Date of Graduation

12-2012

Document Type

Dissertation (PhD)

Program Affiliation

Biomedical Sciences

Degree Name

Doctor of Philosophy (PhD)

Advisor/Committee Chair

Jean-Pierre Issa, M.D.

Committee Member

Michelle Barton, Ph.D.

Committee Member

Ralf Krahe, Ph.D.

Committee Member

Min Gyu Lee, Ph.D.

Committee Member

Bin Wang, Ph.D.

Abstract

DNA methylation at the C5 position of cytosine (5-methylcytosine, 5mC) is a crucial epigenetic modification of the genome and has been implicated in numerous cellular processes in mammals, including embryonic development, transcription, X chromosome inactivation, genomic imprinting and chromatin structure. Like histone modifications, DNA methylation is also dynamic and reversible. However, in contrast to well defined DNA methyltransferases, the enzymes responsible for erasing DNA methylation still remain to be studied. The ten-eleven translocation family proteins (TET1/2/3) were recently identified as Fe(II)/2-oxoglutarate (2OG)-dependent 5mC dioxygenases, which consecutively convert 5mC into 5-hydroxymethylcytosine (5hmC), 5-formylcytosine and 5-carboxylcytosine both in vitro and in mammalian cells. Based on their potent oxidative activities on 5mC, TET proteins have shown great potential as the long-sought DNA demethylases that induce DNA demethylation through multiple potential mechanisms. Here, we show that overexpression of TET1 catalytic domain alone (TET1-CD) but not full length TET1 (TET1-FL) induces global DNA demethylation in HEK293T cells. Genome-wide mapping of 5hmC further reveals a unique regulation pattern of 5mC by TET1-FL, where its 5hmC production is relatively inhibited as local basal DNA methylation level increases. By contrast, overexpression of TET1-CD exhibited a strong positive correlation between 5hmC production and basal DNA methylation level. In support of it, we interestingly found that through CXXC domain TET1 specifically binds hypomethylated but not hypermethylated CpG-rich regions. Moreover, overexpression of TET1-FL specifically decreased DNA methylation levels to certain extent only in hypomethylated CpG sites (methylation level ≤ 10%). To further investigate the effect of TET1, we also developed a lentiviral shRNA mediated TET1 knockdown in HEK293T cells, which originally have a comparable TET1 expression level as human embryonic stem cells. Knockdown of TET1 significantly induced an increase of DNA methylation in the pre-methylated edges, but not unmethylated edges and center regions of CpG islands (CGIs), indicating that TET1 can efficiently maintain the DNA hypomethylated state of CGIs by inhibiting the spreading of de novo DNA methylation from the pre-methylated edges of CGIs. Finally, with the use of the inducible TET1-CD overexpression system in HEK293T cells, we found that knockdown or inhibition of APEX1, the key player of DNA base excision repair pathway (BER), did not impair DNA demethylation induced by TET1-CD overexpression, suggesting that TET-mediated DNA demethylation is independent on BER. Moreover, our results also suggest that the replication-dependent passive pathway is not the primary mechanism for TET-mediated DNA demethylation. In conclusion, our results demonstrated that TET1 is a unique DNA demethylase which cannot significantly change DNA methylation levels, but rather specifically maintains the DNA hypomethylation state in CpG-rich regions by removing aberrant de novo DNA methylation. Moreover, neither BER nor DNA replication is required for TET-mediated DNA demethylation. Future studies focusing on potential 5-carboxylcytosine decarboxylases are necessary to elucidate the underlying mechanism.

Keywords

TET1, 5-hydroxymethylcytosine, DNA methylation, DNA demethylase, CpG island

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