Conversion of Cell Fate Using MYOD1

The road to the discovery of MYOD1 was long and rocky, and incorporated the work of many researchers. Let me tell the story from the perspective of Lassar, the 3^rd author of the ground-breaking paper, and the original author of the idea it was based on. Lassar was interested in how cell fate is determined. At the time, it was known that the transcription factors were probably involved in the process. However, the methods of identifying those factors were very primitive and involved months spent performing column chromatography in a cold-room at 4°C. Lassar was eager to find another method.

Fortunately, he happened to read about the intriguing work of Jones and Taylor, who performed experiments on fibroblasts, which are cells that can be found in connective tissue. The two researchers showed that when embryonic mouse fibroblasts of 10T1/2 line were treated with a DNA demethylating agent called 5-azacytidine, those fibroblasts were converted into adipogenic (fat), chondrogenic (cartilage), or myogenic (muscle) cell lines. 5-azacytidine blocks the activity of DNA methyltransferases, causing the demethylation of CpG residues. Demethylation leads to increased expression of genes that used to be silenced (methylated). The scientists thus theorized that the cell conversions happened because of the activation of regulatory genes. After treating the 10T1/2 cells with 5-azacytidine, those activated (demethylated) transcription regulators induced a different cellular phenotype.

Moreover, other papers by Konieczny and Emerson showed that the conversion from fibroblasts to myoblasts probably happened due to the activation of only one or several loci. This meant that there were only very few possible transcription factors responsible for the conversion, which in turn suggested that identifying myogenic regulator(s) could be a pretty accessible research field.

Therefore, Lassar decided to build his research on the aforementioned scientists' work, aiming to identify the myogenic regulator that converted fibroblasts to myoblasts. He joined the laboratory of Weintraub, the 2^nd author of his future paper, to conduct his research.

Lassar reasoned that the myogenic regulator locus he was looking for would be demethylated and thus active in myogenic cell lines, while in 10T1/2 cells, it would be methylated and silenced. If we assume a single regulatory factor gene is responsible for cell conversion, introducing this gene into those 10T1/2 cells would convert them into myocytes, even without 5-azacytidine demethylating treatment. This hypothesis was sound, but the challenge was to identify the myogenic regulator among thousands of potential candidates.

Lassar struggled greatly with his work, facing many challenges with his experimental design. He spent 3 years treating 10T1/2 fibroblast cells with 5-azacytidine, thus turning them into myogenic cells, then transfecting the genomic DNA from those myogenic cells back into the 10T1/2 fibroblast cells fragment by fragment, trying to find the sequence that induced cell conversion. He later realized that he could change his approach and use a simpler method called subtractive hybridization. The logic is that the myogenic regulator gene is one of the genes expressed in cells converted to myogenic cells, but not expressed in normal 10T1/2 cells. If we want to investigate gene expression, mRNA is what we should be looking at. Therefore, if we isolate the mRNA from 10T1/2 fibroblast cells and cells converted to myogenic cells, RNA fragments present only in the converted cells, but not in the fibroblast cells, contain the myogenic regulator transcript. We can use subtractive hybridization to selectively magnify the fragments present in converted cells only. As part of the procedure, mRNA from the myogenic cells is hybridized with the first-strand cDNA from the fibroblasts immobilized on beads. Only the transcripts present in both myogenic cells and fibroblasts will hybridize with this cDNA, and the mRNA specific to myogenic cells is left suspended in the supernatant. We then use reverse transcriptase to convert this myogenic-cell-specific mRNA to cDNA and perform PCR.

The remaining challenge was to identify the myogenic regulator cDNA in the sample containing hundreds of other cDNAs specific to myogenic cells. It was at that time Lassar was joined by Davis, who was later to become the 1^st author of the famous paper. Together, they managed to find the target cDNA sequence, which, upon being transfected into the 10T1/2 fibroblast cells, converted fibroblasts into myoblasts. That sequence was then confirmed to contain the helix-loop-helix transcription factor, subsequently named myogenic determination gene number 1 (MYOD1). MYOD1 was also capable of converting nerve, fat, and liver cell lines into muscle-like cells, but the conversion frequency was very low, and the resulting cells looked aberrant.

This story illustrates how long and strenuous the process of finding MYOD1 was, even though it was just a single transcription factor. Notably, most of the other cell conversions require more than one transcription factor, making the future search for other factors all the more difficult.

Sources

Content

[1] Lassar A. B. (2017). Finding MyoD and lessons learned along the way. Seminars in cell & developmental biology, 72, 3–9. https://doi.org/10.1016/j.semcdb.2017.10.021

[2] Hochedlinger, K. From MYOD1 to iPS cells. Nat Rev Mol Cell Biol 11, 817 (2010). https://doi.org/10.1038/nrm3018

[3] T. Graf, Historical Origins of Transdifferentiation and Reprogramming, Cell Stem Cell Volume 9, Issue 6, 2 December 2011, Pages 504-516 https://doi.org/10.1016/j.stem.2011.11.012

[4] Davis RL, Weintraub H, Lassar AB. Expression of a single transfected cDNA converts fibroblasts to myoblasts. Cell. 1987 Dec 24;51(6):987-1000. DOI: 10.1016/0092-8674(87)90585-x

[5] Thermo Fisher Scientific. 2020. Subtractive Hybridization. https://www.thermofisher.com/jp/en/home/brands/product-brand/dynal/streptavidin-coupled-dynabeads/subtractive-hybridization.html. [Accessed 5 May 2020].

Graphics

Figure 1: Takahashi, K., Yamanaka, S. A decade of transcription factor-mediated reprogramming to pluripotency. Nat Rev Mol Cell Biol 17, 183–193 (2016). https://doi.org/10.1038/nrm.2016.8