NASP maintains histone H3–H4 homeostasis through two distinct H3 binding modes

Histone chaperones regulate all aspects of histone metabolism. NASP is a major histone chaperone for H3–H4 dimers critical for preventing histone degradation. Here, we identify two distinct histone binding modes of NASP and reveal how they cooperate to ensure histone H3–H4 supply. We determine the structures of a sNASP dimer, a complex of a sNASP dimer with two H3 α3 peptides, and the sNASP–H3–H4–ASF1b co-chaperone complex. This captures distinct functionalities of NASP and identifies two distinct binding modes involving the H3 α3 helix and the H3 αN region, respectively. Functional studies demonstrate the H3 αN-interaction represents the major binding mode of NASP in cells and shielding of the H3 αN region by NASP is essential in maintaining the H3–H4 histone soluble pool. In conclusion, our studies uncover the molecular basis of NASP as a major H3–H4 chaperone in guarding histone homeostasis.     

The H3 chaperone function of NASP is conserved in Arabidopsis

Histones are abundant cellular proteins but, if not incorporated into chromatin, they are usually bound by histone chaperones. Here, we identify Arabidopsis NASP as a chaperone for histones H3.1 and H3.3. NASP interacts in vitro with monomeric H3.1 and H3.3 as well as with histone H3.1–H4 and H3.3–H4 dimers. However, NASP does not bind to monomeric H4. NASP shifts the equilibrium between histone dimers and tetramers towards tetramers but does not interact with tetramers in vitro. Arabidopsis NASP promotes [H3–H4]₂ tetrasome formation, possibly by providing preassembled histone tetramers. However, NASP does not promote disassembly of in vitro preassembled tetrasomes. In contrast to its mammalian homolog, Arabidopsis NASP is a predominantly nuclear protein. In vivo, NASP binds mainly monomeric H3.1 and H3.3. Pulldown experiments indicated that NASP may also interact with the histone chaperone MSI1 and a HSC70 heat shock protein.     

Characterization of the histone H1-binding protein, NASP, as a cell cycle-regulated somatic protein

Nuclear autoantigenic sperm protein (NASP), initially described as a highly autoimmunogenic testis and sperm-specific protein, is a histone-binding protein that is a homologue of the N1/N2 gene expressed in oocytes of Xenopus laevis. Here, we report a somatic form of NASP (sNASP) present in all mitotic cells examined, including mouse embryonic cells and several mouse and human tissue culture cell lines. Affinity chromatography and histone isolation demonstrate that NASP from myeloma cells is complexed only with H1, linker histones. Somatic NASP is a shorter version of testicular NASP (tNASP) with two deletions in the coding region arising from alternative splicing and differs from tNASP in its 5' untranslated regions. We examined the relationship between NASP mRNA expression and the cell cycle and report that in cultures of synchronized mouse 3T3 cells and HeLa cells sNASP mRNA levels increase during S-phase and decline in G(2), concomitant with histone mRNA levels. NASP protein levels remain stable in these cells but become undetectable in confluent cultures of nondividing CV-1 cells and in nonmitotic cells in various body tissues. Expression of sNASP mRNA is regulated during the cell cycle and, consistent with a role as a histone transport protein, NASP mRNA expression parallels histone mRNA expression.     

Expanded binding specificity of the human histone chaperone NASP

NASP (nuclear autoantigenic sperm protein) has been reported to be an H1-specific histone chaperone. However, NASP shares a high degree of sequence similarity with the N1/N2 family of proteins, whose members are H3/H4-specific histone chaperones. To resolve this paradox, we have performed a detailed and quantitative analysis of the binding specificity of human NASP. Our results confirm that NASP can interact with histone H1 and that this interaction occurs with high affinity. In addition, multiple in vitro and in vivo experiments, including native gel electrophoresis, traditional and affinity chromatography assays and surface plasmon resonance, all indicate that NASP also forms distinct, high specificity complexes with histones H3 and H4. The interaction between NASP and histones H3 and H4 is functional as NASP is active in in vitro chromatin assembly assays using histone substrates depleted of H1.     

Depletion of the histone chaperone tNASP inhibits proliferation and induces apoptosis in prostate cancer PC-3 cells

Background  

NASP (Nuclear Autoantigenic Sperm Protein) is a histone chaperone that is present in all dividing cells. NASP has two splice variants: tNASP and sNASP. Only cancer, germ, transformed, and embryonic cells have a high level of expression of the tNASP splice variant. We examined the consequences of tNASP depletion for prostate cancer PC-3 cells.     

Nuclear autoantigenic sperm protein (NASP), a linker histone chaperone that is required for cell proliferation

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.     

Histone acetyltransferase 1 is a succinyltransferase for histones and non‐histones and promotes tumorigenesis

Lysine succinylation (Ksucc) is an evolutionarily conserved and widespread post‐translational modification. Histone acetyltransferase 1 (HAT1) is a type B histone acetyltransferase, regulating the acetylation of both histone and non‐histone proteins. However, the role of HAT1 in succinylation modulation remains unclear. Here, we employ a quantitative proteomics approach to study succinylation in HepG2 cancer cells and find that HAT1 modulates lysine succinylation on various proteins including histones and non‐histones. HAT1 succinylates histone H3 on K122, contributing to epigenetic regulation and gene expression in cancer cells. Moreover, HAT1 catalyzes the succinylation of PGAM1 on K99, resulting in its increased enzymatic activity and the stimulation of glycolytic flux in cancer cells. Clinically, HAT1 is significantly elevated in liver cancer, pancreatic cancer, and cholangiocarcinoma tissues. Functionally, HAT1 succinyltransferase activity and the succinylation of PGAM1 by HAT1 play critical roles in promoting tumor progression in vitro and in vivo. Thus, we conclude that HAT1 is a succinyltransferase for histones and non‐histones in tumorigenesis.     

Epigenetic Hierarchy within the MAGEA1 Cancer-Germline Gene: Promoter DNA Methylation Dictates Local Histone Modifications

Gene MAGEA1 belongs to a group of human germline-specific genes that rely on DNA methylation for repression in somatic tissues. Many of these genes, termed cancer-germline (CG) genes, become demethylated and activated in a wide variety of tumors, where they encode tumor-specific antigens. The process leading to DNA demethylation of CG genes in tumors remains unclear. Previous data suggested that histone acetylation might be involved. Here, we investigated the relative contribution of DNA methylation and histone acetylation in the epigenetic regulation of gene MAGEA1. We show that MAGEA1 DNA hypomethylation in expressing melanoma cells is indeed correlated with local increases in histone H3 acetylation (H3ac). However, when MAGEA1-negative cells were exposed to a histone deacetylase inhibitor (TSA), we observed only short-term activation of the gene and detected no demethylation of its promoter. As a more sensitive assay, we used a cell clone harboring a methylated MAGEA1/hph construct, which confers resistance to hygromycin upon stable re-activation. TSA induced only transient de-repression of the transgene, and did not lead to the emergence of hygromycin-resistant cells. In striking contrast, transient depletion of DNA-methyltransferase-1 in the reporter cell clone gave rise to a hygromycin-resistant population, in which the re-activated MAGEA1/hph transgene displayed not only marked DNA hypomethylation, but also significant reversal of histone marks, including gains in H3ac and H3K4me2, and losses of H3K9me2. Collectively, our results indicate that DNA methylation has a dominant role in the epigenetic hierarchy governing MAGEA1 expression.     

Structural basis for histone H3 recognition by NASP in Arabidopsis

The structural basis for histone recognition by the histone chaperone nuclear autoantigenic sperm protein (NASP) remains largely unclear. Here, we showed that Arabidopsis thaliana AtNASP is a monomer and displays robust nucleosome assembly activity in vitro. Examining the structure of AtNASP complexed with a histone H3 α3 peptide revealed a binding mode that is conserved in human NASP. AtNASP recognizes the H3 N-terminal region distinct from human NASP. Moreover, AtNASP forms a co-chaperone complex with ANTI-SILENCING FUNCTION 1 (ASF1) by binding to the H3 N-terminal region. Therefore, we deciphered the structure of AtNASP and the basis of the AtNASP–H3 interaction.