Nuclear Autoantigenic Sperm Protein (NASP), a Linker Histone Chaperone That Is Required for Cell Proliferation

Richard T Richardson,Oleg M Alekseev,Gail Grossman,Esther E Widgren,Randy Thresher,Eric J Wagner,Kelly D Sullivan,William F Marzluff,Michael G O'Rand

doi:10.1074/jbc.m603816200

Richard T Richardson, Oleg M Alekseev + Show 7 more

Open Access

https://doi.org/10.1074/jbc.m603816200

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Abstract

A multichaperone nucleosome-remodeling complex that contains the H1 linker histone chaperone nuclear autoantigenic sperm protein (NASP) has recently been described. Linker histones (H1) are required for the proper completion of normal development, and NASP transports H1 histones into nuclei and exchanges H1 histones with DNA. Consequently, we investigated whether NASP is required for normal cell cycle progression and development. We now report that without sufficient NASP, HeLa cells and U2OS cells are unable to replicate their DNA and progress through the cell cycle and that the NASP(-/-) null mutation causes embryonic lethality. Although the null mutation NASP(-/-) caused embryonic lethality, null embryos survive until the blastocyst stage, which may be explained by the presence of stored NASP protein in the cytoplasm of oocytes. We conclude from this study that NASP and therefore the linker histones are key players in the assembly of chromatin after DNA replication.

Highlights

Tor that is necessary for progression through S phase [6, 7]
Our results imply that nuclear autoantigenic sperm protein (NASP) orchestrates the supply of linker histones that are necessary for nucleosome remodeling during DNA replication or repair and support the conclusion that H1 linker histones are necessary for normal development [2, 3]
NASP Is Required for Cell Proliferation—HeLa cells were transfected with NASP small interfering RNA (siRNA) or a control (C2), nonspecific siRNA [20] at 0 and 48 h

Summary

EXPERIMENTAL PROCEDURES

Materials—Miscellaneous chemicals were the highest possible molecular biology grade (Sigma). The DNA sequences of the PCR primers used to generate the NASP arms of homology are as follows: 1) long arm upstream primer, 5Ј-AGAGCGGCCGCGGCTATTCCTCAGTTGACACTCAG-3Ј; 2) long arm downstream primer, 5Ј-GCGTTAATTAAATACCTGGCTAACTCCAGAAGTGAC-3Ј; 3) short arm upstream primer, 5Ј-GCGCACGTGATGTTAACTTCTGTCGCTGTGGAGG-3Ј; and 4) long arm downstream primer, 5Ј-GCGGGATCCTAAGAATCCTCCAGCTTTCCAAACAGCCAC-3Ј Homologous recombination with this targeting construct deletes 9682 bp from the NASP gene, which includes exons 6 –11 (see Fig. 4). To isolate DNA for PCR template, individual blastocyst cultures were suspended by pipetting and incubating them in 20 ␮l of 25 mM NaOH/0.2 mM EDTA for 1 h at 95 °C followed by addition of 100 ␮l of 40 mM Tris-HCl. After a brief vortexing, the samples were centrifuged at 1000 ϫ g for 10 min, and the supernatant was removed. After a wash in PBS, cells were fixed a second time in 3% formaldehyde, incubated for 1 h with 4 M HCl [7], and incubated in Alexa Fluor-488-conjugated anti-BrdUrd (1:50) antibody (Molecular Probes)

RESULTS

NASP Is Required for Normal

DISCUSSION