Overexpression of the Linker Histone-binding Protein tNASP Affects Progression through the Cell Cycle

Alekseev, Oleg; Bencic, David C.; Richardson, Richard T.; Widgren, Esther E.; O’Rand, Michael G.

doi:10.1074/jbc.m210352200

Cited by 56 publications

(70 citation statements)

References 19 publications

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“…Chromatography-Immunoaffinity chromatography was carried out as described previously (8). For the preparation of NASP-enriched fractions from testis, lysates of 50 mouse testes (Pel-Freez® Biologicals, Rogers, AR) were prepared in 2 ml of MPER reagent (Pierce) with Protease (catalog number 8340; Sigma) and Proteasome (catalog number 539160; Calbiochem) inhibitors added.…”

Section: Methodsmentioning

confidence: 99%

“…1 The abbreviations used are: H1, linker histone; tNASP, testis/embryo form of NASP; HSP90, heat shock protein 90; NLS, nuclear localization signal; H1t, testis form of H1; HPLC, high pressure liquid chromatography; DTSSP, 3,3Ј-dithiobissulfosuccinimidyl propionate; MALDI, matrix assisted laser desorption/ionization; TOF, time of flight; TPR, tetratricopeptide repeat. tech, Palo Alto, CA) as described previously (8). A tNASP deletion mutant (NASP-⌬TPR; nucleotides 92-1590), that lacked all tetratricopeptide repeats (nucleotides 1591-1891) was PCR-amplified, cloned into pCR® T7/CT-TOPO vector (Invitrogen), expressed, and purified identically to the full-length construct (8).…”

Section: Methodsmentioning

confidence: 99%

“…A tNASP deletion mutant (NASP-⌬TPR; nucleotides 92-1590), that lacked all tetratricopeptide repeats (nucleotides 1591-1891) was PCR-amplified, cloned into pCR® T7/CT-TOPO vector (Invitrogen), expressed, and purified identically to the full-length construct (8). Preparation of the NLS deletion mutant (NASP-⌬NLS) has been described previously (8).…”

Section: Methodsmentioning

confidence: 99%

“…NASP is a histone-binding protein found in all dividing cells in either a somatic/embryo or testis/embryo (tNASP) form (9 -11). Overexpression of tNASP affects progression through the cell cycle, and Alekseev et al (8) recently postulated that a dynamic equilibrium exists between H1-NASP complexes and H1 bound to DNA. Previous studies on NASP (9) demonstrated that in vivo the somatic linker histones H1a-H1e (see Ref.…”

mentioning

confidence: 99%

“…to DNA are bound to the histone-binding protein NASP (8). NASP is a histone-binding protein found in all dividing cells in either a somatic/embryo or testis/embryo (tNASP) form (9 -11).…”

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Association of NASP with HSP90 in Mouse Spermatogenic Cells

Alekseev

Widgren

Richardson

et al. 2005

Journal of Biological Chemistry

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Linker histones (H1s) 1 bind to DNA in the nucleosomes of chromatin and influence the formation of chromatin fibers, gene transcription, nucleosome spacing, chromatin remodeling, and cell cycle progression (1-5). Studies on linker histones in the cell nucleus indicate that there is a constant exchange of H1s with their DNA-binding sites (6, 7) and that H1s not bound to DNA are bound to the histone-binding protein NASP (8). NASP is a histone-binding protein found in all dividing cells in either a somatic/embryo or testis/embryo (tNASP) form (9 -11). Overexpression of tNASP affects progression through the cell cycle, and Alekseev et al. (8) recently postulated that a dynamic equilibrium exists between H1-NASP complexes and H1 bound to DNA. Previous studies on NASP (9) demonstrated that in vivo the somatic linker histones H1a-H1e (see Ref. 12 for mouse histone H1 nomenclature) were co-precipitated with NASP from somatic cell lysates, indicating that NASP is not selective for H1 subtypes.Of the several different subtypes of H1 histones, H1a, b, c, d, and e are found in most cells, whereas H1t is restricted to the testis. H1t is expressed during spermatogenesis, representing as much as 50% of the total H1 histones in pachytene spermatocytes (13-15), and is preceded by tNASP expression in preleptotene spermatocytes. Although their exact function during spermatogenesis is unknown, gene-targeting experiments (16) have shown that the absence of H1t and H1a in double null mice does not compromise spermatogenesis because compensation by H1c, H1d, and H1e most likely maintained normal spermatogenesis despite a reduced H1/nucleosome ratio (16). Under these conditions the major H1 histone binding activity of tNASP could have shifted from H1t to H1c, H1d, and H1e in double null mice. Unfortunately neither the relationship between tNASP, H1t, and other proteins nor the ability of tNASP to transport H1s into the mammalian cell nucleus has been characterized. Therefore we have studied tNASP in spermatogenic cells to understand more about the role of NASP-H1t complexes and now report that H1t binds tNASP in mouse germ cells in a complex in the cytoplasm that contains HSP90, which facilitates the initial loading of linker histones to tNASP. Moreover, tNASP can transport both H1t and somatic linker histones into nuclei. EXPERIMENTAL PROCEDURESAll of the chemicals and reagents used in this study were molecular biology grade. The restriction enzymes were purchased from Roche Applied Science. Purification of plasmid DNA and PCR products were carried out using QIAprep Miniprep and QIAquick PCR purification kits (Qiagen), and sequencing was performed at the University of North Carolina at Chapel Hill automated sequencing facility. Rabbit antihistone H1 (FL-219) polyclonal antiserum and mouse monoclonal IgG 2a anti-HSP90 (F-8) antiserum were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-H1t antibody was a gift from Dr. M. Meistrich (Department of Experimental Radiation Oncology, M. D. Anderson Cancer Center, University ...

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

“…to DNA are bound to the histone-binding protein NASP (8). NASP is a histone-binding protein found in all dividing cells in either a somatic/embryo or testis/embryo (tNASP) form (9 -11).…”

mentioning

confidence: 99%

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Association of NASP with HSP90 in Mouse Spermatogenic Cells

Alekseev

Widgren

Richardson

et al. 2005

Journal of Biological Chemistry

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Mass spectrometry identification of NASP binding partners in HeLa cells

et al. 2005

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Nuclear autoantigenic sperm protein (NASP) is a linker histone binding protein that is cell-cycle regulated. Synchronized HeLa cells are delayed in progression through the G1/S border when transiently transfected to overexpress full-length NASP, but not the histone-binding site (HBS) deletion mutant (NASP-DeltaHBS). The purpose of the current study was to identify possible NASP-associated proteins in HeLa cell nuclei that could elucidate NASP's influence on the cell cycle and chromatin remodeling. For this purpose, we employed a new approach: mass spectrometry identification of initially cross-linked proteins after their separation in a second dimension by reducing SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Of the twelve proteins identified, three appear to be relevant to NASP's function: heat shock protein 90 (HSP90), DNA-activated protein kinase, and ATP-dependent DNA helicase II (70-kDa subunit). Individual protein-protein interactions were tested by immunoprecipitation techniques. This new method can be used for expedited identification of binding partners of different proteins in enriched fractions and as a complementary or alternative strategy to the yeast two-hybrid system and immunoprecipitation methods.

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How Is Epigenetic Information on Chromatin Inherited After DNA Replication?

Nakatani

Tagami

Shestakova

The Histone Code and Beyond

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Overexpression of the Linker Histone-binding Protein tNASP Affects Progression through the Cell Cycle

Cited by 56 publications

References 19 publications

Association of NASP with HSP90 in Mouse Spermatogenic Cells

Association of NASP with HSP90 in Mouse Spermatogenic Cells

Mass spectrometry identification of NASP binding partners in HeLa cells

How Is Epigenetic Information on Chromatin Inherited After DNA Replication?

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