Are you Muhan Hu?

Claim your profile

Publications (2)9.3 Total impact

  • [Show abstract] [Hide abstract]
    ABSTRACT: Background: Base J regulates Pol II transcription. Results: JBP1 and -2 stimulate the first step of base J synthesis: hydroxylation of thymidine. Conclusion: JBP are Fe 2 /2-OG-dependent dioxygenases sensitive to physiologically relevant O 2 tensions. Significance: These results predict that JBPs can act as oxygen sensors regulating trypanosome gene expression and adaption to different host niches. We have recently demonstrated that O-linked glucosylation of thymine in trypanosome DNA (base J) regulates polymerase II transcription initiation. In vivo analysis has indicated that base J synthesis is initiated by the hydroxylation of thymidine by pro-teins (JBP1 and JBP2) homologous to the Fe 2 /2-oxoglutarate (2-OG)-dependent dioxygenase superfamily where hydroxyla-tion is driven by the oxidative decarboxylation of 2-OG, forming succinate and CO 2 . However, no direct evidence for hydroxylase activity has been reported for the JBP proteins. We now demon-strate recombinant JBP1 hydroxylates thymine specifically in the context of dsDNA in a Fe 2 -, 2-OG-, and O 2 -dependent manner. Under anaerobic conditions, the addition of Fe 2 to JBP1/2-OG results in the formation of a broad absorption spec-trum centered at 530 nm attributed to metal chelation of 2-OG bound to JBP, a spectroscopic signature of Fe 2 /2-OG-depen-dent dioxygenases. The N-terminal thymidine hydroxylase domain of JBP1 is sufficient for full activity and mutation of residues involved in coordinating Fe 2 inhibit iron binding and thymidine hydroxylation. Hydroxylation in vitro and J synthesis in vivo is inhibited by known inhibitors of Fe 2 /2-OG-depen-dent dioxygenases. The data clearly demonstrate the JBP enzymes are dioxygenases acting directly on dsDNA, confirm-ing the two-step J synthesis model. Growth of trypanosomes in hypoxic conditions decreases JBP1 and -2 activity, resulting in reduced levels of J and changes in parasite virulence previously characterized in the JBP KO. The influence of environment upon J biosynthesis via oxygen-sensitive regulation of JBP1/2 has exciting implications for the regulation of gene expression and parasite adaptation to different host niches. -D-Glucopyranosyloxymethyluracil (base J) 2 is a hyper-modified DNA base found in eukaryotes. This DNA modifica-tion is evolutionarily conserved within members of the kineto-plastid family, namely Trypanosoma brucei, Trypanosoma cruzi, and Leishmania, where J replaces about 1% of the total T in the genome and is predominantly present in repetitive DNA sequences, such as telomeric repeats (for review, see Ref. 1). However, more recently, we localized a minor fraction of J to chromosome-internal regions coinciding with RNA polymer-ase II (Pol II) transcription initiation and termination sites (2). Loss of base J synthesis at these chromosome-internal regions in T. cruzi led to increased Pol II transcription initiation and corresponding changes in gene expression and parasite viru-lence (3, 4). Thus, base J represents a novel epigenetic modifi-cation of kinetoplastid DNA involved in regulating gene expression. Indirect evidence (for review, see Ref. 5) indicates J is synthe-sized in a two-step pathway (Fig. 1). Step one involves the hydroxylation of thymine in DNA by a thymidine hydroxylase (TH) enzyme, forming 5-hydroxymethyluracil (hmU). This intermediate is then glucosylated by a glucosyltransferase forming base J. Although the glucosyltransferase has not been identified, two proteins involved in the first step (JBP1 and JBP2) (6, 7) have been characterized. Both JBP1 and JBP2 (8, 9) contain a putative TH domain at the N terminus that has led to the designation of these enzymes belonging to the new TET/ JBP subfamily of dioxygenases that require Fe 2 and 2-oxogl-utarate (2-OG) for activity (10, 11). Family members are typi-cally identified on a structural level by the presence of a jelly roll -helix sheet that contains four key conserved residues involved in the binding of Fe 2 and 2-OG and are essential for catalytic activity (see for review, see Ref. 12). Mutation of these conserved residues within the TH domain of JBP1 and JBP2
    Journal of Biological Chemistry 04/2012; 287(24):19886. · 4.65 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: We have recently demonstrated that O-linked glucosylation of thymine in trypanosome DNA (base J) regulates polymerase II transcription initiation. In vivo analysis has indicated that base J synthesis is initiated by the hydroxylation of thymidine by proteins (JBP1 and JBP2) homologous to the Fe(2+)/2-oxoglutarate (2-OG)-dependent dioxygenase superfamily where hydroxylation is driven by the oxidative decarboxylation of 2-OG, forming succinate and CO(2). However, no direct evidence for hydroxylase activity has been reported for the JBP proteins. We now demonstrate recombinant JBP1 hydroxylates thymine specifically in the context of dsDNA in a Fe(2+)-, 2-OG-, and O(2)-dependent manner. Under anaerobic conditions, the addition of Fe(2+) to JBP1/2-OG results in the formation of a broad absorption spectrum centered at 530 nm attributed to metal chelation of 2-OG bound to JBP, a spectroscopic signature of Fe(2+)/2-OG-dependent dioxygenases. The N-terminal thymidine hydroxylase domain of JBP1 is sufficient for full activity and mutation of residues involved in coordinating Fe(2+) inhibit iron binding and thymidine hydroxylation. Hydroxylation in vitro and J synthesis in vivo is inhibited by known inhibitors of Fe(2+)/2-OG-dependent dioxygenases. The data clearly demonstrate the JBP enzymes are dioxygenases acting directly on dsDNA, confirming the two-step J synthesis model. Growth of trypanosomes in hypoxic conditions decreases JBP1 and -2 activity, resulting in reduced levels of J and changes in parasite virulence previously characterized in the JBP KO. The influence of environment upon J biosynthesis via oxygen-sensitive regulation of JBP1/2 has exciting implications for the regulation of gene expression and parasite adaptation to different host niches.
    Journal of Biological Chemistry 04/2012; 287(24):19886-95. · 4.65 Impact Factor