How does Nanog maintain stem cell self-renewal? : A study using Super-Resolution Microscopy

Session

Early Career Symposium & RMS Early Career Committee AGM

Authors

Dr. Aishwarya Sivakumar (1), Dr. Nicholas Mullin (1), Prof. Ian Chambers (1)

Affiliations

1. University of Edinburgh

Keywords

Nanog, SIM, transcription factor, ESCs, chromatin

Abstract text

Embryonic Stem Cells (ESCs) when cultured in vitro rely on the presence of a cytokine called Leukemia Inhibitory Factor (LIF) for their self-renewal. Nanog is a transcription factor that utilizes its homeodomain to bind DNA and, when over-expressed in ESCs, is capable of driving LIF-independent self-renewal. Outwith the homeodomain, Nanog has a low complexity tryptophan repeat (WR) domain through which it dimerizes and also binds to partner proteins such as SOX2. Substitution of the tryptophan residues in the WR with alanine (Nanog-W10A) abolishes the self-renewal function of Nanog. In this study we have used structured illumination microscopy (SIM) to gain insights into the functions of Nanog and Nanog-W10A in the ESC nuclei. We have found that Nanog forms distinct punctae ranging from 120nm to 2μm in size. We further demonstrate that the ability of Nanog to bind the DNA through the homeodomain is crucial for the formation of the larger punctae (>800nm). Surprisingly, we found that despite of the presence of the homeodomain, the ability to form larger punctae is compromised in Nanog-W10A. This highlights the specific importance of protein-protein interactions in the formation of these larger structures. We propose that the formation of larger punctae by Nanog is the key to its ability to bridge long-range genomic interactions (LRIs), which are a feature of the open chromatin state observed in ESCs. Using the current super-resolution microscopy technique, accurate quantitation is restricted to punctae larger than 150nm. Ongoing work using stimulated emission depletion (STED) microscopy will also allow us to quantify the physical characteristics of the smaller Nanog-W10A punctae including size and numbers. This information will help define if the WR is also necessary for the formation of short range chromatin interactions in ESCs. These comparisons between Nanog and Nanog-W10A using super-resolution microscopy will yield crucial insights into how a low complexity domain like the WR contributes to transcription factor function in the maintenance of pluripotency.

References

1. Mullin, 2008. The pluripotency rheostat Nanog functions as a dimer. Biochem. J. 

2. Gagliardi, 2013. A direct physical interaction between Nanog and Sox2 regulates embryonic stem cell self-renewal. EMBO J.

3. Novo, 2016. The pluripotency factor Nanog regulates pericentromeric heterochromatin organization in mouse embryonic stem cells. Genes Dev.

4. Mullin, 2017. Distinct Contributions of Tryptophan Residues within the Dimerization Domain to Nanog Function. J. Mol. Biol.

5. Novo, 2018. Long-Range Enhancer Interactions Are Prevalent in Mouse Embryonic Stem Cells and Are Reorganized upon Pluripotent State Transition. Cell Rep.