Clontech Laboratories, Inc. • www.clontech.com Reprinted from Clontechniques April 2006 CR692118 US (630574)  Use of Suppression Subtractive Hybridization to Identify Osmotic Stress Transcription Factors in Tilapia (Oreochromis mossambicus) Diego F. Fiol, Ph.D. and Dietmar Kültz, Ph.D. Physiological Genomics Group, Department of Animal Science, University of California, Davis, One Shields Avenue, Davis, CA 9566, USA Gills of euryhaline teleosts are excellent models for studying osmotic stress adaptation because they directly contact the aquatic environment and are an important effector tissue during osmotic stress. We acclimated tilapia (Oreochromis mossambi- cus) from fresh water (FW) to seawater (SW); performed suppression subtractive hybridization (SSH) of gill mRNAs; and identified two tran- scription factors, osmotic stress transcription factor 1 (OSTF1) and the tilapia homolog of transcrip- tion factor II B (TFIIB), that are rapidly and transiently induced during hyperosmotic stress. We conclude that OSTF1 and TFIIB are critical elements of osmosensory signal transduction in eu- ryhaline teleosts that mediate osmotic adaptation by means of transcriptional regulation. Introduction Euryhaline teleosts are osmoregulators that maintain plasma osmotic homeostasis largely by extrarenal NaCl transport. In seawater (SW) they actively secrete salt (NaCl), while in fresh water (FW) they actively absorb salt mainly across gill epi- thelium. During acclimation of euryhaline fish from FW to SW, gill epithelium is ex- tensively remodeled to account for altered requirements of ion transport and perme- ability. Remodeling includes changes in turnover of gill epithelial cells, altered dif- ferentiation patterns of gill epithelial cells, and modulation of expression and activity of many ion transporters actively involved in osmoregulation (1). Many adaptive responses to salinity change are based on transcriptional regulation (2–7). However, no specific osmotic stress transcription factors have been identified in teleosts. In the current study we have performed suppression subtractive hybridization (SSH) of gill mRNAs isolated from gill epithelium of euryhaline tilapia (Oreo- chromis mossambicus). Using this approach we identified two novel transcription factors: osmotic stress transcription factor 1 (OSTF1) and the tilapia homolog of general transcription factor II B (TFIIB). Cloning of two transcriptional factors that are regulated by osmolality Total RNA was isolated from gills of fish transferred for 4 hr from freshwater (FW) to seawater (SW) or from FW to FW (handling controls) using Stratagene’s RNA isolation kit. mRNA was purified using the NucleoTrap mRNA Purification Kit (Clontech). A subtracted library of cDNAs enriched in the SW condition was obtained using the SSH-PCR technique via the Clontech PCR-Select™ cDNA Subtraction Kit (Cat. No. 637401). Full- length coding sequences of the original SSH clones were obtained by using degen- erate primers and Clontech’s SMART™ RACE cDNA Amplification Kit (Cat. No. 634914) (Figure 1, Panels A and B). Semi-quantitative PCR using gill epithe- lial mRNA samples from FW- and 4 hr SW-acclimated fish confirms that OSTF1 and TFIIB transcripts are upregulated during hypertonic stress (Figure 1, Panels B and C). A more detailed description of this study is available (8). OSTF1 and TFIIB are immediate early genes during hyperos- motic stress e tilapia clones enriched by SSH were short and located mainly in the 3’ UTR. Despite the short sequences identified by SSH and tilapia being a non-model species, we were able to rapidly obtain the full-length sequences and identify the corresponding clones as OSTF1 and TFIIB by using Smart-RACE technol- ogy. Quantitative real-time PCR (qPCR) showed that osmotic induction of both transcripts is transient and follows a similar time course (Figure 2). us, OSTF1 and TFIIB are coinduced during hyperosmotic stress with induction kinet- ics characteristic of immediate early genes (IEGs). Kinetics of OSTF1 and TFIIB protein abundance following hyperos- motic stress as assessed by immunoblot- ting with specific antibodies is similar to that of mRNA induction with a slight delay (Figure 3). is manifestation of hyperosmotic induction of OSTF1 and TFIIB at the protein level indicates that it is functionally significant. e induction of both transcription factors clearly depends on the degree of increase in environmental salinity (Figure 4). On the contrary, exposure of fish to oxidative stress (1 mM H2O2) or heat shock (36°C) does not increase mRNA abundance of OSTF1 or TFIIB (Figure 4). To confirm that oxidative stress and heat shock represented stressful conditions to the fish we also assayed induction of Hsp70 in the same samples. Our data in- dicate that OSTF1 and TFIIB induction is highly specific for osmotic stress. OSTF1 belongs to the TSC-22/GILZ (Transforming Growth Factor beta-Stimu- lated Clone-22/Glucocorticoid-Induced Leucine Zipper) protein family of leucine zipper-containing transcription factors. ese proteins form homo- and heterodi- mers with other family members (at least 7 in mammals and 5–10 in teleosts). Based on what is known about func- tions of TSC-22/GILZ family members, 244 bp SSH clone  3,300 pb ORF (410–1,078) OSTF full-length clone 1,700 bp 650 bp 810 bp 5' RACE 3' RACE 5' DPP 340 bp SSH clone 0 1,831 pb ORF (29–981) TFIIB full-length clone 600 bp 1,000 bp 5' RACE 3' RACE A B C D OSTF1 FW 4H SW Figure . Isolation of OSTF and TFIIB by suppression subtractive hybridization (SSH). SSH clone 2 (Panel A) and SSH clone 10 (Panel B) were fully extended using PCR-based methods such as RACE (SMART RACE cDNA Amplification Kit) and DPP (degenerated primer PCR). Semi- quantitative RT-PCR from samples of FW fish (control) and fish acclimated to SW for 4 hr are shown in Panels C and D. TFIIB FW 4H SW