Abstract
In many animal species, the sperm DNA is packaged with male germ line–specific chromosomal proteins, including protamines. At fertilization, these non-histone proteins are removed from the decondensing sperm nucleus and replaced with maternally provided histones to form the DNA replication competent male pronucleus. By studying a point mutant allele of the Drosophila Hira gene, we previously showed that HIRA, a conserved replication-independent chromatin assembly factor, was essential for the assembly of paternal chromatin at fertilization. HIRA permits the specific assembly of nucleosomes containing the histone H3.3 variant on the decondensing male pronucleus. We report here the analysis of a new mutant allele of Drosophila Hira that was generated by homologous recombination. Surprisingly, phenotypic analysis of this loss of function allele revealed that the only essential function of HIRA is the assembly of paternal chromatin during male pronucleus formation. This HIRA-dependent assembly of H3.3 nucleosomes on paternal DNA does not require the histone chaperone ASF1. Moreover, analysis of this mutant established that protamines are correctly removed at fertilization in the absence of HIRA, thus demonstrating that protamine removal and histone deposition are two functionally distinct processes. Finally, we showed that H3.3 deposition is apparently not affected in Hira mutant embryos and adults, suggesting that different chromatin assembly machineries could deposit this histone variant.
Highlights
The assembly of nucleosome particles on nuclear DNA is the initial step for the formation of chromatin
The replacement of histones with protamines or other sperm nuclear basic proteins (SNBPs) during the differentiation of post-meiotic spermatids is generally associated with a high level of nuclear condensation, a general shutdown of transcriptional activity, and a state of chromatin that is incompatible with DNA replication [3,4,5]
This process, called sperm chromatin remodelling (SCR), allows the paternal DNA to recover a nucleosomal chromatin and guarantees the ability of the male pronucleus to replicate its DNA in coordination with its female counterpart [3,4,5]
Summary
The assembly of nucleosome particles on nuclear DNA is the initial step for the formation of chromatin. Nucleosome assembly initiates with the formation of a H3-H4 histone tetramer on DNA followed by the addition of two H2A-H2B dimers to form the octameric particle [1,2] This organisation of genomic DNA is remarkably conserved in eukaryotes, sperm cells of many species are characterized by a very different type of chromatin architecture involving nonhistone proteins such as protamines [3]. Once entered in the egg cytoplasm, the fertilizing sperm nucleus must replace its SNBPs with maternally provided histones that are stored in the egg cytoplasm This process, called sperm chromatin remodelling (SCR), allows the paternal DNA to recover a nucleosomal chromatin and guarantees the ability of the male pronucleus to replicate its DNA in coordination with its female counterpart [3,4,5].
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