The APOBEC3 Gene Family as Guardians of Genome Stability in Human Embryonic Stem Cells

The APOBEC3 Gene Family as Guardians of Genome Stability in Human Embryonic Stem Cells

Funding Type: 
SEED Grant
Grant Number: 
RS1-00210
Award Value: 
$717,363
Disease Focus: 
Cancer
Stem Cell Use: 
Embryonic Stem Cell
Status: 
Closed
Public Abstract: 
Statement of Benefit to California: 
Progress Report: 

Year 1

Human embryonic stem cells contain roughly 3 million “jumping genes” or mobile genetic retroelements that comprise up to 45% of human genome. While many of these retroelements have been silenced during evolution by crippling mutations, many remain active and capable of jumping to new chromosomal locations potentially producing disease-causing mutations or cancer. In tissues, mobility of these elements is suppressed by DNA methylation, which inactivates expression of the retroelement RNAs. In sharp contrast, embryonic stem cells exhibit very dynamic changes in DNA methylation, where the methylation patterns are gained and lost at high rates. During periods of low DNA methylation, retroelement RNA expression likely increases. Accordingly, hESCs must deploy other defensive strategies in order to maintain genomic integrity. Recent studies have identified the APOBEC3 family of genes (A3A-A3H) as powerful antiviral factors. These A3s interrupt the conversion of viral RNA into DNA (reverse transcription), a key step also employed by retroelements for their successful retrotransposition. We hypothesized that one or more of the APOBECs function as guardians of genome integrity in hESCs. In the last two years we have found that six out of the seven human A3 genes located in a tandem array on chromosome 22 are expressed in hESCs. A3A, which in prior studies was suggested to exert the greatest anti-retroelement effects, surprisingly is not expressed in hESCs. Further, we find that the A3 proteins decrease when pluripotent cells differentiate into somatic cells suggesting an important function of these A3 proteins in pluripotent hESCs. We established a LINE1 retrotransposition assay in hESCs that allows us to visualize genetic jumping of this class of “marked” retroelements via flow cytometry. Using this assay we have found that LINE1 elements effectively jump in hESCs. To test our central hypothesis, namely that A3 proteins guard the genome in hESCs, we have established experimental conditions for RNAi knock-down of all expressed A3 genes. By combining the knock-down and the retrotransposition assay we demonstrated that the knock-down of one member of the A3 protein family leads to a 3.5-fold increase in LINE1 retrotranspositon. This finding highlights a protective role for the A3 family of cytidine deaminases that helps safeguard the genome integrity of hESCs.

© 2013 California Institute for Regenerative Medicine