A new mouse gene, SRG3, related to the SWI3 of Saccharomyces cerevisiae, is required for apoptosis induced by glucocorticoids in a thymoma cell line - PubMed (original) (raw)

Comparative Study

S H Jeon et al. J Exp Med. 1997.

Abstract

We isolated a new mouse gene that is highly expressed in thymocytes, testis, and brain. This gene, SRG3, showed a significant sequence homology to SWI3, a yeast transcriptional activator, and its human homolog BAF155. SRG3 encodes 1,100 amino acids and has 33-47% identity with SWI3 protein over three regions. The SRG3 protein contains an acidic NH2 terminus, a myb-like DNA binding domain, a leucine-zipper motif, and a proline- and glutamine-rich region at its COOH terminus. Rabbit antiserum raised against a COOH-terminal polypeptide of the SRG3 recognized a protein with an apparent molecular mass of 155 kD. The serum also detected a 170-kD protein that seems to be a mouse homologue of human BAF170. Immunoprecipitation of cell extract with the antiserum against the mouse SRG3 also brought down a 195-kD protein that could be recognized by an antiserum raised against human SWI2 protein. The results suggest that the SRG3 protein associates with a mouse SWI2. The SRG3 protein is expressed about three times higher in thymocytes than in peripheral lymphocytes. The expression of anti-sense RNA to SRG3 mRNA in a thymoma cell line, S49.1, reduced the expression level of the SRG3 protein, and decreased the apoptotic cell death induced by glucocorticoids. These results suggest that the SRG3 protein is involved in the glucocorticoid-induced apoptosis in the thymoma cell line. This implicates that the SRG3 may play an important regulatory role during T cell development in thymus.

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Figures

Figure 1

Figure 1

Amino acid sequence of the SRG3 gene predicted from cDNA sequence. The predicted leucine-zipper motif is indicated by asterisks, and the myb-like tryptophan repeat is indicated by the closed triangles. The regions showing highest homology to the yeast SWI3 are underlined. These sequence data are available from EMBL/GenBank/DDBJ under accession number U85614.

Figure 2

Figure 2

The comparison of amino acid sequences of the SRG3 with SWI3 and its human homologues, BAF155 and BAF170. The YFK7, another yeast homologue of SWI3, is also presented. The regions showing highest homology are shown by rectangles with distinctive fillings (A). The SRG3 and human homologues of SWI3 protein contain the proline- and glutamine-rich domains that lack in the yeast SWI3. Amino acid comparisons of the three regions (Region I, II, and III) are shown in B and C. The three homologous regions of SRG3 and SWI3 protein displayed 33–47% identity and 61–64% similarity. The amino acids that are identical to consensus sequences are indicated as dots (C).

Figure 3

Figure 3

Immunoblotting and immunoprecipitation of the SRG3 protein. The overexpressed COOH-terminal part of SRG3 gene in Escherichia coli system was used to immunize rabbits to produce the polyclonal antiserum. When thymus and lymph node extract were blotted with the SRG3 antiserum, bands at 155 and 170 kD were detected (A). When the extract was blotted with the hSWI2 antiserum, a band at 195 kD (B, top) was detected. After immunoprecipitating the extract with the SRG3 antiserum, the precipitates were blotted with the SRG3 antiserum (B, bottom) or the hSWI2 antiserum (B, top), displaying the 155- and 195-kD bands, respectively. Immunoprecipitation with preimmune serum and blotting with the SRG3 and hSWI2 antiserum dose not show any band (B). TCL, total cell lysate; IP:Pre, immunoprecipitation with the pre immune serum; IP: anti-SRG3, immunoprecipitation with the SRG3 antiserum.

Figure 6

Figure 6

Effect of SRG3 expression on GR mediated apoptosis. (A) Expression of the antisense RNA to SRG3 in thymoma cell line, S49.1, reduced the level of SRG3 protein. The pRcASRG3 plasmid expressing 2.9 kb of XbaI fragment (bases 1–2829) of SRG3 gene in anti-sense orientation under the control of CMV promoter was transfected into the S49.1 cells. The expression of the SRG3 protein in transfected cells was detected by immunoblotting using the SRG3 antiserum. lane 1, S49.1; lane 2, vector only; lane 3, pRcASRG3 transfectant, clone A; lane 4, pRcASRG3 transfectant, clone B. (B) Effects of SRG3 expression on apoptotic cell death induced by glucocorticoid treatment. The 10 mM of hydrocortisone was treated for 72 h (lanes 1–4) and the DNAs of each cell were electrophoresed on 2% agarose gel containing ethidium bromide. The DNA fragmentation was reduced as SRG3 expression level was reduced. Lane C, control (untreated S49.1); lane 1, S49.1; lane 2, vector only; lane 3, pRcASRG3 transfectant, clone A; lane 4, pRcASRG3 transfectant clone B. (C) FACS® analysis of the DNA contents of the cells transfected with vector only (a and c) and pRcASRG3 transfectant, clone B (b and d). The subdiploid peak (closed and open arrowheads) indicates apoptotic cells induced by glucocorticoid treatment (c and d).

Figure 4

Figure 4

Northern blot analysis of SRG3 gene expression in different organs (A) and cell types (B). The same amount (15 μg) of total RNAs isolated from various tissues were analyzed by probing with a 1.8-kb HindIII fragment (bases 653–2361). (A) The lanes represent thymus (1), spleen (2), brain (3), lymph nodes (4), testis (5), and lung (6). Two transcripts of about 5 and 3.5 kb in size were expressed highly in thymus (lane 1), brain (lane 3), and testis (lane 5). (B) Both T and B cell expressed the SRG3. Lane 1, thymus; lane 2, T cells; lane 3, B cells. Normal T and B cells were separated from spleen and lymph nodes by magnetic activated cell sorter (miniMACS). The purity of the separated population was tested by probing the RNA blot with TCF-1 (closed arrowhead). Both B and T cells expressed about the same levels of SRG3, as judged by the control β-actin probe (open arrowhead).

Figure 5

Figure 5

Size fractionation of the SRG3 and SWI–SNF protein complexes. After total thymocytes extract was separated on glycerol density gradient sedimentation, the gradients were fractionated and immunoblotted with the SRG3 and hSWI2 antisera. The SRG3 protein was fractionated as separated complexes (300 kD) which are different from SWI2 complexes (2 MD). The blue–dextran (2 MD), thyroglobulin (669 kD), β-amylase (200 kD), and alcohol–dehydronase (150 kD) were used as standard molecular mass size markers.

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