Identification Of 5-hydroxymethylcytosine Binding Protein And Its Regulation Of DNA Demethylation

The genomic DNA of metazoan carries the whole genetic information required to develop from a zygote into a complete organism.In order to sustain the lineage stability,DNA encoded genetic information should be faithfully transmitted to the offsprings.On the other side,genomic DNA must be feasible to give rise to all the tissues and cell types which are characterized by distinct morphologies,functions and gene expression patterns.Thus,genomic DNA should be capable of modifications and packaging.DNA methylation is recognized as one of the most stable epigenetic modifications,which is not easy to reverse once established,due to the high energistic obstacle of the carbon-carbon bond.The DNA methylation patterns of mother cell could be transmitted to daughter cells during mitosis in a semi-conservative manner through the maintenance DNA methyltransferase DNMT1.Therefore,DNA methylation enriches the diversity of the genomic information without altering the DNA sequences.Recent studies discovered that TET family enzymes could successively oxidize 5-methylcytosine(5mC)to 5-hydroxymethylcytosine(5hmC),5-formylcytosine(5fC)and 5-carboxylcytosine(5caC),which may lead to eventual demethylation.Oxidative derivatives of 5mC and TET proteins occupy distinct chromatin regions.In mouse embryonic stem cells(ESCs),TET1 and TET2 mostly localize to the gene promoters arounding transcription starting sites(TSSs),but 5hmC is enriched at gene distal regulatory regions.Furthermore,5fC and 5caC tend to accumulate at those distal regulatory regions in TDG-depleted mouse ESCs,indicating there are multiple mechanisms controlling the distributions and fates of the oxidative derivatives of 5mC in the genome,and a dynamic regulation of these modifications may play a role in regulating gene expression.However,many more mechanistic and functional details currently unknown need to be elucidated.In this study,we identified 5hmC binding proteins using quantitative mass spectrometry-assisted DNA affinity puficiation using mouse ESCs,and investigated their genome-wide association and interplay with 5hmC and TET family proteins.First,the pluripotent factors SALL1 and SALL4 preferentially associate with 5hmC in vitro.Second,in mouse ESCs,SALL1 and SALL4 form a protein complex and primarily colocalize at distal regulatory regions.Thrid,the chromatin association of SALL1 and SALL4 is largely TET1 dependent,suggesting that TET 1-mediated DNAhydroxymethylation contributes to recruitment or stabilizing the genomic localization of SALL1 and SALL4.Fourth,SALL1 and SALL4 binding sites are generally devoid of 5hmC,but enriched for 5caC in TDG-depleted mouse ESCs,indicating dynamic DNA oxidation reactions at those sites.Fifth,knockout of Sall4 but not Salll results in an ectopic accumulation of 5hmC at the original binding sites,with a decreased enrichment of 5caC while TDG is depleted simultaneously,indicating the chromatin association of SALL4 facilitates further oxidation of 5hmC at its binding sites.Sixth,the direct interaction between TET1 and SALL4 may helps to recruit TET1 to the SALL4 occupied distal regulatory regions.In conclusion,we propose that TET1-mediated DNA hydroxymethylation assists SALL1 and SALL4 binding,which in turn promotes the further oxidation of 5hmC.