My group has developed several experimental systems to study epigenetic control of mammalian retrotransposons. Using a marked L1 retrotransposon, we are studying the mechanism of variegation and silencing of L1 reporters that are newly inserted via a retrotransposition-dependent mechanism in tissue culture cells. We have also studied epigenetic control of native L1 retrotransposons pre-existing in the human and mouse genomes, and the relationship between such control and L1 expression in various cells. Our experiments have utilized techiques includingDNA methylation analysis of both DNA strands by hairpin bisulfite sequencing, chromatin immunoprecipitation, and short inhibitory RNAs. These experiments, as a test of the """"""""genome defense model"""""""", have shown that DNA methylation does not by itself account for exceptionally strong negative regulation of L1 transcription in somatic tissues.We have successfully sequenced and analyzed several full Serial Analysis of Gene Expression (SAGE) long-tag libraries to characterize global transcript levels, including those corresponding to repetitive and transposable elements, in somatic cells with altered epigenetic controls. The libraries completed to date include numerous transcripts that are differentially expressed at very high ratios. Differentially expressed transcripts have been identified and validated by independent techniques including Northern blotting and quantitative polymerase chain reaction (qPCR) experiments. We are now further characterizing how altered expression may be related to changes in epigenetic control.

Agency
National Institute of Health (NIH)
Institute
Division of Basic Sciences - NCI (NCI)
Type
Intramural Research (Z01)
Project #
1Z01BC010628-02
Application #
7292918
Study Section
(MCGP)
Project Start
Project End
Budget Start
Budget End
Support Year
2
Fiscal Year
2005
Total Cost
Indirect Cost
Name
Basic Sciences
Department
Type
DUNS #
City
State
Country
United States
Zip Code