We used IP-MS to identify TET1 interacting proteins and gain insight into the biological functions of this protein in oligodendrocytes. Stringent filtration steps including comparation with oligodendrocytes transcriptome and with CRAPome database were applied to exclude non OL lineage proteins and potential contaminant proteins during IP-MS. Taking the list of TET1 interactors in OPCs and OLs, we next used GO and pathway analysis to link TET1 interacting proteins to putative biological functions and pathways, which revealed that TET1 may be involved in protein homeostasis (protein localization, protein stability and assembly), myelin sheath and molecular metabolic and catabolic process. Notably, the results revealed gene enrichment for mitochondrion organization, nucleotide binding and cell redox homeostasis in OPCs, and translational initiation, RNA metabolism, and MAPK family signaling cascades in OLs.
Further analysis of the protein-protein interaction (PPI) networks for TET1 showed that in OPCs, proteins associated with TET1 were involved in cell homeostasis or protein synthesis. Oligodendrocytes display a strict vesicular transport system including protein folding, protein sorting, formation of carrier vesicles, vesicle transport along elements of the cytoskeleton, and vesicle targeting/fusion . The synchronization and coordinate of vesicle transport are essential to maintain the structural and functional organization of oligodendrocytes. In addition, many genes closely related to neurological diseases, such as Got1, ApoE, Gm2a, have been found to interact with TET1 in OPCs [27–30] ; and Cst3 is associated with dementia in Lewy body disease (24) and Alzheimer's Disease (25). Different from OPCs, PPI networks for TET1 are relatively simple in OL, including ATP activity, nuclear pore outer ring and regulation of DNA metabolic process. These TET1-associated terms suggest unknown functional settings for TET1 in OLs as a supplement to the GO functional annotation analyses. Together, our results provide novel perspectives into distinctive functions beyond transcriptional regulation role for TET1 in oligodendrocyte biology.
Although there are no reports regarding the involvement of TET1 in most of above biological processes, some studies can explain our observations to some extent. For example, one of the TET1-IP products in OPC, Calpain, a protein belonging to the family of calcium-dependent, non-lysosomal cysteine proteases, could mediate TET1 degradation in mouse embryonic stem cells (ESCs) . Studies have suggested putative involvement of TETs in the formation of 5hmC in mitochondria DNA (mtDNA), which is consistent with the mitochondria associated proteins (e.g. Abcb6, Acly) in TET1-IP products from OPC cultures. In purified cerebellum granule neuron cultures, TET1 and TET2 presence not only in the nucleus but also in the mitochondrial fraction identified by Western Blot assay ; mouse 3T3-L1 cells treated with histone deacetylase inhibitor show reduced 5hmC content in mtDNA and decreased mitochondrial TET1 expression . We anticipate that future studies extending the role of TETs beyond genomic DNA, i.e., into the field of mitochondrial epigenetics, will likewise reveal functional diversity for TET family proteins in the central nervous system.
Regarding the transcriptional functions, interacting partners of TETs may also contribute to their recruitment to specific genomic regions. In mouse ESCs, the pluripotency factor NANOG physically interacts with TET1, and NANOG depletion results in reduced TET1 binding at NANOG-bound regions . Similarly, PR domain zinc finger protein 14 (PRDM14) , Polycomb repressive complex 2 (PRC2) and LIN28A  have also been reported to interact with and recruit TET proteins in mouse ESCs. TET1 could promote glycosylation of chromatin by binding to O-N acetyl glucose transferase (OGT) and mediate posttranscriptional modification. A recent study indicates that EGR1 interacts and recruits TET1 to its target binding sites . Collectively, these results imply that the interacting partners of TETs, in many cases key transcription factors of the cells studied, contribute to TETs recruitment into target genes. Further analysis is needed to determine whether the interaction per se mediates the recruitment or instead the interacting partner helps to establish a favorable chromatin environment for TET binding of DNA.
TET proteins are iron (II)/αketoglutarate (Fe (II)/α-KG)-dependent dioxygenases. The core catalytic domain at the carboxyl terminus is comprised of a double-stranded βhelix (DSBH) domain and a cysteine-rich domain. Full-length TET1 have a CXXC zinc finger DNA binding domain at amino terminus; however, the CXXC domain of TET1 has no DNA binding activity and is dispensable for its catalytic activity in vivo. This implies that other proteins are involved in DNA binding of TET1, a necessary step to promote the conversion of 5-mC to 5-hmC. Interestingly, mouse TET1 preferentially exists in an Nterminus-truncated form (known as TET1s) in somatic tissues but exists in its full-length form (known as TET1e) in early embryos. TET1s, which does not have a CXXC domain and the other Nterminal sequence, has reduced global chromatin binding compared with TET1e and confers weaker demethylation activity in cells. Therefore, it is important to further investigate the function and mechanism of individual forms of TET1 in different cell types.
In our study, both Olig2 and HDAC1 were shown to interact with TET1 in oligodendrocytes. HDAC1 has been identified to be recruited specifically by TET1 in male germline stem cells  and this complex binds to key genes to regulated histone acetylation and gene expression. Therefore, we speculate that in oligodendrocytes, TET1 may play a role of recruitment with HDAC1 to affect histone acetylation, which may further influence chromosome structure and gene transcription activation. As one of the OL lineage specific TFs, Olig2 belongs to the basic helix-loop-helix (bHLH) transcription factor family and is necessary for oligodendrocyte development. All bHLH transcription factors function in a dimeric state as homodimers or as heterodimers with another bHLH protein. Once in contact with the promoter or enhancer elements of a target, bHLH homodimers and heterodimers serve as scaffolding upon which a multimeric complex of transcriptional coregulator proteins can be assembled. Olig2 has been shown to interact with NKX2.2  and histone acetyl transferase p300 , all suggesting the transcriptional activator role of Olig2 in OL development. Our identification of TET1 as novel Olig2 co-factor thus provide further clue for Olig2 function in modulating oligodendrocytes development.
Overall, the comprehensive analysis of endogenous TET1 interactome highlights many novel partners with interesting roles and provide a basis for further functional investigations of TET1 in oligodendrocytes biology and related disease.