Generation of SENP1−/− mice
The SENP1+/− ES cell line XG001 was obtained from BayGenomics. XG001 cells were generated by using a gene trap protocol with the trapping construct pGT1Lxf containing the intron from the engrailed-2
gene upstream of the gene encoding the β-galactosidase/neomycin-resistance fusion protein (see http://baygenomics.ucsf.edu
). The vector was inserted into intron 8 of the SENP1 locus. A male chimeric mouse was generated from the ES cell line. C57 BL6 mice were obtained from The Jackson Laboratory.
Plasmids and antibodies
The Epo enhancer (Epo-Luc) and the HIF1α binding site mutant (mEpo-Luc) were provided by Dr. H.M. Sucov. HRE-Luc was provided by Dr. J. Yi. pGal4-VP16 and pGal4-ODD(344–698)-VP16 were provided by Dr. P.J. Ratcliffe. Flag-VHL was provided by Dr. T. Kamitani. VHL(1–213), VHL(54–213), VHL(63–155), and VHL(156–213) were provided by Dr. M. Ohh. HA-SUMO-1, Flag-SENP1, and Flag-SENP1 catalytic mutant were previously described (1, 2). RGS-HIF1α, RGS-HIF1αK391R, RGS-HIF1αK477R, RGS-HIF1αSM (K391R, K477R), RGS-HIF1α PM (P402A, P564A), pET-ODD (344–698)PM(p402A, P564A), and pET-ODD (344–698)PM-SUMO1 were generated using standard cloning procedures and PCR-based mutagenesis. We used antibodies against Flag (M2, Sigma), HA (HA-7, Sigma), Myc (Santa Cruz), SUMO-1 (Zymed), SUMO-2/3 (gifted from Dr. Mike Matunis), mouse HIF1α (Novus), and human HIF1α (BD).
Histopathologic and immunohistochemistry analysis
Embryos were fixed in 10% buffered formalin (Sigma) and embedded in paraffin. Five-micrometer-thick sections were stained with hematoxylin and eosin. For proliferation studies, the sections were stained with Ki67-specific antibodies (Dako). Apoptotic cells were detected in sections using the TUNEL stain (TACS TdT DAB kit, R&D).
Cells were prepared from the livers of E13.5 embryos in α-MEM (GIBCO–BRL) and counted in the presence of 3% acetic acid, which lysed erythrocytes. Cell suspensions were mixed with MethoCult M3334 to detect BFU-e or MethoCult M3434 to detect CFU-e (StemCell Technologies). Cells were plated in 35-mm dishes and cultured at 37°C in an atmosphere containing 5% CO2. For the CFU-e assay, benzidine-positive CFU-e colonies were scored on day 3. For the BFU-E assay, benzidine-positive BFU-e colonies were scored on day 8.
Single-cell suspensions were obtained from E13.5 wild-type and mutant fetal livers. Cell suspensions were first incubated on ice with rat anti-mouse CD16/CD32 (PharMingen) to block non-specific binding to Fc receptors. Subsequently, cells were incubated with rat anti-mouse PE-conjugated anti-c-kit and anti-CD44 and FITC–conjugated anti-CD34 and anti-Ter-119 (all from PharMingen). Appropriate isotype control antibodies were used. Cell surface expression of different markers was analyzed in a Becton Dickinson FACScan using CellQuest software. For the TUNEL assay, cells were stained using a TUNEL kit from Roche and then analyzed by flow cytometry.
Two 21-nucleotide SENP1 siRNAs (si-1: AACTACATCTTCGTGTACCTC; si-2: CTAAACCATCTGAATTGGCTC) were synthesized (Dharmacon). The same sequence of the si-1-inverted orientation was used as a non-specific siRNA control. The SENP1 and non-specific siRNA oligos were inserted into a pSuppressorNeo vector (IMGENEX Corporation) according to manufacturer’s instructions. Hep 3B cells were transfected with the siRNA plasmid using lipofectamine 2000 (Invitrogen). RCC4 and RCC4/VHL (purchased from ECACC) were infected by retrovirus based SENP1si-1 virus particles produced from pSuppressorNeo-SENP1si-1 and selected by G418. Silencing efficiency of the siRNA was confirmed by performing real-time PCR (for Hep 3B) or RT-PCR (for RCC4 and RCC4/VHL) to examine SENP1 expression. TaqMan Master Mix Reagents (Applied Biosystems) were utilized for quantitative real-time PCR (QRT-PCR) reaction. The TaqMan ABI PRISM 7000 Sequence Detector System (PE Applied Biosystems) was used for the analysis. Primers for SENP1 (forward: 5′-TTGGCCA GAGTGCAAATGG-3′; reverse: 5′-TCGGCTGTTTCTTGA TTTTTGTAA-3′) and the housekeeping 18S rRNA (ABI) were utilized.
In vitro SUMOylation assay
In vitro SUMOylation kit was purchased from LAE Biotech International (Rockville, MD). The reaction was carried out at 37°C, for 1 hr with the mixture including E1 (150 ng), E2 (5 μg), SUMO-1 (5 μg), ATP (2 mM), and GST-ODD (344–698)PM (300 ng).