Deposition of newly synthesized histones: hybrid nucleosomes are not tandemly arranged on daughter DNA strands

Density labeling procedures have been utilized to study the dynamics of histone-histone interactions in vivo. Cells were labeled for 60 min with dense amino acids, and the label was chased for up to 22 h (two replication events for these cells). Nuclei were isolated and treated with formaldehyde to stabilize the histone-histone interactions with a covalent cross-link that produces an octameric complex of two each of H3, H2B, H2A, and H4. This complex was then extracted from the DNA and analyzed on density gradients. The results indicate that new H3,H4 deposits as a tetramer and does not dissociate in the subsequent chases. New H2A,H2B deposited as a dimer and also does not dissociate in subsequent chases. These new histones form hybrid octamers with old histones. On the basis of the new:old ratio in the hybrid octamers, we propose that additional old H2A,H2B from elsewhere in the genome interacts with tetramers of new H3,H4 to form the newly synthesized nucleosomes. It is also observed that 5% of the cross-linked complexes produced by formaldehyde are octamer-octamer (dioctamer). Upon analysis of the density of the dioctamer, the hybrid octamers were found adjacent to octamers that were homogeneous with respect to containing normal density histones. Control experiments are presented to demonstrate that the octamer-octamer cross-links are a product of intrastrand and not interstrand interactions between nucleosomes. These same control experiments also indicate that these procedures do not induce histone exchange during the preparative procedure prior to density gradient analysis. The significance of these results with regard to the dynamics of histone-histone interactions at the replication fork and the potential role in the maintenance of differentiation is discussed.     

Deposition of newly synthesized histones: new histones H2A and H2B do not deposit in the same nucleosome with new histones H3 and H4

We have developed procedures to study histone-histone interactions during the deposition of histones in replicating cells. Cells are labeled for 60 min with dense amino acids, and subsequently, the histones within the nucleosomes are cross-linked into an octameric complex with formaldehyde. These complexes are sedimented to equilibrium in density gradients and octamer and dioctamer complexes separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. With reversal of the cross-link, the distribution of the individual density-labeled histones in the octamer is determined. Newly synthesized H3 and H4 deposit as a tetramer and are associated with old H2A and H2B. Newly synthesized H2A and H2B deposit as a dimer associated with old H2A, H2B, H3, and H4. The significance of these results with respect to the dynamics of histone interactions in the nucleus is discussed. Control experiments are presented to test for artifactual formation of these complexes during preparative procedures. In addition, reconstitution experiments were performed to demonstrate that the composition of these octameric complexes can be determined from their distribution on density gradients.     

Preferential association of newly synthesized histones with replicating SV40 DNA.

The assembly of newly synthesized histones into nucleosomes during replication of SV40 minichromosomes in vivo was studied. Infected cells were labeled with 35S-methionine for a time shorter than that required to complete a round of viral DNA replication. Mature and replicating SV40 minichromosomes were extracted and separated by zonal sedimentation, and their histone content was analyzed by polyacrylamide gel electrophoresis (SDS and acidic urea). We show that the pulse-labeled histones associate preferentially with the replicating DNA.     

The role of nuclear serine proteases in the degradation of newly synthesized histones and ribosomal proteins

To examine whether serine proteases of rat liver chromatin are also involved in the degradation of newly synthesized and unbound ribosomal proteins and histones, like the nuclear thiol protease which we reported previously (Tsurugi, K. & Ogata, K. (1979) Eur. J. Biochem. 101, 205-213), in vivo experiments were carried out with serine protease inhibitor, PMSF. The following results were obtained. When normal rats received an intraperitoneal injection of PMSF (10 mg per 100 g body weight), nuclear serine proteases were inhibited almost completely for at least 90 min. PMSF did not affect the synthesis of proteins and RNAs of ribosomes and other subcellular fractions. The effects of PMSF treatment in vivo on the degradation of newly synthesized ribosomal proteins and histones in regenerating rat liver pretreated with a low dose of actinomycin D, which preferentially inhibited rRNA synthesis, were examined by using the double-isotope method. It was found that PMSF treatment did not affect their degradation. On the other hand, administration of E-64, a thiol protease inhibitor, to partially hepatectomized rats inhibited the degradation of those proteins markedly. From these results, it is concluded that the nuclear thiol protease, but not serine proteases, is preferentially involved in the degradation of newly synthesized ribosomal proteins and histones which are not associated with rRNA and DNA, respectively.     

Fate of newly synthesized histones shortly after interruption of DNA replication

The synthesis and association of histones with chromatin were studied using MH-134SC cells in suspension culture. Cultures containing approximately equal numbers of cells were pulse-labeled with [3H]lysine at various times after the interruption of DNA synthesis with hydroxyurea. Each culture was mixed with a fixed volume of a culture generally labeled with [14C]lysine at the time of harvesting. Acid-soluble proteins extracted from different subcellular fractions of cells labeled under various conditions were compared by electrophoresis on polyacrylamide gels containing acetic acid and urea. All types of chromatin histones were labeled nearly equally as [14C]marker histones by a 15 min pulse under normal conditions, except that a considerable portion of pulse-labeled H4 was in highly acetylated forms. Addition of hydroxyurea at the start of the pulse markedly reduced the labeling of H3 and H4, but affected the labeling of the other histones only slightly. When DNA synthesis was inhibited before the start of the pulse, labeling of all histones decreased significantly. The addition of hydroxyurea was found to cause transient accumulation of newly synthesized proteins in the cytoplasmic soluble fraction; these were characterized as H3 and H4 from their metabolic properties and their electrophoretic mobilities on sodium dodecyl sulfate-polyacrylamide gels. The results suggest that association of newly synthesized H3 and H4 histones is closely coupled with ongoing DNA replication. The implications of the results for the mechanism of formation of new nucleosomes are discussed.     

Divinyl sulfone as a cross-linking reagent for oligomeric proteins

The kinetics of the nucleophilic addition reactions of divinyl sulfone to amino groups of glycine and model proteins was studied in aqueous solution at 30 degrees C. The rate constants for glycine, bovine serum albumin, and alpha 1-casein were (4.84 +/- 0.58) x 10(-1), (2.97 +/- 0.31) x 10(-2), and (2.38 +/- 0.49) x 10(-2) M-1s-1, respectively. Divinyl sulfone was proposed as a cross-linking reagent for the qualitative detection of protein association in solution. The cross-linking capacity of divinyl sulfone was compared to that of 1,3,5-triacryloylhexahydro-s-triazine.     

Fate of newly synthesized histones in G1 and G0 cells

We have shown that quiescent cells as well as those in the G1 phase of the cell cycle synthesize histones at a reduced but significant rate. Now, we show that the histones synthesized during G0 and G1 are stably incorporated into nuclei soon after synthesis. Micrococcal nuclease digestion of nuclei isolated from cells in G0 and G1 revealed that the specific histone variants synthesized in these different physiological states are found associated with DNA as nucleosomes. Nucleosomes were separated by polyacrylamide gel electrophoresis in a reducing buffer so that histone spot morphology, particularly that of the H3s was improved.     

Replication-coupled chromatin assembly of newly synthesized histones: distinct functions for the histone tail domains

The maintenance of the genome during replication requires the assembly of nucleosomes with newly synthesized histones. Achieving the deposition of newly synthesized histones in chromatin implies their transport from the cytoplasm to the nucleus at the replication sites. Several lines of evidence have revealed critical functions of the histone tail domains in these conserved cellular processes. In this review, we discuss the role of the amino termini of the nucleosome building blocks, H2A/H2B and H3/H4, in different model systems. The experimental data showed that H2A/H2B tails and H3/H4 tails display distinct functions in nuclear import and chromatin assembly. Furthermore, we describe recent studies exploiting the unique properties of the slime mold, Physarum polycephalum , that have advanced understanding of the function of the highly conserved replication-dependent diacetylation of H4.     

Photochemical cross-linking of histones to DNA nucleosomes.

Ultraviolet (UV)-induced cross-linking was utilized in order to identify histone-DNA interacting regions in the chromatin repeating unit. Fractionated mononucleosomes which contained 185 base pairs of DNA and a full complement of the histones, including histone H1, were irradiated with light of lambda greater than 290nm in the presence of a photosensitizer. Equimolar amounts of histones H2A and H2B were found, by two independent labeling experiments, to be cross-linked to the DNA. Based on previous finding that the UV irradiation specifically cross-links residues which are in close proximity, irrespective of the nature of the amino acid side chain or the nucleotide involved, our results indicate that the four core histones are not positioned equivalently with respect to the DNA. This arrangement allows histones H2A and H2B to preferentially cross-link to the DNA. A water soluble covalent complex of DNA and histones was isolated. This complex was partially resistant to mild nuclease digestion, it exhibited a CD spectrum similar to that of chromatin, and was found to contain histone H1. These results are compatible with a model which suggests that histone H1, though anchored to the linker, is bound to the DNA at additional sites. By doing so it spans the whole length of the nucleosome and clamps together the DNA fold around the histone core.     

Chaperone mediated autophagy contributes to the newly synthesized histones H3 and H4 quality control

Although there are several pathways to ensure that proteins are folded properly in the cell, little is known about the molecular mechanisms regulating histone folding and proteostasis. In this work, we identified that chaperone-mediated autophagy (CMA) is the main pathway involved in the degradation of newly synthesized histones H3 and H4. This degradation is finely regulated by the interplay between HSC70 and tNASP, two histone interacting proteins. tNASP stabilizes histone H3 levels by blocking the direct transport of histone H3 into lysosomes. We further demonstrate that CMA degrades unfolded histone H3. Thus, we reveal that CMA is the main degradation pathway involved in the quality control of histone biogenesis, evidencing an additional mechanism in the intricate network of histone cellular proteostasis.     

Degradation from the endoplasmic reticulum: disposing of newly synthesized proteins

We have characterized a pre-Golgi, proteolytic pathway for rapid degradation of newly synthesized T cell receptor (TCR) subunits which is insensitive to drugs that block lysosomal proteolysis. The site of degradation in this pathway is either part of or closely related to the endoplasmic reticulum (ER). This "ER" degradative pathway very likely plays an important role in many cells in the removal of unassembled or incompletely assembled membrane protein complexes from the secretory pathway. It is the sole pathway followed by TCR alpha chains and alpha-beta complexes in transfected fibroblasts. In T cells treated with ionophores, which disrupt transport of the TCR from the ER to the Golgi, all newly synthesized alpha, beta, and delta chains are destroyed by this pathway. A variety of biochemical and morphological techniques have been used to distinguish the "ER" degradative pathway from an alternative, lysosomal pathway.     

Architectural editing: determining the fate of newly synthesized membrane proteins

Many integral membrane proteins exist on the plasma membrane as part of multicomponent complexes. In addition to correctly transporting newly synthesized proteins from their site of synthesis in the endoplasmic reticulum to the plasma membrane, the cell must possess mechanisms to ensure that the complexes expressed on the cell surface are accurately assembled. The cell appears to accomplish this feat by superimposing a set of constraints on the newly synthesized membrane proteins whereby the structure and state of assembly of the protein determine its intracellular fate. These processes impose a dramatic level of post-translational regulation on the expression of surface membrane protein complexes. By and large, the cell uses these mechanisms to dispose of, or "edit out," newly synthesized proteins that are not correctly assembled or folded. This review will describe current views of the processes of architectural editing, with an emphasis on the regulation of cell surface expression of the multicomponent T-cell antigen receptor complex.     

Chaperone-assisted folding of newly synthesized proteins in the cytosol

The way in which a newly synthesized polypeptide chain folds into its unique three-dimensional structure remains one of the fundamental questions in molecular biology. Protein folding in the cell is a problematic process and, in many cases, requires the assistance of a network of molecular chaperones to support productive protein foldingin vivo. During protein biosynthesis, ribosome-associated chaperones guide the folding of the nascent polypeptide emerging from the ribosomal tunnel. In this review we summarize the basic principles of the protein-folding process and the involved chaperones, and focus on the role of ribosome-associated chaperones. Our discussion emphasizes the bacterial Trigger Factor, which is the best studied chaperone of this type. Recent advances have determined the atomic structure of the Trigger Factor, providing new, exciting insights into the role of ribosome-associated chaperones in co-translational protein folding.