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

Abstract

Nuclear DNA in the male gamete of sexually reproducing animals is organized as sperm chromatin compacted primarily by sperm-specific protamines. Fertilization leads to sperm chromatin remodeling, during which protamines are expelled and replaced by histones. Despite our increased understanding of the factors that mediate nucleosome assembly in the nascent male pronucleus, the machinery for protamine removal remains largely unknown. Here we identify four Drosophila protamine chaperones that mediate the dissociation of protamine-DNA complexes: NAP-1, NLP, and nucleophosmin are previously characterized histone chaperones, and TAP/p32 has no known function in chromatin metabolism. We show that TAP/p32 is required for the removal of Drosophila protamine B in vitro, whereas NAP-1, NLP, and Nph share roles in the removal of protamine A. Embryos from P32-null females show defective formation of the male pronucleus in vivo. TAP/p32, similar to NAP-1, NLP, and Nph, facilitates nucleosome assembly in vitro and is therefore a histone chaperone. Furthermore, mutants of P32, Nlp, and Nph exhibit synthetic-lethal genetic interactions. In summary, we identified factors mediating protamine removal from DNA and reconstituted in a defined system the process of sperm chromatin remodeling that exchanges protamines for histones to form the nucleosome-based chromatin characteristic of somatic cells.

Thioredoxin-dependent disulfide bond reduction is required for protamine eviction from sperm chromatin

“…The Drosophila thioredoxin DHD specifically reduces intermolecular disulfide bonds in protamines and facilitates their eviction from DNA We observed previously that, in our defined system, protamine chaperones occasionally failed to efficiently remove protamines from MSC (Emelyanov et al 2014). It is possible that the reaction was inhibited by oxidized and oligomerized protamines that accumulated during substrate storage (Supplemental Fig.…”

“…When we examined the oligomerization of Prot B after loading on plasmid DNA to form the model sperm chromatin (MSC) (Emelyanov et al 2014), we also observed dimers (Fig. 1C).…”

“…Upon loading on DNA, Drosophila protamines undergo spontaneous oxidation that leads to their oligomerization Recombinant Drosophila Prot B (16.5 kDa) was purified to >95% homogeneity (Emelyanov et al 2014). SDS-PAGE in the absence of β-mercaptoethanol (βME) reveals that it can form dimers in solution (Fig.…”

“…Crude Drosophila extract (S-190) has been observed to consistently outperform purified protamine chaperones in MSC remodeling reactions in vitro (Emelyanov et al 2014). In addition to protamine chaperones, the extract may contain other activities that facilitate protamine eviction; for instance, factors that reduce disulfide bonds and disrupt protamine oligomers.…”

“…Drosophila male-specific transcripts of protamine A (ProtA) and ProtB encode protamine-like proteins that exhibit homology with mammalian protamines and constitute the major protein component of sperm chromatin (Raja and Renkawitz-Pohl 2005;Alvi et al 2013;Kanippayoor et al 2013). After egg fertilization, sperm chromatin undergoes remodeling (Loppin et al 2015), whereupon protamines are expelled from DNA by the combined action of a family of protamine chaperones (Emelyanov et al 2014) and replaced by core histones in a process dependent on the histone chaperone HIRA (Loppin et al 2005) and the ATP-driven motor protein CHD1 (Konev et al 2007).…”