Ex vivo and in vivo biologic effects of C/ebpα sumoylation. (A-B) Alignment of conserved amino acid sequences of SUMO attachment of C/ebpα (A) and Gata1 (B). H.s indicates homo sapiens; M.s, Mus musculus; D.r, Danio rerio. Arrowheads indicate the evolutionarily conserved lysine residue within the consensus. (C-D) Sumoylation of C/ebpα (C) and Gata1 (D) is implicated in transcriptional repression. Luciferase activity assays were performed in 293T cells using various C/ebpα or Gata1 constructs indicated. The Renilla plasmid was used as an internal control. (E-F) Luciferase activity assays of transcriptional interplay between C/ebpα and Gata1. Note that whereas C/ebpα functions normally in the presence of Gata1 (F), it strongly inhibits the transcriptional activity of Gata1 (E). SD is derived from 3 independent transfection experiments. *P < .05; **P < .01. (G-R) In vivo rescue assay of C/ebpα mutants. Whereas SUMO2-C/ebpα (I and O) and POZ-C/ebpα (K and Q) fusions restore normal expression patterns of mpo and gata1 in SUMO-deficient embryos, SUMO2-ΔC/ebpα (J and P) becomes incapable, in agreement with its default in transcriptional activation (panel C lines 7 and 8). Ub-C/ebpα, serving as a negative control, does not rescue SUMO-deficient phenotypes (L and R). Ub indicates ubiquitin.