TET1-Mediated 5hmC Deposition Establishes a Labile Chromatin Landscape Necessary for SOX9 Binding

Cytosine modifications are important epigenetic regulators of development and disease. Methylated cytosine (5mC) can be oxidized initially to 5hmC (5-hydroxymethylcytosine) by the Ten-eleven-translocation (TET) enzymes and further to 5fC and 5caC leading to demethylation via DNA repair. Although 5mC is associated with gene silencing, stable 5hmC has been associated with gene activation. The simplest mode of sensing these cytosine modifications is through the direct biochemical effect on binding of transcription factors. Surprisingly, we identified that loss of TET1 greatly inhibits Sox9 cistrome causing a delay in endochondral ossification in the TET1 mutant mice, even though the Sox9 binding motif is devoid of cytosines. We found that loss of TET1-mediated 5hmC leads to a reduction of nucleosome lability that inhibits Sox9 binding. An alternative model of sensing the cytosine modifications is therefore through the combined effects of the 5mC, 5hmC, 5fC and 5caC modifications on the chromatin accessibility and nucleosomal lability. To date, 5hmC deposition has only been observed with simultaneous 5mC loss, suggesting that 5hmC is merely a by-product of demethylation. In contrast, we see no evidence of demethylation at SOX9 target genes. Our study provides the first example where gain of 5hmC deposition occurs in a hypomethylated context and is required for chromatin lability, underscoring its function as a true epigenetic mark and providing insights into how Sox9 senses this modification as an indirect reader.