Human ALKBH4 interacts with proteins associated with transcription

Abstract

The Fe(II)- and 2-oxoglutarate (2OG)-dependent dioxygenase AlkB from E. coli is a demethylase which repairs alkyl lesions in DNA, as well as RNA, through a direct reversal mechanism. Humans possess nine AlkB homologs (ALKBH1-8 and FTO). ALKBH2 and ALKBH3 display demethylase activities corresponding to that of AlkB, and both ALKBH8 and FTO are RNA modification enzymes. The biochemical functions of the rest of the homologs are still unknown. To increase our knowledge on the functions of ALKBH4 and ALKBH7 we have here performed yeast two-hybrid screens to identify interaction partners of the two proteins. While no high-confidence hits were detected in the case of ALKBH7, several proteins associated with chromatin and/or involved in transcription were found to interact with ALKBH4. For all interaction partners, the regions mediating binding to ALKBH4 comprised domains previously reported to be involved in interaction with DNA or chromatin. Furthermore, some of these partners showed nuclear co-localization with ALKBH4. However, the global gene expression pattern was only marginally altered upon ALKBH4 over-expression, and larger effects were observed in the case of ALKBH7. Although the molecular function of both proteins remains to be revealed, our findings suggest a role for ALKBH4 in regulation of gene expression or chromatin state.

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 ALKBH4^H169A/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 , .//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-ENL_YEATS, (E) ALKBH4-ECFP and ENL_C-EYFP and (F) ALKBH4-ECFP and EYFP-p300_BP, 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.

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 (ALKBH4^H169A/D171A), compared to the equivalent, non-induced cells. Signal intensities of bands corresponding to methylated histones were quantified using ImageJ , and normalized to the total histone H3 load, but no effect of ALKBH4 overexpression was detected (not shown).