ( 3 marks) This experiment is significant as it may prove to be a milestone in development towards potential genetic approach to chromosome therapy, as a way to address Down syndrome. This is because the first step in developing of any clinical gene therapy is to find a way to functionally correct the genetic defect in the cell; which through this study is now achievable. Additionally, through the means of this experiment, author strives to provide a system to study Down syndrome cell pathology. This is important, as previously, disruptions in cellular pathway causing this condition, was poorly understood. This advancement will assist in development of better drugs to address this condition. Lastly, this new technique of using Xist gene to repress the expression of the extra copy of the chromosome and to reverse its effect on the cell can be extended to other trisomy chromosomal disorders, such as trisomy 13 and 18. Also, the insertion of Xist gene, opens doors to insert triple the size of previously synthesised transgene in a host, as Xist gene is one of the largest transgene to be inserted in a host. 1 8. Provide at least two other approaches the authors could undertake to improve on this study? ( 2 marks) Authors can further improve this by investigating whether this autosomal silencing is hereditary. This would ensure that the future generation do not carry this extra chromosome in silenced form, and if they did, possible further implications could be investigated. Additionally, authors can further investigate, the implications of possible inheritance of this single chromosome, as it may cause nullsomy or monosomy, depending on whether the chromosome remains silenced. Lastly, author may further investigate possible ways this silencing can become unstable, as it may prove to have deleterious effects in long run. This is because author talked about stability of this silencing but did not talk about the ways this silencing could become unstable.

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Jeevika Goyal 1209306 WORK CITED:

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Tutorial Assignment 2

December 2nd, 2013

Jiang J, Jing Y, Cost GJ, Chiang JC, Kolpa HJ, Cotton AM., Carone DM, Carone BR, Shivak DA, Guschin D Y, Pearl JR, Rebar E J, Byron M, Gregory PD, Brown CJ, Urnov F D, Hall L L, Lawrence JB. Translating dosage compensation to trisomy 21. Nature. July 17th,2013; 500(7462):296300.
2

University of Massachusetts Medical School. Lawerence Lab. ttp://www.umassmed.edu/cellbio/lawrencelab/pi.aspx. Accessed December 2, 2013.
3

Clemson CM, Mcneil JA, Willard HF, Lawrence JB. XIST RNA paints the inactive X chromosome at interphase: evidence for a novel RNA involved in nuclear/chromosome structure. J Cell Biol. 1996;132(3):259-75.
4

National Down Syndrome Society. What is Down syndrome. http://www.ndss.org/DownSyndrome/What-Is-Down-Syndrome/. Accessed October 21, 2013.
5

Julian. Down Syndrome. http://blog.gretchenmather.com/2011/08/05/what-is-down-syndrome3-types-faqs-recent-photos/. Accessed December 2, 2013.

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Sigma Aldrich. ZFN Technology. http://www.sigmaaldrich.com/technicaldocuments/articles/biowire/zfn-technology.html. Accessed December 2, 2013.

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Kellner S, Kikyo N. Transcriptional regulation of the Oct4 gene, a master gene for pluripotency. Histol Histopathol. 2010;25(3):405-12.
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UniProt. Po5F1_HUMAN. http://www.uniprot.org/uniprot/Q01860. Accessed December 2, 2013.

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