Functional Studies of MP62 During Male Chromatin Decondensation in Sea Urchins Claudio Iribarren, 1,2 Viviana Hermosilla, 1 Violeta Morin, 1 and Marcia Puchi 1,3 * 1 Department of Biochemistry and Molecular Biology, Universidad de Concepcio´n, Concepcio´n, Chile 2 Department of Basical Sciences, Universidad Santo Tomas, Concepcio´n, Chile 3 Department of Biological Sciences, Universidad Andre´s Bello, Concepcio´n, Chile ABSTRACT In amphibians, sperm histone transition post-fertilization during male pronucleus formation is commanded by histone chaperone Nucleoplasmin (NPM). Here, we report the first studies to analyze the participation of a Nucleoplasmin-like protein on male chromatin remodeling in sea urchins. In this report, we present the molecular characterization of a nucleoplasmin-like protein that is present in non fertilized eggs and early zygotes in sea urchin specie Tetrapygus niger. This protein, named MP62 can interact with sperm histones in vitro. By male chromatin decondensation assays and immunodepletion experiments in vitro, we have demonstrated that this protein is responsible for sperm nucleosome disorganization. Furthermore, as amphibian nucleoplasmin MP62 is phosphorylated in vivo immediately post-fertilization and this phosphorylation is dependent on CDK-cyclin activities found after fertilization. As we shown, olomoucine and roscovitine inhibits male nucleosome decondensation, sperm histone replacement in vitro and MP62 phosphorylation in vivo. This is the first report of a nucleoplasmin-like activity in sea urchins participating during male pronucleus formation post-fecundation. J. Cell. Biochem. 114: 1779– 1788, 2013. ß 2013 Wiley Periodicals, Inc. KEY WORDS: CHROMATIN REMODELING; SPERM HISTONES; MP62; NUCLEOPLASMIN/NUCLEOPHOSMIN; SEA URCHINS; FERTILIZATION I n sea urchins, after fertilization, the male nucleus decondenses and transform into male pronucleus with several biochemical and molecular changes. Specifically, sperm specific histones (SpH) are selectively lost post-fertilization as male chromatin decondenses and fuses with female chromatin, at this moment zygote chromatin is condensed only by maternally inherited histones (Cleavage-Stage histones, CS) [reviewed in Imschenetzky et al., 2003]. Over a decade ago, we have demonstrated that the lost of SpH is commanded by nuclear proteolytic activities found post-fertilization, in this context we have characterized a cysteine protease, named SpH-protease, which specifically degrade male histones leaving CS histones intact [Imschenetzky et al., 1997]. This protease activity is regulated selectively by post-translational modifications present on its substrates: it is inhibited by SpH phosphorylation [Morin et al., 1999a] and poly-ADP ribosylation of CS histones [Morin et al., 1999b]. This proteolytic activity is vital for proper development, since its inhibition by E64d, a general inhibitor of cysteine proteases [Monardes et al., 2005] and the microinjection of antibodies specifically directed against SpH-protease [Puchi et al., 2006] blocks the lost of SpH and initial cell divisions in the zygote. We have demonstrated that SpH-protease is unable to degrade SpH when they are forming nucleosomes. Furthermore, we have described a nucleosome disassembly activity that liberates SpH from male chromatin for proper degradation by SpH-protease [Iribarren et al., 2008]. It is unclear which kind of activity is responsible for SpH liberation from male nucleosomes prior degradation. In batracians, the histones transitions are commanded by Nucleoplasmin. This histone chaperone promotes sperm nucleosome remodeling after fertilization together with N1/N2 complex. In this model, nucleoplasmin assembles nucleosome cores by addition of H2A– H2B dimmers to previously formed H3–H4 tetramers [reviewed by Philpott et al., 2000]. In sea urchins, there has not been characterized a nucleoplasmin-like activity using Xenopus laevis nucleoplasmin- isolation and purification protocols [Stephens et al., 2002]. But, with increased amounts in protein sequence data bases, Eirin-Lopez et al. Journal of Cellular Biochemistry ARTICLE Journal of Cellular Biochemistry 114:1779–1788 (2013) 1779 Grant sponsor: FONDECYT; Grant number: 24090019; Grant sponsor: Universidad de Concepcio´n DIUC; Grant numbers: 208.037.008-1.0, 212.037.016-1.0; Grant sponsor: ECOS/CONICYT; Grant number: C07B05. *Correspondence to: Marcia Puchi, Departamento de Ciencias Biolo´ gicas, Facultad de Ciencias Biolo´ gicas, Universidad Andre´s Bello, Concepcio´ n, Chile. E-mail: marcia.puchi@unab.cl Manuscript Received: 27 June 2012; Manuscript Accepted: 12 February 2013 Accepted manuscript online in Wiley Online Library (wileyonlinelibrary.com): 26 February 2013 DOI 10.1002/jcb.24520  ß 2013 Wiley Periodicals, Inc.