SAF-2, a Splice Variant of SAF-1, Acts as a Negative Regulator of Transcription* Received for publication, June 25, 2002, and in revised form, September 20, 2002 Published, JBC Papers in Press, September 20, 2002, DOI 10.1074/jbc.M206299200 Bimal K. Ray‡, Ryan Murphy, Papiya Ray, and Alpana Ray From the Department of Veterinary Pathobiology, University of Missouri, Columbia, Missouri 65211 Serum amyloid A-activating factor-1 (SAF-1), a Cys 2 His 2 -type zinc finger transcription factor, regulates inflammation-induced expression of serum amyloid A protein that is linked to the pathogenesis of reactive amyloidosis, rheumatoid arthritis, and atherosclerosis. Here we report the identification of a novel splice vari- ant, SAF-2, of the SAF family bearing strong sequence similarity to SAF-1. The N-terminal 426 amino acids of both SAF-1 and SAF-2 are identical containing two poly- alanine tracts, one proline-rich domain, and six zinc fingers. However, the C terminus of SAF-2 containing two additional zinc fingers is different from SAF-1, which indicates the capability of different biochemical function. We show that SAF-2 interacts more avidly with the SAF-binding element, but its transactivation poten- tial is much lower than SAF-1. Furthermore, co-expres- sion of SAF-2 markedly suppresses SAF-1-regulated pro- moter function. Finally, we show that the level of SAF-2 protein is reduced during many inflammatory condi- tions, whereas the SAF-1 protein level remains un- changed. Together, these data suggest that the relative abundance of SAF-2 plays a critical role in the fine tuned regulation of inflammation-responsive genes that are controlled by SAF-1. Persistent high levels of serum amyloid A (SAA) 1 protein is linked to various pathophysiological conditions, including am- yloidosis, rheumatoid arthritis, and atherosclerosis (1, 2). Ab- errant transcriptional induction of SAA in response to inflam- mation is regulated by a group of transcription factors in which SAF-1 plays a major role (3, 4). Consequently, mutation of the SAF-1 DNA-binding element of the SAA gene reduces its tran- scription by as much as 80% in several nonhepatic cells (4). Many inflammatory agents including LPS, PMA, and cytokines like IL-1 and IL-6, which trigger SAA overexpression, induce both the DNA binding and transactivation potential of SAF-1 (3–7). These studies showed that SAF-1 could play a critical role in all SAA-linked pathological conditions. Recently, SAF-1 is shown to be involved in the regulation of the -fibrinogen gene, whose abnormal expression is associated with myocardial infarction and stroke (8). Human and mouse homologs of SAF-1, called MAZ (9) and Pur-1 (10) respectively, have been identified as a regulator of expressions of c-myc (9), insulin (10), serotonin 1A receptor (11), CD4 (12), PNMT (13), and CLC-K1 (14) genes. All of these observations suggest that the SAF-1/ MAZ/Pur-1 transcription factor is involved in controlling ex- pression of genes associated with diverse cellular processes. Its activation in different tissues in response to diverse physiolog- ical conditions apparently determines its ability to regulate expression of different genes. Critical unanswered questions are how transcriptional prop- erties of SAF-1 are regulated. In general, activities of many transcription factors are regulated either by controlling expres- sion of the genes coding these factors at the transcriptional level or by modification of the proteins at post-translational level, most often by phosphorylation (15, 16). Some transcrip- tion factors are regulated via interaction of another protein that has a regulatory role, such as that seen in the case of NF-B/IB association (17). Alternative splicing is another im- portant mechanism for regulating activity of a transcription factor in which the same gene can be used to generate splice variants with different functional activities (reviewed in 18 – 20). Wilms tumor gene product, a Cys 2 -His 2 zinc finger con- taining transcription factor (21), cAMP-responsive element- binding protein modulator (22), and signal transducer and activator of transcription, STAT3 (23), represent a few exam- ples of many known transcription factors that contain multiple splice variants with distinct functional properties. Here we describe alternative splicing of SAF transcripts that yields a novel splice variant, designated as SAF-2, with an additional exon that is normally embedded within a large in- tron. Insertion of this exon in SAF-2 mRNA results in the formation of SAF-2 protein containing a C-terminal region that is different from SAF-1. Compared with SAF-1, SAF-2 protein has higher DNA binding but reduced transactivation ability. Furthermore, overexpression of SAF-2 suppresses SAF-1-reg- ulated promoter function. Interestingly, during inflammation, the level of SAF-2 protein is considerably reduced, whereas the SAF-1 protein level remains unchanged. These unique proper- ties of SAF-2 strongly support the notion that SAF-2 plays a critical role in fine-tuning the regulation of all SAF-1-con- trolled genes. EXPERIMENTAL PROCEDURES Isolation of a Splice Variant—HeLa cDNA library in gt-11 (a gift from M. Blanar) was screened with a rabbit cDNA probe of SAF-1 (3). Five positive clones were selected and subcloned into the plasmid pTZ19U and sequenced. Human genomic DNA library in EMBL3 (Clontech) was screened with the SAF-1 cDNA probe, and three inde- pendent positive clones were selected. Regions of the phage DNA span- ning the SAF-1 gene were sequenced. * This work was supported in part by National Institutes of Health Grant R01 DK49205 and funds from the College of Veterinary Medi- cine, University of Missouri. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. The nucleotide sequence(s) reported in this paper has been submitted to the GenBank TM /EBI Data Bank with accession number(s) AF489858. ‡ To whom correspondence should be addressed: Dept. of Veterinary Pathobiology, University of Missouri, Columbia, MO 65211. Tel.: 573- 882-4461; Fax: 573-884-5414; E-mail: rayb@missouri.edu. 1 The abbreviations used are: SAA, serum amyloid A; SAF, SAA- activating factor; MAZ, Myc-associated zinc finger protein; CAT, chlor- amphenicol acetyltransferase; MAP kinase, mitogen-activated protein kinase; LPS, lipopolysaccharide, PMA, phorbol 12-myristate 13-acetate; EMSA, electrophoretic mobility shift assay; IL, interleukin; RT, reverse transcriptase; ESE, exonic splicing enhancer; RPA, RNase protection assay. THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 277, No. 48, Issue of November 29, pp. 46822–46830, 2002 © 2002 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. This paper is available on line at http://www.jbc.org 46822 by guest on December 13, 2016 http://www.jbc.org/ Downloaded from