BIR-1, a Caenorhabditis elegans homologue of Survivin, regulates transcription and development Marta Kostrouchova*†, Zdenek Kostrouch†‡, Vladimir Saudek§, Joram Piatigorsky¶, and Joseph Edward Rall†ʈ *Laboratory of Molecular Biology and Genetics and ‡Laboratory of Molecular Pathology, Institute of Inherited Metabolic Disorders, First Faculty of Medicine, Charles University, CZ-128 01 Prague 2, Czech Republic; †Diabetes Branch, National Institute of Diabetes and Digestive and Kidney Diseases, and ¶Laboratory of Molecular and Developmental Biology, National Eye Institute, National Institutes of Health, Bethesda, MD 20892 Contributed by Joseph Edward Rall, February 7, 2003 bir-1,aCaenorhabditis elegans inhibitor-of-apoptosis gene homol- phorylation of histone H3 on serine 10 (15), a phosphorylation ogous to Survivin is organized in an operon with the transcription that is involved in promoter activation (6, 9, 10, 13). Together, cofactor C. elegans SKIP (skp-1). Because genes arranged in oper- these data suggested that BIR-1 may have a transcriptional ons are frequently linked functionally, we have asked whether function unrelated to cell division. BIR-1 also functions in transcription. bir-1 inhibition resulted in In this study, we report evidence that BIR-1 is a transcriptional multiple developmental defects that overlapped with C. elegans regulator for numerous target genes. We show that the loss of SKIP loss-of-function phenotypes: retention of eggs, dumpy, move- function of bir-1 results in phenotypes that partially overlap with ment defects, and lethality. bir-1 RNA-mediated interference de- CeSKIP and CHR3 (nhr-23) loss of function. We also show that creased expression of several gfp transgenes and the endogenous several transgenes (elt-2::gfp, hlh-1::gfp, and hlh-2::gfp) and two genes dpy-7 and hlh-1. Immunoblot analysis revealed decreased endogenous genes (dpy-7 and hlh-1) are inhibited as a conse- phosphoacetylated histones in bir-1 RNA-mediated interference- quence of bir-1 loss of function. Western blot analysis using treated worms. In a heterologous transfection system, BIR-1 aug- antibodies against phosphoacetylated histone H3 (S10-P K14-Ac ments thyroid hormone-regulated transcription and has an addi- and K9-Ac S10-P) shows that bir-1 inhibition in worms negatively tive effect with SKIP. These results show that BIR-1 functions in the affects phosphoacetylation of histone H3. Finally, we demon- regulation of transcription and development. strate that BIR-1 acts as coactivator in a heterologous transfec- tion system, and bir-1-transfected cells have increased phos- odulation of specific gene transcription depends on or- phoacetylated histones H3. Thus our results show that BIR-1, a Mchestrated events involving the modification of chromatin member of the inhibitor-of-apoptosis gene family, functions in and cooperation between RNA polymerase II and supporting transcription regulation and is involved in regulation of devel- factors. A large number of events participate in this process opment. including the assembly of RNA polymerase II active complex on particular promoters, cooperation and modification of TFII Methods transcription factors, and interactions with numerous cofactors Strains. The following strains expressing GFP fusion proteins (for review see ref. 1). Modification of chromatin proteins such were used: JM 63 (elt-2::gfp) integrated line (20, 21); PD7963 as acetylation, methylation, and phosphorylation further influ- (hlh-1::gfp) integrated line; PD8097 (hlh-2 ::gfp) (22, 23); ences transcription activation or repression and represents an- egl-15::gfp (24), Nde::gfp (25), and hlh-8::gfp (26); his2B::gfp (27), other level of regulation (2–6). Regulation of gene transcription a gift from G. Seydoux (Johns Hopkins University, Baltimore); includes complex chromatin reorganization. During this process, and PD 6904 (let-858::gfp), a gift from W. Kelly (Emory Uni- histones in promoter regions as well as proteins involved in versity, Atlanta). As a wild type, C. elegans Bristol strain was used transcription complex are modified covalently. Modification of and maintained as described (28). histones H3 and H4 includes methylation, acetylation, and phosphorylation of N-terminal residues. A growing number of bir-1 RNA-Mediated Interference (RNAi). The RNAi was introduced cofactors and specific enzymes with methyltransferase, acetyl- by feeding bacteria producing double-stranded (ds)RNA to transferase, and aminotransferase activity were shown recently worms (feeding method) and microinjection of dsRNA into the also to precede acetylation and contribute to the activation of gonad of young adult hermaphrodites (microinjection method) transcription (3, 7, 8). Phosphorylation of histones is part of (29, 30). mitotic chromosome condensation but was shown recently also The full-length bir-1 genomic sequence was amplified by PCR to precede acetylation and contribute to the activation of and cloned into the L4440 vector. The plasmid (4851) was transcription (9–13). transformed into HT115 Escherichia coli and induced by isopro- The Caenorhabditis elegans baculoviral inhibitor-of-apoptosis ␤ repeat protein 1 (bir-1) gene in C. elegans encodes a homolog of pyl- -D-galactoside as described (30). The same construct was the human gene Survivin (14, 15). Survivin in mammals func- used for preparation of dsRNA and used for microinjections as tions as an inhibitor of apoptosis (16). In C. elegans, bir-1 has only described (29, 31). The injected larvae were transferred into new been linked thus far to spindle midzone formation and cytoki- plates at 12-h intervals. The embryos were scored after 12 h of nesis (14, 15). Our previous work noted that bir-1 is expressed incubation, and the larvae were followed for the next 3–7 days. from an operon with the transcription cofactor SKI-binding protein (SKIP; skp-1) (17). In C. elegans, genes expressed from Abbreviations: BIR-1, Caenorhabditis elegans baculoviral inhibitor-of-apoptosis repeat an operon are often functionally linked (18, 19), suggesting that protein 1; SKIP, SKI-binding protein; CeSKIP, C. elegans SKIP; RNAi, RNA-mediated inter- bir-1 may also have a transcriptional function related to C. ference; ds, double-stranded; mRXR␣, murine retinoid X receptor ␣; THR, thyroid hormone elegans SKIP (CeSKIP). CeSKIP is indispensable for C. elegans receptor; DTC, distal tip cell; T3, triiodothyronine; TSA, Trichostatine A. development, and its loss-of-function phenotype partially over- §Present address: Lead Generation Informatics, Avensis Pharma, Vitry-sur-Seine F-94400, France. laps with that of nuclear hormone receptor CHR3 (nhr-23) (17). ʈTo whom correspondence should be addressed at: Laboratory of Molecular Biology and CeSKIP and bir-1 are expressed strongly during embryonic Genetics, Institute of Inherited Metabolic Disorders, First Faculty of Medicine, Charles Univer- development and continue to be expressed in certain postmitotic sity, Ke Karlovu 2, CZ-128 01 Prague 2, Czech Republic. E-mail: marta.kostrouchova@ cells to adulthood (17). Furthermore, bir-1 can mediate phos- lf1.cuni.cz. 5240–5245 ͉ PNAS ͉ April 29, 2003 ͉ vol. 100 ͉ no. 9 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0730770100 Downloaded by guest on October 1, 2021 The reporter genes elt-2::gfp, hlh-1::gfp, hlh-2::gfp, hlh-8::gfp, with ratios: 400 ng of reporter plasmid, 150 ng of human THR␤1, his2B::gfp, and let-858::gfp were used to assay the expression of mRXR␣, flag-SKIP, and bir-1, and 15 ng of control cytomega- transgenes. Animals were fed bacteria producing bir-1 dsRNA or lovirus-Renilla luciferase vector. Empty vectors and plasmid transformed with empty vector. For transcription induction, the pBluescript SK(Ϫ) were used to substitute for receptor, bir-1,or plates contained 4 mM isopropyl-␤-D-galactoside, and both flag-SKIP. Plasmid pBluescript was also used to maintain the cultures were induced with 4 mM isopropyl-␤-D-galactoside for total amount of DNA constant (1 ␮g or 1,115 ng in some 4 h. Approximately 50 animals from each transgenic line were experiments) and 3 ␮l of FuGENE. Where possible, all plasmids screened for expression of particular transgene with an Olympus were mixed first as a master mix and split after mixing to (Tokyo) BX60 microscope. minimize inequalities. All experiments were done at least twice and in triplicate or quadruplicate. After preparation of trans- Gene-Expression Assays. The animals were fed with bacteria fection suspensions and appropriate incubations, the medium producing bir-1 dsRNA or empty vector. Approximately 10,000 with hormone-depleted serum and with gentamycin was added, worms were used for each experiment. The worms were grown mixed, and 1 ml of transfection medium was overlaid over cells. from synchronized L1 stage for one or two generations on 2% After a 24-h incubation, the medium was removed and ex- agarose plates and collected as young adults just when some changed for fresh medium supplemented with 10Ϫ6 M triiodo- started to lay embryos. RT-PCR was performed from total RNA thyronine (T3, Sigma) dissolved in ethanol as 10Ϫ3 M stock as described (32, 33). The dpy-7, elt-2, and hlh-1 genes were solution or with ethanol only. Trichostatine A (TSA) was assayed. These experiments were done independently three dissolved also in ethanol and used at final concentration 10 nM. times in duplicate, and each was assayed by PCR several times. Cells were harvested 24 h later and used for the dual luciferase For Western blot analysis, synchronized L1 stage animals were method (Promega) as recommended by the manufacturer. The grown for two generations on nematode growth medium plates human telomerase RNA ␤1, mRXR␣, F2 thyroid-responsive and collected as young adults. Protein extracts were prepared by construct, and DR4 construct were kind gifts from Paul Yen, and using standard protocol with Laemmli buffer. Protein content ME-TRE was a kind gift from Keiko Ozato (National Institute was estimated by using the BCA protein assay kit (Pierce), and of Child Health and Human Development, National Institutes of 20 ␮g of protein were loaded per lane. For immunodetection of Health, Bethesda).