Roles of DNA hydroxymethylation versus DNA formylation and carboxylation in neural stem cells [CUT&Tag]

TET enzymes are highly expressed in neural stem cells (NSCs). They oxidize 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC) in a step-wise manner. In this study we have generated NSCs that only lack the Tet formylation and carboxylation activities (Tet-TFoCa) and have compared their multipotency, transcriptome and epigenome to NSCs that lacked all three Tet activities (Tet-TMut). Our findings reveal unique roles for DNA hydroxymethylation vs DNA formylation and carboxylation in multipotency of NSCs. 2 wild type, 2 lines Tet-FoCa and 2 Tet-TMut NSCslines were harvested and H3K27ac genomic distribution was mapped by CUTnTag. Cells were fixed, bound to ConA beads, incubated with anti-H3K27ac antibody, and treated with pre-loaded pA-Tn5 adapter complex. Libraries were prepared with NEBNext HiFi 2x PCR Master mix and sequencing was performed at the Albert Einstein College of Medicine epigenomics core on Illumina NextSeq 500 platform. Details of CUT&Tag procedure and data analyses are described in detail in the methods section of the manuscript.