The AlkB family of hydroxylases; unique roles in DNA, RNA and histone hydroxylation/demethylation.

This project is partly based upon the intriguing phenotype and breeding pattern of Alkbh1, Alkbh4 and Alkbh7 (AlkB homolog 1, 4 and 7) targeted mice. Alkbh1 targeting in mice cause sex-ratio distortion and asymmetric left-right eye development. Alkbh1 is highly expressed in embryonic stem (ES) cells and the promoter of the gene is occupied by Oct4 and Nanog, two essential regulators of early development and ES cell identity. Furthermore, there are some remarkable similarities between the different AlkB ho mologs. First, it has been proposed that the very low pI of Alkbh1, 4 and 7 makes them likely candidates for being histone demethylases. We have shown that these three homologs are considerably upregulated in pachytene, the third stage of the prophase of male meiosis. Alkbh7 targeting, like Alkbh1 targeting, cause dramatic sex-ratio distortion. Alkbh4 targeting cause early embryonic lethality and the conditional model is at this time being breeding with an inducible cre-line. No obvious phenotypes are ass ociated with 1st generation mice with Alkbh5 and Alkbh6 targeting. This application does not include Alkbh2, 3 and 8. We have succeeded in generating gene-targeted models for all these five Alkb homologs, both as constitutive and conditional knockouts. Th is will allow us to perform, in collaboration with outstanding international collaborators, unique experiments to determine the role of the AlkB homologs in processes related to ES- and germ cell specification. Furthermore, we have recently succeeded in p urifying a histone H2A variant that differ, between wild-type and Alkbh1 targeted mice, in that the Alkbh1 H2A variant contains an additional dimethyl group. Thus, we also describe various approaches for using gene-targeted mice for identifying the bioche mical properties of the Alkb homologs. It is worth noticing that these hydroxylases share homology to the TET proteins and might well hydroxylate 5-meC to 5-hmeC. cells.