Schematic representation of yeast two-hybrid high confidence hits involved in transcription.

<p>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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049045#pone.0049045-Hunter1" target="_blank">[73]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049045#pone.0049045-Sigrist1" target="_blank">[74]</a>.//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.</p

The genes most differentially expressed in either direction identified in ALKBH7 over-expressing cells compared to parental non over-expressing cells.

<p>The genes most differentially expressed in either direction identified in ALKBH7 over-expressing cells compared to parental non over-expressing cells.</p

Ectopic ALKBH4 expression does not change H3K79 methylation levels <i>in vivo</i>.

<p>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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049045#pone.0049045-Schneider1" target="_blank">[69]</a>, and normalized to the total histone H3 load, but no effect of ALKBH4 overexpression was detected (not shown).</p

High confidence^a ALKBH4 interacting proteins identified by the yeast two-hybrid system.

a<p>PBS (predicted biological score) of A or B. PBS (calculated according to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049045#pone.0049045-Formstecher1" target="_blank">[75]</a>) represents the probability of an interaction to be non-specific, and refers to an e-value with defined thresholds to rank the results in the high-to-low confidence categories A–D.</p>b<p>SID (selected interaction domain) refers to the amino acid region shared by all prey fragments matching the same reference protein,</p>c<p>(+) Low confidence hit (PBS D),</p>d<p>EIF3CL. Grey shade, transcription related interactants. YEATS, Yaf9 ENL AF9 TAF14 Sas5; DBD, DNA binding domain; PHD, plant homeodomain; PET, Prickle Espinas Testin; LIM, Lin-11 Isl-1 Mec-3; PCI, Proteasome, COP9, Initiation factor-3; PTP, protein tyrosine phosphatase.</p

Differentially expressed genes (q-value <5, fold change >1.35) identified in ALKBH4 over-expressing cells compared to parental non over-expressing cells.

1<p>The <i>ALKBH4</i> probe was not detected due to annealing to the UTR of the gene. Sorted by fold change.</p

Effects of ectopic expression of ALKBH4 or ALKBH7 on global gene expression.

<p>(<b>A</b>) Quantitative RT-PCR analysis of relative <i>ALKBH4</i> 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 <i>β-actin</i> ± S.D. (<b>B</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. (<b>C</b>) 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 (<b>D</b>) MetaCore (GeneGo Inc.) analysis of molecular pathways significantly (False discovery rate (FDR) <0.05) enriched with genes affected by ectopic ALKBH7 expression. DOX, doxycycline.</p