Other Genetic Studies

ES Cell Self-renewal

To understand the molecular basis of embryonic stem cell (ES cell) self-renewal, we carried out a genome-wide RNAi screen in mouse ES cells and identified more than 100 novel candidate genes that are important for self-renewal (227). In collaboration with the laboratory of Stu Orkin, we found that two of these identified genes, Cnot3 and Trim28, form a novel module in the self-renewal transcription network. This screen demonstrated the power of forward genetics in the systematic study of stem cell biology, and further characterization of the candidate genes will provide new insights into both pluripotency and reprogramming.

In addition to the study in ES cells, we also designed and constructed shRNA vectors and libraries for virus-based long-term gene silencing in mammalian cells. In particular we now have vectors that work well in mouse cells and even highly regulated tet-inducible systems that carry the reverse Tet activator, rtTA, on a lentiviral backbone so that the regulator can be introduced with the shRNA of choice or even an entire library.

Regulation of Mitochondrial Abundance

Mitochondria serve a critical role in physiology and disease.  The genetic basis of mitochondrial regulation in mammalian cells has not yet been detailed.  We performed a large-scale RNA interference screen in C2C12 myocytes to systematically identify genes that affect mitochondrial abundance and function (241).  This screen revealed previously unrecognized roles for over 150 proteins in mitochondrial regulation.  We found that increased Wnt signals are a potent activator of mitochondrial biogenesis, and the resultant increases in oxidative DNA damage mediate accelerated cellular senescence in mammalian cells.  The signaling protein insulin receptor substrate-1 (IRS-1), was discovered here to be a transcriptional target of Wnt and is rapidly induced in this setting.  It accumulates in the nucleus and drives activation of a known mitochondrial regulator, the transcription factor Myc.  This scheme demonstrates a temporally defined transcriptional cascade controlled by induction of a coactivator.  Our results identify a key component of the mitochondrial regulatory apparatus with a potential link to cellular aging and other degenerative processes associated with disease.