The AlkB family of hydroxylases; roles in embryonic stem cell identity and epigenetic reprogramming of the germ line.
The conservation of most of the human AlkB homologues across the animal kingdom clearly suggests fundamental and important functions. Very few macromolecular demethylases have been identified, and the Alkbh proteins likely represent completely novel and highly interesting functions. We believe that gene-targeting in mice is required for illuminating their precise molecular role. Such analysis might also be very relevant for studying regulation during embryo development and stem cell differentiation. Our in vivo experiments will profit considerably if we, or others, are able to reveal the exact biochemical activities of these AlkB homologs. However, biochemical properties clarified by in vitro assays must be verified in a genetic model to establish its biological relevance.
During the last year we have succeeded in identifying the substrate of two more AlkB homologs. Alkbh5 was published in Molecular Cell in December 2012 and we are currently finalizing the work on Alkbh7. Both these enzymes reverse modifications in RNA and we believe that reversible RNA modification, and their role in disease, might be a completely new research field for the future (The Alkbh5 publication was Editors Choice in Science January 2013).
Based on our studies on hydroxylation, several patents have been obtained and two licencing contracts.
The AlkB family of hydroxylases; roles in embryonic stem cell identity and epige
We focus our activity on 9 genes (Alkbh1-9) in the mammalian genome encoding novel hydroxylases (they can add an OH-group to a methyl(CH3)-group on a macromolecule.
The conservation of most of the human AlkB homologues across the animal kingdom clearly suggests fundamental and important functions. Very few macromolecular demethylases have been identified, and the Alkbh proteins likely represent completely novel and highly interesting functions. We believe that gene-targeting in mice is required for illuminating their precise molecular role. Certainly, this strategy has already revealed intriguing phenotypical abnormalities associated with Alkbh1 deficiency. The proposed project will focus on the characterization of ES cell and germ cells generated from gene-targeted mice by a broad range of strategies. A transcriptional portrays in fractionated sperm from wild type and homozygous Alkbh1, Alkbh4 and Alkbh7 mice will be carried out by microarray and ChIP-Chip technology. Such analysis might also be very relevant for studying regulation during embryo development and stem cell differentiation. In vitro approaches include biochemical characterization of recombinant proteins for activities against histone peptides and “fully” methylated endogenous histones, as well as for structural analysis. Our in vivo experiments will profit considerably if we, or others, are able to reveal the exact biochemical activities of these AlkB homologs. However, biochemical properties clarified by in vitro assays must be verified in a genetic model to establish its biological relevance.
In addition to the papers published in 2011, wee have detected novel mechanisms for controlling cell division by removal of a singel methyl-group. Additionally, our early studies on certain Alkb homologs involved in DNA repair have been continued through international collaborations and we show that Alkb homologs have unique roles in cancer, cell division and fertility. We have submitted four major studies for publication.
AlkB hydroxylases; roles in embryonic stem cell identity
In has been challenging to identify the thrue substrates for Alkbh1-8. Through own research and fruitful collaborations we are in very good progress to resolve the biochemical properties of these enzymes. Some of the homologs are vital and gene knockouts cause disease, early lethality and sterility.
Eschericia coli AlkB is a 2-oxoglutarate- and iron-dependent dioxygenase that reverses alkylated DNA damage by oxidative demethylation. In has been challenging to identify the thrue substrates for Alkbh1-8. We have generated mice lacking Alkb homologs and we have unravelled their biological functions. Through own research and fruitful collaborations we are in very good progress to resolve the biochemical properties of these enzymes. Some of the homologs are vital and gene knockouts cause disease, early lethality and sterility.
Mouse AlkB homolog 1 (Alkbh1) is one of eight members of the newly discovered family of mammalian dioxygenases (Nordstrand et al., 2010 PloS One). Both Alkbh1-/- and heterozygous Alkbh1+/- offspring are born at a greatly reduced frequency. Additionally, the sex-ratio is considerably skewed against female offspring, with one daughter born for every three to four sons. Most mechanisms that cause segregation distortion, act in the male gametes and affect male fertility, consistent with the apoptosis revealed in testes from Alkbh1-/- adults. The skewing of the sexes appears to be of paternal origin, and might be set in the pachythene stage of meiosis during spermatogenesis, in which Alkbh1 is upregulated more than 10-fold. The deficiency of Alkbh1 also causes misexpression of Bmp2, 4 and 7 at E11.5 during embryonic development. This is consistent with the incompletely penetrant phenotypes observed, particularly recurrent asymmetric left–right eye defects and craniofacial malformations. Genetic and phenotypic assessment suggests that Alkbh1 mediates gene regulation in spermatogenesis, and that Alkbh1 is essential for normal sex-ratio distribution and embryonic development in mice.
We have identified the role of Alkbh8 in more detail (Songe-Møller et al., 2010 MCB; van den Born et al., 2011 Nature Comm). We have generated for this study Alkbh8-targeted mice that lack exons critical for both the MT and AlkB activities of Alkbh8. The mice did not display any overt phenotype, but tRNA from these mice was completely devoid of mcm5U, mcm5s2U, and mcm5Um, and the relevant tRNA isoacceptors instead contained the acid form 5-carboxymethyluridine (cm5U) and/or the amide forms ncm5U/ncm5s2U. Furthermore, we show that recombinant ALKBH8 and TRM112 form a heterodimeric complex capable of catalyzing the methyl esterification of cm5U and cm5s2U to mcm5U and mcm5s2U, respectively. In agreement with the involvement of mcm5Um in selenoprotein synthesis, we observed a reduced level of Gpx1 in the Alkbh8-/- mice, and tRNASec from these mice showed a reduced ability to decode the UGA stop codon to Sec.
Some of the other homologs have extremely interesting properties in totipotent and pluripotent stem cells and work is in progress for the completion of these studies.
The DNA dioxygenase ALKBH2 protects Arabidopsis thaliana against methylation damage.
Nucleic Acids Res 2012 Aug;40(14):6620-31. Epub 2012 apr 24
PMID: 22532610 - Inngår i doktorgradsavhandlingen
ALKBH1 is a histone H2A dioxygenase involved in neural differentiation.
Stem Cells 2012 Dec;30(12):2672-82.
PMID: 22961808 - Inngår i doktorgradsavhandlingen
Pull-down of 5-hydroxymethylcytosine DNA using JBP1-coated magnetic beads.
Nat Protoc 2012 Feb;7(2):340-50. Epub 2012 jan 26
The presence of 5-hydroxymethylcytosine at the gene promoter and not in the gene body negatively regulates gene expression.
Biochem Biophys Res Commun 2011 Jul;411(1):40-3. Epub 2011 jun 15
PMID: 21703242 - Inngår i doktorgradsavhandlingen
A novel method for the efficient and selective identification of 5-hydroxymethylcytosine in genomic DNA.
Nucleic Acids Res 2011 Apr;39(8):e55. Epub 2011 feb 7
ALKBH8-mediated formation of a novel diastereomeric pair of wobble nucleosides in mammalian tRNA.
Nat Commun 2011;2():172. Epub 2011 feb 1
PMID: 21285950 - Inngår i doktorgradsavhandlingen
Mice lacking Alkbh1 display sex-ratio distortion and unilateral eye defects.
PLoS One 2010;5(11):e13827. Epub 2010 nov 3
PMID: 21072209 - Inngår i doktorgradsavhandlingen
Mammalian ALKBH8 possesses tRNA methyltransferase activity required for the biogenesis of multiple wobble uridine modifications implicated in translational decoding.
Mol Cell Biol 2010 Apr;30(7):1814-27. Epub 2010 feb 1
PMID: 20123966 - Inngår i doktorgradsavhandlingen
Continuous and periodic expansion of CAG repeats in Huntington's disease R6/1 mice.
PLoS Genet 2010;6(12):e1001242. Epub 2010 des 9
Histone H3 lysine 27 methylation asymmetry on developmentally-regulated promoters distinguish the first two lineages in mouse preimplantation embryos.
PLoS One 2010;5(2):e9150. Epub 2010 feb 10
PMID: 20161773 - Inngår i doktorgradsavhandlingen