The nucleosome assembly protein Nap1 regulates chromatin stability and nuclear division in Tetrahymena thermophila.

Chromatin stability is essential for maintaining genome integrity and gene regulation in eukaryotic cells. Nucleosome assembly protein 1 (Nap1) is a key regulator of chromatin dynamics across different organisms. The ciliate Tetrahymena thermophila contains two functionally distinct nuclei within a single cell: the diploid germline micronucleus (MIC), which undergoes mitosis and meiosis, and the polyploid somatic macronucleus (MAC), which divides amitotically. However, the function of Nap1 in this evolutionarily distant protist remains unclear. Here, we show that Nap1 localizes predominantly to the perinuclear region of the MAC during vegetative growth and is also detectable in the MAC, MIC, and cytoplasm. Truncation of the nuclear export signal (NES) increases accumulation of Nap1TrN67 in the MAC. During sexual development, Nap1 localizes to the cytoplasm, parental MAC, and developing new MAC, while Nap1TrN67 shows enhanced enrichment in the new MAC. Nap1 also partially colocalizes with the nuclear pore protein Nup98 at the MAC envelope. Knockdown of NAP1 impairs proliferation, disrupts amitotic MAC division, and induces nuclear extrusion bodies. Moreover, NAP1 deficiency causes abnormal meiotic progression. Co-immunoprecipitation coupled with mass spectrometry showed that Nap1 associates with nuclear pore complex proteins, histones, and DNA replication/repair factors. Direct binding of Nap1 to H2A-H2B and the ribosomal protein Rps6 was confirmed by pull-down assays. These findings establish Nap1 as a multifunctional protein required for nuclear envelope integrity, nuclear division, and chromatin stability in Tetrahymena.