This web page was produced as an assignment for Genetics 677, an undergraduate course at UW-Madison.
Figuring out the nuts and bolts of Nanog
Figure 1: Image modified from references 5-7 to illustrate the use of chemical genetics, global gene and protein analysis in attempt to understand the mechanics of cell immortality through Nanog.
There are several categories of experiments to be carried out to decipher the function and methods to control this marker of immortality, Nanog.
Firstly, experiments should focus on function analysis.
This will include investigations on phosphorylation as a possible activation mechansim for Nanog. This could be done by mutating or removing individual phosphorylation sites predicted by NetPho 2.0. Mass spectrometry of purified Nanog protein, mutated and wildtype, could be done to confirm if phosphorylation takes place by mass comparison. Function analysis could be performed by providing mutated Nanog protein to Nanog Knockout mice models and determining if rescue of Nanog function occurs.
Next immunoprecipitation of Nanog and other proteins of the protein interaction network should be carried out. This will experimentally prove the interaction of Nanog and the proteins found via text-mining on STRING. In addition, parts of Nanog protein should be truncated in various combinations to determine which is the interactive domains of Nanog; for example, the N-termini, C-termini and homeodomain both individually and in combination.
Also, chromatin immunoprecipitation and cDNA microarray hybridization (ChIP-array) will be useful to determine possible protein and Nanog DNA interactions and transcription regulation on a genome-wide level. By understanding what protein regulates Nanog transcription, possible methods could be derived to control the expression level of Nanog on a transcriptional level.
Then, experiments should continue efforts on global analysis.
Chemical genetics will also be useful in seeking ways to control Nanog protein expression level - a useful pharmaceutical application may be to alleviate Gestational Trophoblastic Disease or breast and/or testicular cancers. By making use of Nanog protein structural information and screening for possible chemical molecules present only after blastocyst stages, it may be possible to discover Nanog chemical regulators. These chemical regulators may be tested in model organisms or embryonic stem cell cultures to develop pharmaceutical products that may be useful for alleviating GTD or cancers by reducing expression level of Nanog.
Transcriptome or microarray studies on proteins of Nanog protein interaction network like Oct3/4, Sox2, Klf4, should be done while manipulating expression levels of Nanog in model organisms or pluripotent stem cells. Reverse experiments should also be done, microarray studies of Nanog genetic and protein expression levels should be carried out when manipulating individual protein levels like Gata 6, Oct 3/4, Wnt11, and while in combination. It will be interesting to decipher which proteins actually have a direct impact on Nanog transcript or protein expression level. By uncovering these regulatory proteins, on a transcriptional and translational level, we will be able to control the expression of Nanog and thus freely control cell immortality and prevent overexpression of Nanog.
And finally, making use of model organisms to understand in vivo reprogramming and Nanog function.
By understanding the relationship of model organisms and human Nanog through phylogenetic analysis, comparative in vivo functional studies of Nanog can be performed. In C. Elegans, tab-1 protein could be studied and analogies could be drawn to functional behavior of Nanog in humans. Similarly, Drosophila, the popular fly model, has Brain-specific-homeobox (Bsh) that could be studied and used to understand Nanog function and possible regulatory mechanisms. Protein interactions of tab-1 and Bsh may be extrapolated to hunt for possible protein partners of Nanog. In addition, these orthologues may be used to determine domains that are functionally essential for Nanog.
Other than animal models, Nanog has a great advantage of being expressed in embryonic stem cells, or the inner cell mass of a blastocyst. As embryonic stem cell lines are readily available in in vitro conditions, the human cell lines could be used to perform in vitro functional studies of Nanog gene or protein.
By making use of model organisms and performing functional studies of Nanog, methods to regulate Nanog may be developed and mechanisms of Nanog function may be uncovered. In the understanding of Nanog as a marker of cell immortality, the gap to synthetically manipulate cell immortality at will will be drawn closer. The importance of understanding cell immortality may just be the key that opens the gate to cell therapy and regenerative medicine for tomorrow.
Figure 2: Image modified from references 1-4 to illustrate the common model organisms used in Biological experiments; in a clockwise manner: C. Elegans, Drosophila, Mus muculus and Gallus gallus.
References
1. Social Fiction. Retrieved April 18, 2009, from http://www.socialfiction.org/img/Enlarged_c_elegans.PNG
2. Brembs. net Retrieved April 19, 2009, from http://brembs.net/learning/drosophila/fly_down.jpg
3. Retrieved April 18, 2009, from http://64.207.144.142/movabletype/PETA2Daily/archives/kick1.PNG
4. Transhuman Express. Retrieved April 18, 2009, fromhttp://images.forbes.com/images/2001/07/26/mice_400x300.jpg
5. DCAD. Retrieved April 19, 2009 from http://dcad.com.pl/image/dna1.jpg
6. DNASTAR. Retrieved April 19, 2009 from http://www.dnastar.com/media/linegraphLrg.jpg
7. School of Information Technology. Unversity of Sydney. Irena Koprinska. Retrieved April 19, 2009 from http://www.cs.usyd.edu.au/%7Eirena/arrayimage.jpg
Ka Yi, Ling ([email protected])
Page last updated 05/12/09
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