Specific interactions of histone H1 and a 45 kilodalton nuclear protein with a putative matrix attachment site in the distal promoter region of a cell cycle-regulated human histone gene

Protein-DNA interactions within the promoter of a cell cycle-regulated human H4 histone gene were examined by binding of 5'-end-labeled DNA segments to Western blots of nuclear protein fractions. Specific protein interactions were observed with DNA segments located between -500 bp and -1,070 bp upstream of the ATG initiation codon and included a histone H1 binding segment flanked on both sides by binding sites for a 45 kD nuclear protein. This region of the gene contains a DNase I-sensitive site in the center (-720 to -820 bp), and sequence analysis revealed the presence of scaffold attachment sequences in the two flanking segments. Topoisomerase II consensus sequences and in vitro topoisomerase II cleavage sites were also detected in the two flanking segments. Our results suggest that the 45 kd nuclear protein may preferentially interact with these two segments of the H4 histone gene to mediate association with the nuclear matrix. The presence of negative regulatory elements in this putative matrix attachment region provides a basis for the speculation that such nuclear proteins are associated with alterations in gene-matrix interaction that are functionally related to gene expression.     

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.     

Characterization of the chicken histone H1 gene complement. Generation of a complete set of vertebrate H1 protein sequences

Sequence analysis of four chicken H1 histone genes described here completes the characterization of the full complement of six H1 genes in the chicken genome. Each of the six genes codes for a different H1 protein sequence, and these range in size from 217 to 224 amino acids. The proteins are distinct in sequence from the H1-related chicken H5 protein and appear to be analogous to the standard somatic mammalian H1 subtypes. The protein sequence data deduced from the genes represent the first complete set of vertebrate H1 protein sequences. Comparison of the chicken H1 gene noncoding sequences with each other and with H1 gene sequences from other organisms reveals conservation of an H1 gene-specific element, a G-rich element, and histone gene-specific 3' elements. Additional sequences are conserved between H1 genes of the chicken and other vertebrates. Comparisons also reveal variation in promoter and 3' elements between chicken genes that could play a role in the differential expression of H1 gene protein products.     

Identification of the linker histone H1 as a protein kinase Cepsilon-binding protein in vascular smooth muscle.

A variety of anchoring proteins target specific protein kinase C (PKC) isoenzymes to particular subcellular locations or multimeric signaling complexes, thereby achieving a high degree of substrate specificity by localizing the kinase in proximity to specific substrates. PKCepsilon is widely expressed in smooth muscle tissues, but little is known about its targeting and substrate specificity. We have used a Far-Western (overlay) approach to identify PKCepsilon-binding proteins in vascular smooth muscle of the rat aorta. Proteins of approximately 32 and 34 kDa in the Triton-insoluble fraction were found to bind PKCepsilon in a phospholipid/diacylglycerol-dependent manner. Although of similar molecular weight to RACK-1, a known PKCepsilon-binding protein, these proteins were separated from RACK-1 by SDS-PAGE and differential NaCl extraction and were not recognized by an antibody to RACK-1. The PKCepsilon-binding proteins were further purified from the Triton-insoluble fraction and identified by de novo sequencing of selected tryptic peptides by tandem mass spectrometry as variants of the linker histone H1. Their identity was confirmed by Western blotting with anti-histone H1 and the demonstration that purified histone H1 binds PKCepsilon in the presence of phospholipid and diacylglycerol but absence of Ca(2+). The interaction of PKCepsilon with histone H1 was specific since no interaction was observed with histones H2A, H2S or H3S. Bound PKCepsilon phosphorylated histone H1 in a phospholipid/diacylglycerol-dependent but Ca(2+)-independent manner. Ca(2+)-dependent PKC was also shown to interact with histone H1 but not other histones. These results suggest that histone H1 is both an anchoring protein and a substrate for activated PKCepsilon and other PKC isoenzymes and likely serves to localize activated PKCs that translocate to the nucleus in the vicinity of specific nuclear substrates including histone H1 itself. Since PKC isoenzymes have been implicated in regulation of gene expression, stable interaction with histone H1 may be an important step in this process.     

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.     

Evidence for isopentenyladenine modification on a cell cycle-regulated protein

We have prepared antibodies that recognize isopentenyladenosine (i6A), a modified nucleoside derived from mevalonic acid (MVA). In immunoblot assays, affinity-purified anti-i6 A antibodies specifically bound to a 26-kDa protein (i6A26) in Chinese hamster ovary cells. Anti-i6A recognition of i6A26 was blocked with i6A but not adenosine or isopentenol. Employing immunoblot analysis we have quantitated the level of i6A26 in cells expressing various rates of DNA synthesis. The cellular content of i6A26 was reduced 4-fold in quiescent cells cultured in the absence of serum. When serum-deprived cells were stimulated to enter the cell cycle, the amount of i6A26 increased in the cells during the G1 phase. However, when synchronized cells were stimulated with serum-containing medium in the presence of mevinolin (an inhibitor of cellular MVA synthesis), we observed impaired G1 expression of i6A26 and delayed onset of S phase DNA synthesis. Mevinolin addition to asynchronously growing cells resulted in low rates of cellular DNA synthesis and suppressed levels of i6A26 which were reversed by coincubation with MVA. The ability of MVA to restore DNA synthesis and the cellular content of i6A26 in mevinolin-treated cells showed similar MVA concentration and time dependences. Regenerating liver tissue also exhibited elevated levels of i6A26. Thus, the expression of i6A26 correlates with cellular proliferation and growth. We speculate that i6A26 contains isopentenyladenine moieties and mediates isoprenoid regulation of DNA synthesis. Isopentenyladenylated proteins may also function in cytokinin regulation of proliferation and differentiation in plants.     

Homology model building of Hho1p supports its role as a yeast histone H1 protein

Biochemical studies to date have not been able to identify the linker histone H1 protein in the budding yeast Saccharomyces cerevisiae. Database homology searching against the complete yeast genome has identified a gene, HHO1, (or YPL127C, formerly LPI17) which encodes a protein that has two regions that show similarity to the pea histone H1 globular domain. To determine whether Hho1p can assume the shape of an H1 protein, homology model building experiments were performed using the structure of chicken histone H5 globular domain as the basis for comparison. A statistically significant match between each of the two globular domains of Hho1p and the chicken histone H5 structure was obtained, and probability values indicate that there is a less than 1 in 100 chance that such a match would be the result of a random event. These findings support the proposal that Hho1p acts as an "H1 dimer" and could be responsible for the decreased linker DNA length observed between nucleosomal core particles.     

The histone demethylases Jhdm1a/1b enhance somatic cell reprogramming in a vitamin-C-dependent manner

Reprogramming of somatic cells into induced pluripotent stem cells (iPSCs) resets the epigenome to an embryonic-like state. Vitamin C enhances the reprogramming process, but the underlying mechanisms are unclear. Here we show that the histone demethylases Jhdm1a/1b are key effectors of somatic cell reprogramming downstream of vitamin C. We first observed that vitamin C induces H3K36me2/3 demethylation in mouse embryonic fibroblasts in culture and during reprogramming. We then identified Jhdm1a/1b, two known vitamin-C-dependent H3K36 demethylases, as potent regulators of reprogramming through gain- and loss-of-function approaches. Furthermore, we found that Jhdm1b accelerates cell cycle progression and suppresses cell senescence during reprogramming by repressing the Ink4/Arf locus. Jhdm1b also cooperates with Oct4 to activate the microRNA cluster 302/367, an integral component of the pluripotency machinery. Our results therefore reveal a role for H3K36me2/3 in cell fate determination and establish a link between histone demethylases and vitamin-C-induced reprogramming.     

TAK1-binding protein 1 is a pseudophosphatase

TAB1 [TAK1 (transforming growth factor-beta-activated kinase 1)-binding protein 1] is one of the regulatory subunits of TAK1, a protein kinase that lies at the head of three pro-inflammatory kinase cascades. In the current study we report the crystal structure of the N-terminal domain of TAB1. Surprisingly, TAB1 possesses a fold closely related to that of the PPM (Mg2+- or Mn2+-dependent protein phosphatase) family as demonstrated by the close structural similarity with protein phosphatase 2C alpha. However, we were unable to detect any phosphatase activity for TAB1 using a phosphopeptide or p-nitrophenyl phosphate as substrate. Although the overall protein phosphatase 2C alpha fold is conserved in TAB1, detailed structural analyses and mutagenesis studies show that several key residues required for dual metal-binding and catalysis are not present in TAB1, although binding of a single metal is supported by soaking experiments with manganese and isothermal titration calorimetry. Thus, it appears that TAB1 is a 'pseudophosphatase', possibly binding to and regulating accessibility of phosphorylated residues on substrates downstream of TAK1 or on the TAK1 complex itself.     

Differential affinity of mammalian histone H1 somatic subtypes for DNA and chromatin

Background  

Histone H1 is involved in the formation and maintenance of chromatin higher order structure. H1 has multiple isoforms; the subtypes differ in timing of expression, extent of phosphorylation and turnover rate. In vertebrates, the amino acid substitution rates differ among subtypes by almost one order of magnitude, suggesting that each subtype might have acquired a unique function. We have devised a competitive assay to estimate the relative binding affinities of histone H1 mammalian somatic subtypes H1a-e and H1° for long chromatin fragments (30–35 nucleosomes) in physiological salt (0.14 M NaCl) at constant stoichiometry.     

The centrosomal protein C-Nap1 is required for cell cycle-regulated centrosome cohesion

Duplicating centrosomes are paired during interphase, but are separated at the onset of mitosis. Although the mechanisms controlling centrosome cohesion and separation are important for centrosome function throughout the cell cycle, they remain poorly understood. Recently, we have proposed that C-Nap1, a novel centrosomal protein, is part of a structure linking parental centrioles in a cell cycle-regulated manner. To test this model, we have performed a detailed structure-function analysis on C-Nap1. We demonstrate that antibody-mediated interference with C-Nap1 function causes centrosome splitting, regardless of the cell cycle phase. Splitting occurs between parental centrioles and is not dependent on the presence of an intact microtubule or microfilament network. Centrosome splitting can also be induced by overexpression of truncated C-Nap1 mutants, but not full-length protein. Antibodies raised against different domains of C-Nap1 prove that this protein dissociates from spindle poles during mitosis, but reaccumulates at centrosomes at the end of cell division. Use of the same antibodies in immunoelectron microscopy shows that C-Nap1 is confined to the proximal end domains of centrioles, indicating that a putative linker structure must contain additional proteins. We conclude that C-Nap1 is a key component of a dynamic, cell cycle-regulated structure that mediates centriole-centriole cohesion.     

dBigH1, a second histone H1 in Drosophila,and the consequences for histone fold nomenclature

Recently, Pérez-Montero and colleagues (Developmental cell, 26: 578–590, 2013) described the occurrence of a new histone H1 variant (dBigH1) in Drosophila. The presence of unusual acidic amino acid patches at the N-terminal end of dBigH1 is in contrast to the arginine patches that exist at the N- and C-terminal domains of other histone H1-related proteins found in the sperm of some organisms. This departure from the strictly lysine-rich composition of the somatic histone H1 raises a question about the true definition of its protein members. Their minimal essential requirements appear to be the presence of a lysine- and alanine–rich, intrinsically disordered C-terminal domain, with a highly helicogenic potential upon binding to the linker DNA regions of chromatin. In metazoans, specific targeting of these regions is further achieved by a linker histone fold domain (LHFD), distinctively different from the characteristic core histone fold domain (CHFD) of the nucleosome core histones.