The transcription factor Krox20 is an E3 ligase that sumoylates its Nab coregulators

Abstract

Covalent attachment of small ubiquitin-like modifier (SUMO) to proteins regulates many processes in the eukaryotic cell. This reaction is similar to ubiquitination and usually requires an E3 ligase for substrate modification. However, only a few SUMO ligases have been described so far, which frequently facilitate sumoylation by bringing together the SUMO-conjugating enzyme Ubc9 and the target protein. Ubc9 is an interaction partner of the transcription factor Krox20, a key regulator of hindbrain development. Here, we show that Krox20 functions as a SUMO ligase for its coregulators--the Nab proteins--and that Nab sumoylation negatively modulates Krox20 transcriptional activity in vivo.

Figure 1

The zinc-finger domain of Krox20 mediates interaction with Ubc9. (A) Growth of yeast transformed with the indicated constructs was tested on non-selective and selective media. Bait and prey constructs were based on Gal4 DNA-binding domain (G4DB) and Gal4 activation domain (G4AD) vectors, respectively. (B) Pull-down experiments were carried out with immobilized purified glutathione S-transferase (GST) or a GST–Ubc9 fusion and in vitro translated, radioactively labelled Krox20. (C) Deletion constructs of Krox20 were tested for interaction with Ubc9 by yeast two-hybrid screening as indicated in A. TA, transactivation domain; ZF, zinc finger.

Figure 2

Krox20 mediates Nab sumoylation. (A–F) 293T cells were transfected with Flag–Nab or Flag–RanGAP1-C-ter (Flag–Ran) expression vectors and the constructs indicated at the top of each panel. Flag-tagged proteins were detected by western blot. Black arrowheads indicate non-modified proteins, and other arrowheads indicate modified proteins. Bottom panels correspond to inputs of the indicated proteins. (A,B) Nab is sumoylated in the presence of Krox20, and sumoylation is enhanced if low amounts of histidine-tagged small ubiquitin-like modifier 1 (His–SUMO1) or green fluorescent protein (GFP)–SUMO1 are transfected. (C) Nab is specifically sumoylated by Krox20, as the addition of NeuroM does not affect Nab sumoylation state; however, dominant-negative Ubc9 (C93S) interferes with Nab sumoylation. (D) The Nab K379RK517R (KR2) protein is not sumoylated. (E) Krox20 I268F and Nab Q64RH95Q mutants, affected in their ability to interact with each other, are ineffective as ligase and target in the sumoylation reaction, respectively. (F) Sumoylation of RanGAP1-C-ter is not altered by the presence of Krox20 or the I268F mutant. (G–I) Sumoylation of endogenous Nab was analysed in P19 cells in pull-down experiments with anti-Nab antibodies. (G) Endogenous Nab is sumoylated following His–SUMO1 transfection and serum stimulation, which results in the expression of Krox20. (H) Sumoylation of endogenous Nab was also observed after transfection of a Krox20 expression construct under normal growth conditions. (I) Nab sumoylation was prevented by transfection of Krox20 short interfering RNA (siRNA; si), but not by a control GFP siRNA, under serum stimulation conditions. Upper panels in G–I show pulled His–SUMO1–Nab, revealed with Nab antibodies, whereas lower panels show 1.5% input of the indicated proteins. Note that Krox20 antibodies also reveal a nonspecific upper band. (J) In vitro sumoylation assays with 300 ng of purified Flag-tagged Nab were carried out in the presence or the absence of mature SUMO (SUMO1GG) and 15 ng of Krox20 as indicated. Sumoylated products corresponded to 52.96%±2.74 (mean±s.d.) of loaded protein, as determined by chemiluminescence measurement from three independent western blots. HA, haemagglutinin epitope tag.

Figure 3

Sumoylation of Nab contributes to SUMO recruitment to chromatin and modulates Krox20 transcriptional activity. (A) Levels of small ubiquitin-like modifier 1 (SUMO1) associated with the Id4 promoter were determined by chromatin immunoprecipitation experiments in P19 cells transfected with Flag-tagged wild-type Nab or the KR2 mutant or not transfected (−). Levels of SUMO were determined on cells growing normally (white bars) or subjected to serum stimulation (black bars). Fold increase in SUMO levels were normalized to the value determined in non-transfected untreated cells. Flag levels were also determined as a control (grey bars) and fold increase was normalized to the value of Nab-transfected cells. (B) A lacZ reporter construct responsive to Krox20 was tested in P19 cells cotransfected with the indicated expression constructs. Relative units of β-galactosidase (β-gal) were normalized to the value of cells cotransfected with Krox20 alone (100%). Values are means of three independent experiments±s.d.

Figure 4

Nab sumoylation regulates Krox20 target genes in vivo. Flat-mounted chick hindbrains were analysed by (A) in situ hybridization using a Ubc9 antisense RNA probe or by (B) immunofluorescence using Ubc9 and EphA4 antibodies. (C–J) The neural tube of chick embryos was electroporated with constructs expressing the proteins indicated at the top of each panel. Green fluorescent protein (GFP) was used to monitor electroporation. Electroporated hindbrains were processed for (C–J) immunofluorescence using EphA4 antibodies, or (L,M) in situ hybridization using EphA4, Hoxb1 and Krox20 RNA probes. Arrowheads in L delimit rhombomere (r) 4. Electroporations were performed on the left side of embryos. (K) Fluorescence signals of EphA4 hybridization in C, E, G and I were measured using the MetaMorph software. For that, regions of the same area encompassing r3–r5 were defined on both electroporated and control sides. (N) Size of r4 was determined by measuring the Hoxb1-positive area using the ImageJ application, and intensity of the Krox20 hybridization signal was measured with the MetaMorph software on inverted grey-scale-converted images. Relative levels were normalized with respect to those on the control side (−). Values correspond to the mean±s.d. of 5–8 samples from three independent experiments. Statistical significance was analysed using Student's t-test: (K) ^*P=0.13, ^**P<0.025, ^***P<0.005 and ^****P<0.001; (N) ^*P<0.025 and ^**P<0.005. SUMO, small ubiquitin-like modifier.