ASB4 is highly differentially expressed in the vascular lineage during ES cell differentiation (Diff). (A) Supervised hierarchical cluster of genes with a ≥1.5-fold absolute mean difference in 84-h Flk1⁺ cells from embryoid bodies. The Flk1 cluster containing ASB4 is shown at the right. This experiment was described in detail by Wang et al. (), and the complete data set is available online through the Gene Expression Omnibus (series record GSE3757, http://www.ncbi.nlm.nih.gov/geo) and through the University of North Carolina Microarray Database (http://genome.unc.edu). Experiments were performed in quadruplicate. (B) RT-PCR analysis of ASB4 expression levels in Flk1⁺ cells from differentiated embryoid bodies. GAPDH was used as a loading control. US, unsorted.

ASB4 associates with a ubiquitin ligase complex. (A) HEK-293T cells were transfected with Flag-ASB4, Flag-ASB4ΔSOCS (flag-ΔSOCS), or the EV pCMV. Lysates were immunoprecipitated (IP) with anti-Flag-agarose beads and subjected to immunoblotting (IB) with anti-Flag, -Cul2, -Cul5, -elongin B, and -Roc1 antibodies. Asterisks denote nonspecific bands. (B) COS7 cells were infected with Flag-ASB4 adenovirus or GFP-alone adenovirus (GFP). Lysates were immunoprecipitated and immunoblotted as described for panel A. (C) HEK-293T cells were transfected with Flag-ASB4, cultured for 20 h, and then treated with the proteasome inhibitor MG-132 at 40 μM for 4 h before protein harvesting. Lysates were immunoprecipitated as described for panel A and immunoblotted with antiubiquitin (ub) and anti-Flag antibodies. (D) HEK-293T cells were cotransfected with Flag-ASB4 and myc-ASB4, immunoprecipitated with anti-myc agarose beads, and immunoblotted.

ASB4 mRNA expression in embryonic and adult tissues. (A) Northern blot analysis of ASB4 expression levels in whole embryos and dissected extraembryonic tissues. (B) Northern blot analysis of ASB4 expression levels in adult tissues. BM, bone marrow. Ethidium bromide staining of 28S rRNA was used as a loading control.

Localization of ASB4 mRNA during mouse embryogenesis. Whole-mount in situ hybridization with DIG-labeled RNA probes for ASB4 was performed on E9.5 to E11.5 mouse embryos. (A) E9.5 embryo. Arrow, allantois; arrowhead, forelimb; bar, 500 μm. (B) E9.5 embryo at high magnification. Arrow, rostral capillary plexus; arrowheads, branchial arch capillary plexi; bar, 50 μm. (C) E9.5 embryo probed with sense probe as a negative control. Bar, 500 μm. (D) E9.5 yolk sac. Arrows, yolk sac vessels; bar, 750 μm. (E) E10.5 embryo. Arrow, umbilical vessels; bar, 800 μm. (F) E11.5 embryo. Arrow, caudal intersomitic vessels; bar, 1 mm. (G) Transverse section of E9.5 embryo heart. Arrow, endocardium; arrowheads, dorsal aorta and intersomitic vessel; bar, 80 μm. (H) Sagittal section of E9.5 embryo heart. v, ventricle; arrow, pro-epicardium/septum transversum. (I) Transverse section of E10.5 embryo liver. HL, hind limb; FL, forelimb; arrow, liver; arrowhead, umbilical vessels; bar, 150 μm. (J) Schematic of section location in panels G and I. Antisense probes were used for all images except that in panel C (sense probe). Purple staining denotes a positive signal. In some cases (G, H, and I), stained whole embryos were paraffin embedded and sectioned for microscopic examination.

ASB4 binds to FIH through a conserved motif in AR6. (A) Yeast wheel assay. Yeast cells transformed with different combinations of bait (ASB4ΔSOCS) and prey (FIH) constructs were spread onto a high-stringency selective plate lacking tryptophan, leucine, histidine, and adenine. p53 and T were used as positive controls. V1, empty bait vector (pGBKT7). V2, empty prey vector (pGADT7). (B) HEK-293T cells were transfected with either Flag-ASB4 or Flag-ASB1. Lysates were immunoprecipitated (IP) with Flag antibody-conjugated agarose beads and immunoblotted (IB) with anti-Flag and anti-FIH antibodies. (C) Sequence alignment of mouse ASB4 and other known FIH hydroxylation substrates. The consensus sequence shows high conservation of E244, V245, N246, A247, and flanking leucine residues. Residue numbers are indicated above the ASB4 sequence. (D) Schematic of ASB4 with the N-terminal variable region (NTV), nine tandem ARs (poorly conserved repeats are indicated by parentheses), and a C-terminal SOCS box. Asparagine 246 is contained in the loop of AR6. Schematics of N-terminally Flag-tagged constructs are denoted below. (E) HEK-293T cells were transfected with Flag-ASB4, Flag-ASB1, and the Flag-ASB4 deletion mutants described in panel D. Protein lysates were immunoprecipitated with anti-Flag-agarose and immunoblotted with anti-FIH antibody. (F) HEK-293T cells were transfected with various Flag-ASB4 point mutants and myc-FIH. Lysates were immunoprecipitated with Flag antibody-conjugated agarose beads and immunoblotted with anti-Flag and anti-myc antibodies. WCL, whole-cell lysate.

ASB4 is hydroxylated on asparagine 246 by an oxygen-dependent mechanism. (A) Structural model of ASB4 AR6 and -7 (yellow ribbon backbone) positioned above the FIH (gray stick backbone)/HIF peptide (purple ribbon backbone) crystal structure (PDB accession no. 1H2K). Space-filled EVNA residues are colored orange. Space-filled FIH residues contributing to HIF binding are colored yellow. Leucines and other possible hydrophobic interacting residues are colored blue. (B) COS7 cells were infected with Flag-ASB4 adenovirus for 24 h. Lysates were immunoprecipitated with Flag antibody-conjugated agarose beads. Beads were eluted with Flag-tagged peptide and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Coomassie staining. Flag-ASB4 bands were subjected to in-gel trypsin digestion and MALDI-TOF MS analysis. The 1,329-Da peak represents the unhydroxylated form of the asparagine-containing peptide, and the 1,345-Da peak (asterisk) represents the hydroxylated form of the peptide. (C) The 1,329- and 1,345-Da peptides were analyzed by MS/MS. A 16-Da shift was observed with the y4-y8 ions but not with the y2 ions, consistent with hydroxylation at the asparagine residue. (D) HEK-293T cells transiently transfected with Flag-ASB4 were cotransfected with myc-FIH or siRNA targeting FIH or GAPDH (negative control) or cultured under hypoxic conditions (1%). Flag-ASB4 protein was isolated and analyzed as for panel B, and the peak area ratio of unhydroxylated (1,329 Da) to hydroxylated (1,345 Da) peptide was calculated to evaluate the percentage of hydroxylated ASB4. In parallel, whole-cell lysates were subjected to immunoblotting (IB) by FIH antibody. Asterisks indicate nonspecific bands.

ASB4 overexpression in ES cells increases the percentage of Flk1⁺ cells and vascular commitment via an oxygen-dependent mechanism. ES cells were electroporated with constructs encoding cytomegalovirus promoter-driven 3×Flag-ASB4, unhydroxylatable ASB4 mutants (ΔAR6 and EVN→ATA), or the vector alone (EV), and stably expressing clones were selected for by culture in G418 for 14 days. Positive clones were isolated, expanded, and used for differentiation experiments as previously described. (A and B) FACS analysis of 96-h differentiated embryoid bodies (EBs) shows a drastic increase in Flk1⁺ cells in ASB4-expressing clones but not in clones expressing unhydroxylatable mutants. Results in panel A are representative of three independent experiments. Results in panel B represent averages of three independent clones in three independent differentiation experiments. Flk1⁺ cells in ASB4-expressing clones were normalized to EV clones in each experiment. (C) Real-time RT-PCR analysis of differentiated embryoid bodies was used to determine the lineage commitment of ASB4-expressing ES cells. Compared with EV cells, 96-h differentiated ASB4-expressing ES cells show increased expression of a marker of mesoderm commitment (brachyury [T]) and of a marker of early vascular lineage commitment (Flk1). At day 6, ASB4-expressing ES cells show decreased expression of hematopoietic lineage markers (Gata1, Scl/Tal), and at day 8, they show increased expression of vascular markers (Tie2, VE-cadherin [VE-Cad]). Results represent averages of three independent biologic replicates. (D and E) Embryoid bodies stably expressing either ASB4 or EV were differentiated under hypoxic conditions (1% oxygen) and analyzed via FACS and real-time PCR as described for panels B and C.