SRP189657 PRJNA529472 GSE128951 5-hydroxymethylcytosine Promotes Productive Transcription and Inhibits Spurious Promoters Covalent DNA modifications play vital roles during animal development. While 5-methylcytosine (5mC) is important for imprinting, X chromosome inactivation and transcriptional repression, the function of 5-hydroxymethylcytosine (5hmC) is less well defined. Recent studies indicate that 5hmC can be stable and long-lived in some cell types, suggesting roles beyond a DNA demethylation intermediate. Here we show that oxidation of 5mC to 5hmC enhances transcription of a specific repertoire of genes. 5hmC upregulates transcription by counteracting the repressive effects of promoter-proximal 5mC, while supressing the ability of gene body resident spurious transcription initiation sites to produce mature transcripts. By contrast, the equivalent hypomethylation results in chaotic gene expression potentiating a broad collection of genes and cryptic promoters. Our observations provide an explanation why some post-mitotic cell types favour high 5hmC in the genomes, while equivalent global hypomethylation is rarely observed. Overall design: 94 samples in total. 4 hmC-Seal: 2 conditions, 2 biological replicates, total genomic DNA as an input DNA. 4 MeDIP: 2 conditions, 2 biologial replicates, total genomic DNA as an input DNA. 4 EpiJET: 2 conditions, 2 biologial replicates, total genomic DNA as an input DNA. 63 RNA-Seq: 6 conditions, pair-wise comparison was performed within one experiment containg 3 replicates. 9 PRO-Seq: 3 conditions, 3 biologial replicates. 9 PRO-Cap: 3 conditions, 3 biologial replicates. GSE128951