The regulation of catalase gene expression in mouse muscle cells is dependent on the CCAAT-binding factor NF-Y Dan Luo and Thomas A. Rando * Neurology Service and GRECC, VA Palo Alto Health Care System, Palo Alto, CA 94304, USA Department of Neurology and Neurological Sciences, Stanford University School of Medicine, Room A-343, Stanford, CA 94305-5235, USA Received 22 February 2003 Abstract Catalase is an antioxidant enzyme whose expression is transcriptionally regulated and tissue-specific. The level of expression determines, in part, the susceptibility of a cell to oxidative stress. Skeletal muscle is a tissue that experiences high levels of oxidative stress during normal metabolic activity, so the expression of antioxidant enzymes is critical to preventing cellular damage. To study the transcriptional regulation of the catalase gene in mouse muscle cells, the 5 0 -flanking region of the mouse catalase gene was isolatedfromgenomicDNA.Thetranscriptionalactivityofthe5 0 -flankingregionwasinvestigatedintransientlytransfectedmurine myoblasts using a promoter-less luciferase reporter vector and site-directed mutagenesis. Strikingly, we found that nearly all of the transcriptional activity was restricted to the final 191bp of the greater than 2.5kb of the 5 0 -flanking region examined. Of the po- tentialconsensusbindingsitesfortranscriptionalregulatorswithinthis191-bpregion,weidentifiedtwoCCAATboxesandnoother consensus sites that were important for the transcriptional activity of this promoter. Gel shift and super shift assays indicated that the transcription factor NF-Y bound to both CCAAT boxes. Furthermore, co-transfection of reporter constructs with NF-Y ex- pression vectors into Drosophila SL2 cells demonstrated NF-Y-mediated transcriptional activation of the catalase gene. Interest- ingly, there were no nearby sites that appeared to interact with either NF-Y binding sites, and thus it appears that NF-Y acts as a bona fide transcription factor for catalase gene expression in mouse muscle cells. These data provide the first examination of the regulationofthemousecatalasegeneandindicateuniqueaspectsofitsregulationthatmaypertaintothetissue-specificpatternsof expression. Ó 2003 Published by Elsevier Science (USA). Keywords: Catalase; CCAAT box; GC box; Mouse; Myoblast; NF-Y; Transcription Catalase is a cytoplasmic enzyme that catalyzes the reductionofH 2 O 2 toH 2 OandO 2 [1]therebypreventing theaccumulationofH 2 O 2 andthetoxicconsequencesof thereactionofH 2 O 2 with other cellular components [2]. The catalase gene has been isolated from humans [3,4], rats [5], mice [6], and yeast [7,8]. Several common fea- tures found in housekeeping genes have been discovered inthe5 0 -flanking region of catalase genes from different species. For example, the promoter of the catalase gene isTATA-box-lessandcontainsseveralGCandCCAAT boxes[9].However,therearealsocleardifferencesinthe 5 0 -flanking sequences of the catalase gene among three mammalian species [6], suggesting species-specific regu- lation.Furthermore,althoughcatalaseisexpressedinall tissues, the levels of activity differ among tissues. For example, in mouse, there is a high level of activity in kidney and liver and a very low level in brain and con- nective tissue [10,11]. Such findings suggest that the expression of the catalase gene is regulated in a tissue- specific manner. Several groups have explored the transcriptional regulation of catalase gene. Transcriptional activity of the 5 0 -flanking region of the rat catalase gene has been studied in transfected cells and in transgenic mice [12]. The results showed that the expression of the catalase geneistissue-specific.Forexample,themRNAleveland activity of catalase are higher in the human hepatoma celllineHepG2thanintheporcinekidneyepithelialcell line LLCPK1 and the human glioma cell line U-138 Biochemical and Biophysical Research Communications 303 (2003) 609–618 www.elsevier.com/locate/ybbrc BBRC * Corresponding author. Fax: 1-650-858-3935. E-mail address: rando@stanford.edu (T.A. Rando). 0006-291X/03/$ - see front matter Ó 2003 Published by Elsevier Science (USA). doi:10.1016/S0006-291X(03)00397-8