SUMMARY Ten-Eleven Translocation (Tet) family of dioxygenases offers a new mechanism for dynamic regulation of DNA methylation and has been implicated in cell lineage differentiation and oncogenesis. Yet their functional roles and mechanisms of action in gene regulation and embryonic development are largely unknown. Here, we report that Xenopus Tet3 plays an essential role in early eye and neural development by directly regulating a set of key developmental genes. Tet3 is an active 5mC hydroxylase regulating the 5mC/5hmC status at target gene promoters. Biochemical and structural studies further reveal a novel DNA binding mode of the Tet3 CXXC domain that is critical for specific Tet3 targeting. Finally, we show that the enzymatic activity and CXXC domain are crucial for Tet3’s biological function. Together, these findings define Tet3 as a novel transcription factor and reveal a molecular mechanism by which the 5mC hydroxylase and DNA binding activities of Tet3 cooperate to control target gene expression and embryonic development.

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Corneal epithelium removed from underlying extracellular matrix (ECM) extends numerous cytoplasmic processes (blebs) from the formerly smooth basal surface. If blebbing epithelia are grown on collagen gels or lens capsules in vitro, the basal surface flattens and takes on the smooth contour typical of epithelium in contact with basal lamina in situ . This study examines the effect of soluble extracellular matrix components on the basal surface. Corneal epithelia from 9-to 11-d-old chick embryos were isolated with trypsin-collagenase or ethylenediamine tetraacetic acid, then placed on Millipore filters (Millipore Corp ., Bedford, Mass .), and cultured at the medium-air interface. Media were prepared with no serum, with 10% calf serum, or with serum from which plasma fibronectin was removed . Epithelia grown on filters in this medium continue to bleb for the duration of the experiments (12-24 h) . If soluble collagen, laminin, or fibronectin is added to the medium, however, blebs are withdrawn and by 2-6 h the basal surface is flat . Epithelia grown on filters in the presence of albumin, IgG, or glycosaminoglycans continue to bleb . Epithelia cultured on solid substrata, such as glass, also continue to bleb if ECM is absent from the medium . The basal cell cortex in situ contains a compact cortical mat of filaments that decorate with S-1 myosin subfragments ; some, if not all, of these filaments point away from the plasmalemma. The actin filaments disperse into the cytoplasmic processes during blebbing and now many appear to point toward the plasmalemma . In isolated epithelia that flatten in response to soluble collagens, laminin, and fibronectin, the actin filaments reform the basal cortical mat typical of epithelia in situ . Thus, extracellular macromolecules influence and organize not only the basal cell surface but also the actin-rich basal cell cortex of epithelial cells.The basal cell surface and cortical cytoskeleton of the embryonic corneal epithelium are intimately related to the underlying extracellular matrix (ECM) . The cortical cytoskeleton forms a dense mat next to the basal epithelial plasmalemma, and the latter is relatively smooth or flat in configuration . The basal plasmalemma seems to be connected to the lamina densa of the underlying basal lamina (basement membrane) by extracellular filaments that traverse the lamina rara externa (1). The laminae rarae externa and interna of the corneal, and most, if not all, other epithelial basal laminae (2-5) contain proteoglycan granules rich in glycosaminoglycans (GAG), such as heparan and/or chondroitin sulfates (HS, CS) and hyaluronic acid (HA). Laminin and fibronectin occur in the laminae rarae of the glomerular basement membrane (4), and both are found in the embryonic avian corneal basal lamina (Sugrue and Hay, unpublished observations) . Type IV collagen is also present in the corneal basement membrane (6) . Type IV and other col-

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