TET1 Isoform Switch Regulates DNA Demethylation And Mouse Development

The 5-methylcytosine in DNA(5mC)is a classic epigenetic modification,which plays crucial roles in regulating gene transcription,development and is responsible for genome imprints transmitted through generations.TET family proteins are so far the only enzymes known to mediate active DNA demethylation by oxidizing 5mC.As previously reported,TET1 is critical for properly erasing imprints in mouse primordial germ cells through active demethylation pathway.However,it still remains elusive how exactly TET proteins find substrates in the genome for 5mC oxidation.In this thesis,by carefully investigating the transcriptome of various mouse tissues,we unexpectedly found that there are two distinct TET1 isoforms expressed in different developmental stages.The full-length TET1 isoform(TET1e)is restricted to early embryos,embryonic stem cells(mESCs)and primordial germ cells(PGCs).By contrast,a short TET1 isoform is preferentially expressed in somatic tissues.Further investigation revealed these two isoforms are transcribed from individual promoters,and the transcription factor OCT4,SOX2 and NANOG are critical to activate the expression of TET1 e.By comparing the structures of these two isoforms,we found TET1 s lacks 653 amino acid in N-terminus including CXXC domain,which mediates the targeted binding of TET1.Interestingly,though lacking CXXC domain,TET1 s still binds to similar targets enriched in CpG islands and promoters as TET1 e.In contrast,the N-terminus of TET1 significantly promotes its global chromatin binding.By carefully examining various mutated TET1 proteins,we found that besides CXXC domain,the first 1-131 amino acid of TET1 N-terminus can also strongly promote TET1's global chromatin binding.We named this new domain as “BC domain”(Before CXXC domain).Then,using CRIPSR/Cas9 mediated genome editing,we constructed mESCs in which the N-terminus of TET1 e has been deleted and TET1 s is exclusively expressed.In the mutated mESCs,we detected a lower level of 5-hydroxymethylcytosine and a higher DNA methylation level compared with its parental line.Our data also reveals TET1 mediated DNA methylation is not correlated with the targeted binding detected by ChIPseq,but rather correlated with global chromatin affinity.Finally,we constructed mouse model exclusively expresses TET1 s using similar method in mESCs.We found that in the primordial germ cells of mutated mice,imprints cannot be properly erased and established.Such defects in gene imprints will pass to the next generation and result in their decrease body size and partial lethality.In summary,we have identified two TET1 isoforms in mouse and investigated their differences in molecular structure and functions.We have also give an explanation for the stage specific expression of TET1 e during mouse development.