Abstract

BackgroundNASP (Nuclear Autoantigenic Sperm Protein) is a linker histone chaperone required for normal cell division. Changes in NASP expression significantly affect cell growth and development; loss of gene function results in embryonic lethality. However, the mechanism by which NASP exerts its effects in the cell cycle is not understood. To understand the pathways and networks that may involve NASP function, we evaluated gene expression in HeLa cells in which NASP was either overexpressed or depleted by siRNA.MethodsTotal RNA from HeLa cells overexpressing NASP or depleted of NASP by siRNA treatment was converted to cRNA with incorporation of Cy5-CTP (experimental samples), or Cy3-CTP (control samples). The labeled cRNA samples were hybridized to whole human genome microarrays (Agilent Technologies, Wilmington, Delaware, USA). Various gene expression analysis techniques were employed: Significance Analysis of Microarrays (SAM), Expression Analysis Systematic Explorer (EASE), and Ingenuity Pathways Analysis (IPA).ResultsFrom approximately 36 thousand genes present in a total human genome microarray, we identified a set of 47 up-regulated and 7 down-regulated genes as a result of NASP overexpression. Similarly we identified a set of 56 up-regulated and 71 down-regulated genes as a result of NASP siRNA treatment. Gene ontology, molecular network and canonical pathway analysis of NASP overexpression demonstrated that the most significant changes were in proteins participating in organismal injury, immune response, and cellular growth and cancer pathways (major "hubs": TNF, FOS, EGR1, NFκB, IRF7, STAT1, IL6). Depletion of NASP elicited the changed expression of proteins involved in DNA replication, repair and development, followed by reproductive system disease, and cancer and cell cycle pathways (major "hubs": E2F8, TP53, FGF, FSH, FST, hCG, NFκB, TRAF6).ConclusionThis study has demonstrated that NASP belongs to a network of genes and gene functions that are critical for cell survival. We have confirmed the previously reported interactions between NASP and HSP90, HSP70, histone H1, histone H3, and TRAF6. Overexpression and depletion of NASP identified overlapping networks that included TNF as a core protein, confirming that both high and low levels of NASP are detrimental to cell cycle progression. Networks with cancer-related functions had the highest significance, however reproductive networks containing follistatin and FSH were also significantly affected, which confirmed NASP's important role in reproductive tissues. This study revealed that, despite some overlap, each response was associated with a unique gene signature and placed NASP in important cell regulatory networks.

Highlights

  • Nuclear autoantigenic sperm protein (NASP) (Nuclear Autoantigenic Sperm Protein) is a linker histone chaperone required for normal cell division

  • Significance Analysis of Microarrays (SAM) analysis SAM analysis resulted in identification of groups of genes that were significantly differentially expressed after overexpression or depletion of NASP in HeLa cells at least two fold in all four experiments

  • Overexpression of tNASP in HeLa cells significantly affects the expression level of 54 genes (0.14% of 39,064 genes included in the microarray): 47 (0.12%) were up-regulated and 7 (0.02%) were down-regulated

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Summary

Introduction

NASP (Nuclear Autoantigenic Sperm Protein) is a linker histone chaperone required for normal cell division. Changes in NASP expression significantly affect cell growth and development; loss of gene function results in embryonic lethality. The mechanism by which NASP exerts its effects in the cell cycle is not understood. Nuclear autoantigenic sperm protein (NASP) is highly expressed in all dividing cells including embryonic and malignant tissues as either tNASP (testicular/embryonic isoform) or sNASP (somatic/embryonic isoform). In embryonic and transformed cell lines both sNASP and tNASP are expressed and tNASP is present in a variety of malignant tumors [1]. The NASP expression profile could be used to establish the "poor prognosis signature" which consists of genes regulating cell cycle, invasion, metastasis and angiogenesis [4]. NASP has been reported as a serologic marker for ovarian cancer, which could be suitable for clinical testing in high-risk populations [5]

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