Drosophila TAP/p32 is a core histone chaperone that cooperates with NAP-1, NLP, and nucleophosmin in sperm chromatin remodeling during fertilization

The DNA of metazoan somatic cells is packaged into a compact nucleoprotein complex termed chromatin (Van Holde 1988; Wolffe 1998). Chromatin fiber is comprised of highly conserved repetitive units (nucleosomes) that contain an octamer of four core histones and 145–147 base pairs (bp) of DNA wrapped around the octamer in 1.65 turns of a left-handed superhelix (Luger et al. 1997). Nucleosomes are assembled in vivo in an ATP-dependent fashion through a concerted and sequential action of core histone chaperones and motor proteins that belong to the Snf2 family of DNA-dependent ATPases (Gorbalenya and Koonin 1993; Eisen et al. 1995; Mello and Almouzni 2001; Akey and Luger 2003; Haushalter and Kadonaga 2003; Piatti et al. 2011; Torigoe et al. 2013). For instance, Drosophila ACF/CHRAC can mediate chromatin assembly in conjunction with histone chaperone NAP-1 (Varga-Weisz et al. 1997; Ito et al. 1999). Other known ATP-dependent chromatin assembly factors include RSF, CHD1, ATRX, and ToRC/NoRC (LeRoy et al. 1998, 2000; Lusser et al. 2005; Lewis et al. 2010; Emelyanov et al. 2012). The most abundant chromatin component in male germline cells is protamines—small positively charged arginine- and cysteine-rich proteins (Balhorn 2007). During spermiogenesis, protamines replace 85%–95% of DNA-bound histones in the nucleus to achieve a higher density of sperm nuclear DNA (Ward and Coffey 1991; Rathke et al. 2014). Crystalline-like sperm chromatin structure is sixfold more compact than metaphase chromosomes and renders sperm DNA enzymatically inert (Balhorn 1982). At fertilization, the oocyte remodels the condensed sperm chromatin into a transcriptionally competent chromatin of the male pronucleus (McLay and Clarke 2003). During, this process, protamines are expelled and replaced with oocyte-supplied histones, which are then organized into nucleosomes. Sperm chromatin remodeling (SCR) is controlled by biochemical activities in the early oocyte, but components of these activities remain largely unknown (McLay and Clarke 2003). However various protein factors have been implicated in SCR, including core histone chaperones from Xenopus (Nucleoplasmin, N1, HIRA, and TAF-I) and Drosophila (NAP-1, p22, DF31, HIRA, and Yemanuclein) (Dilworth et al. 1987; Philpott and Leno 1992; Kawasaki et al. 1994; Crevel and Cotterill 1995; Ito et al. 1996a; Matsumoto et al. 1999; Loppin et al. 2001; Ray-Gallet et al. 2002; Orsi et al. 2013). In mammals, members of nucleoplasmin/nucleophosmin family proteins (NPM1–3) function in sperm chromatin decondensation in vitro (Okuwaki et al. 2012). In addition, Npm2 knockout female mice exhibit fertility defects consistent with a role of NPM2 in nuclear and nucleolar chromatin organization (Burns et al. 2003). It was also suggested that sperm chromatin decondensation is ATP-dependent (Wright and Longo 1988). Drosophila melanogaster sperm cells contain two major protamines (A and B) encoded by male-specific transcripts Mst35Ba and Mst35Bb, respectively (Ashburner et al. 1999; Jayaramaiah Raja and Renkawitz-Pohl 2005). The Drosophila maternal effect mutant sesame (ssm) prevents male pronucleus formation (Loppin et al. 2001). ssm encodes the histone variant H3.3-specific chaperone HIRA (Tagami et al. 2004), postulated to be required for replication-independent deposition of histones in the male pronucleus during sperm decondensation (Loppin et al. 2005; Bonnefoy et al. 2007). In eggs from homozygous ssm females, maternal histones are not deposited in the chromatin of male pronuclei, preventing normal mitosis and resulting in the development of gynogenetic haploid embryos and embryonic stage lethality. A similar phenotype is observed in null mutants of the gene encoding ATP-dependent chromatin assembly factor CHD1 (Konev et al. 2007). Thus, CHD1 and HIRA act cooperatively and are required for nucleosome assembly during SCR. Intriguingly, protamines are efficiently expelled from the DNA of nascent male pronuclei in Chd1 and ssm eggs (Jayaramaiah Raja and Renkawitz-Pohl 2005; Konev et al. 2007), suggesting that protamine removal and histone deposition are functionally distinct steps. In this study, we used a biochemical approach to identify specific protein components of the Drosophila egg machinery that promote the dissociation of protamine–DNA complexes of sperm chromatin. These factors turn out to be two known core histone chaperones (NAP-1 and NLP), a homolog of mammalian nucleophosmin, and a novel Drosophila histone chaperone (TAP/p32). These putative “protamine chaperones” facilitate SCR independently of CHD1 and HIRA, which mediate nucleosome assembly in nascent male pronuclei. Of note, TAP/p32 is specifically required to expel Drosophila protamine B from sperm chromatin in vitro, whereas NAP-1, NLP, and nucleophosmin share roles in removal of protamine A. We also provide in vivo evidence that TAP/p32 functions in Drosophila egg SCR. In conclusion, we characterized protein factors that mediate the first obligatory step of SCR (protamine dissociation) and reconstituted the complete SCR reaction (reorganization of protamine-containing sperm chromatin into core histone-containing nucleosome arrays) in a purified defined system in vitro.