Table 1.
High confidencea ALKBH4 interacting proteins identified by the yeast two-hybrid system.
Figure 1.
Schematic representation of yeast two-hybrid high confidence hits involved in transcription.
Individual prey fragment clones and the resulting selected interaction domains (SIDs) reported to bind ALKBH4 are indicated above each protein; black lines, placenta library; orange lines, fetal brain library; green lines, placenta library screened with ALKBH4H169A/D171A; red dashed lines, SIDs. Grey boxes indicate protein domains. Proteins and domains are drawn to scale according to the InterPro (version 4.8) and PROSITE (release 20.68) databases [73], [74].//indicates regions omitted for simplicity. ZnF, C2H2 zinc finger; HD, homeodomain; YEATS, Yaf9 ENL AF9 Taf14 Sas5; TAZ, transcription adaptor putative zinc finger; BRD, bromodomain; PHD plant homeodomain; HAT histone acetyl transferase; DBD, DNA binding domain. Bar, 100 aa.
Figure 2.
Subcellular localization of ALKBH4 and its co-localization with transcription-associated proteins.
(A) Subcellular localization of ALKBH4-EYFP in HeLa cells. Co-expression of (B) ALKBH4-ECFP and AF9-EYFP, (C) ALKBH4-EYFP and ECFP-ENL, (D) ALKBH4-ECFP and EYFP-ENLYEATS, (E) ALKBH4-ECFP and ENLC-EYFP and (F) ALKBH4-ECFP and EYFP-p300BP, as analyzed by confocal fluorescence microscopy. Insets are enlargements of boxed areas.
Figure 3.
Effects of ectopic expression of ALKBH4 or ALKBH7 on global gene expression.
(A) Quantitative RT-PCR analysis of relative ALKBH4 levels in HEK293 cells stably transfected with a construct for DOX-inducible over-expression of ALKBH4-FLAG, either treated with DOX (2 µg/ml) or untreated. Results are presented as mean fold change of three independent replicates normalized to β-actin ± S.D. (B) Ectopic ALKBH4 protein levels in DOX-induced and non-induced cells, as determined by Western blot analysis. Ectopic ALKBH4 was detected using an antibody against the FLAG-tag introduced at the C-terminus of ALKBH4. GAPDH expression levels are included as loading control. (C) Microarray analysis of gene expression in cells over-expressing either ALKBH4 or ALKBH7 vs. non-overexpressing cells. The number of genes whose expression is altered at least 2.0-fold (ALKBH7) or 1.35-fold (ALKBH4) is indicated (D) MetaCore (GeneGo Inc.) analysis of molecular pathways significantly (False discovery rate (FDR) <0.05) enriched with genes affected by ectopic ALKBH7 expression. DOX, doxycycline.
Table 2.
Differentially expressed genes (q-value <5, fold change >1.35) identified in ALKBH4 over-expressing cells compared to parental non over-expressing cells.
Table 3.
The genes most differentially expressed in either direction identified in ALKBH7 over-expressing cells compared to parental non over-expressing cells.
Figure 4.
Ectopic ALKBH4 expression does not change H3K79 methylation levels in vivo.
As analyzed by Western blotting, all three methylation states (mono- di- and tri-methylation) of the H3K79 residue remained similar in histones purified from stable HEK293 transfectants after doxycycline-dependent over-expression of either ALKBH4 or an enzymatically inactive mutant (ALKBH4H169A/D171A), compared to the equivalent, non-induced cells. Signal intensities of bands corresponding to methylated histones were quantified using ImageJ [69], and normalized to the total histone H3 load, but no effect of ALKBH4 overexpression was detected (not shown).