Synthesis of H1 histones by BHK cells in G1

The synthesis of histones and DNA was examined in BHK cells arrested in G1 by isoleucine starvation and in cells progressing into the S phase upon isoleucine refeeding. Approximately 2–3% of the cells were not arrested in G1 and synthesized DNA. The rate of synthesis of DNA and nucleosomal histones observed in cells starved for isoleucine could be accounted for by the presence of these asynchronous cells. Synthesis of H1 histones by cells in G1, however, was 3 times that of the nucleosomal histones and approximately 15% of the rate of H1 histone synthesis in mid-S. Upon entry into S, the histones were synthesized in the same molar ratio in which they are present in chromatin. The possible biological significance of H1 histone synthesis in G1 cells and its implications for the regulatory mechanisms controlling histone synthesis are discussed.     

Coordinate regulation of histone mRNAs during growth and differentiation of rat myoblasts

To determine whether histone genes are coordinately regulated, histone mRNA concentrations were measured in exponentially growing L6 myoblasts, S-phase synchronized myoblasts and in differentiating myoblasts. The levels of various histone mRNA subspecies declined rapidly and coordinately once myoblasts were given the signal to differentiate. mRNA levels were reduced on average to 1-5% of the amount observed in exponentially growing cells by 48 h after the signal to differentiate. The reductions occurred in concert with the cessation of DNA synthesis as the cells differentiated. Inhibition of DNA synthesis by treating myoblasts with Ara-C or hydroxyurea resulted in a histone mRNA half-life of 10-13 min for each of the histones examined. One example of non-coordinate regulation was observed however among the H4 mRNA subspecies in S-phase synchronized cells. The levels of two major subspecies of H4 mRNA increased coordinately in S-phase compared to levels observed in cells growing exponentially. A third subspecies of H4 mRNA on the other hand was found to decline by 50%. These studies suggest that the majority of histone mRNA subspecies are under coordinate control, although one exception has been noted among the subspecies of histone H4.     

Molar proportions and relative rates of synthesis of histones in rat testis

The molar proportions and relative rates of synthesis of histones in normal and hypophysectomized rat testis seminiferous epithelial cells were determined. After hypophysectomy the molar proportions of histones H1, H2B and (H2A + protein A24) in seminiferous epithelial cells of rat testis increased while their corresponding variants TH1-x, TH2B-x and X₂ decreased, but the molar proportions of major-class histones (i.e., sum of subfractions) remained relatively constant and similar to the proportions in somatic cells. The apparent molar proportions of the labeled histones, determined immediately after 2-h periods of [³H]leucine incorporation, were much higher relative to H4 than the proportions of total histones determined by dye binding. The values, however, approached the molar proportions of total histones when rats were killed 11 days after the [³H]leucine injection. Two-dimensional gel electrophoresis confirmed that the high initial molar proportions relative to H4 by [³H]leucine incorporation were not due to the possible contamination by highly-labeled non-histone proteins. The specific activity of histone H4 relative to the specific activity of DNA, determined immediately after 3-h periods of [³H]leucine and [¹⁴C]thymidine incorporations was similar to the value when rats were killed 13 days after the injections. It is proposed that histones of seminiferous epithelial cells are synthesized disproportionally relative to H4 and in excess of the quantities required for polynucleosome assembly. The excess histones are subsequently displaced or degraded slowly.     

Changes in histone H3 composition and synthesis pattern during lymphocyte activation

Freshly isolated human lymphocytes were found to synthesize histones at a significant rate even though no DNA was being synthesized. The synthesis pattern of histone variants in resting lymphocytes is similar to that found in other quiescent cells and different from that found in S-phase cells. For this reason, the histone synthesis in resting lymphocytes cannot be attributed to contamination by S-phase cells. Stimulation by the mitogen phytohemagglutinin resulted in a dramatic switch in the histone H3 variant synthesis pattern as well as a readily apparent change in the histone H3 mass pattern. Thus, the chromatin of activated lymphocytes has a different histone H3 variant composition than resting or quiescent lymphocytes. It is suggested that the proportion of H3.3 in the mass pattern of the chromatin of a cell may be related solely to how long that cell has been quiescent. Inducing resting lymphocytes to synthesize DNA by UV irradiation did not qualitatively change the histone variant synthesis pattern. No S-phase H3 variants were induced by the repair process. Furthermore, the quantity of histone synthesized neither increased nor decreased after treatment with UV light.     

Relationship of H1(0) histone to differentiation and cancer

The H1(0) histone was first described by Panyim and Chalkley in 1969 as a new electrophoretic band found with histones of non-replicating tissues. Tissues which are active in DNA replication such as ascites tumor cells or thymus cells were reported to lack this band. In this respect the H1(0) histone differs from the bulk of histones which are generally maintained in a constant ratio with respect to each other and to DNA. An inverse relationship between H1(0) histone levels and growth rate was suggested by the decrease in H1(0) histone concentration during regeneration of the pancreas and liver. The synthesis of H1(0) is unusual but not unique in that, unlike the major histone species, it is not restricted to the S phase of the cell cycle. Although there is a general trend for the levels of H1(0) histone to be lower in neoplastic than normal tissues, exceptions have been observed. Compounds such as sodium butyrate and dimethylsulfoxide, which can induce differentiated properties in neoplastic cells, can bring about the accumulation of increased amounts of H1(0) histones. The relative magnitude of these effects exhibits cell-type specificity. There are two H1(0) histone subtypes (a and b) with ratios which differ according to the tissue examined and whose relative importance is not known. The levels of H1(0) histone appear to be more closely related to the degree of differentiation than to the proliferative activity of cells.     

Histone acetyltransferase-1 regulates integrity of cytosolic histone H3-H4 containing complex

Amounts of soluble histones in cells are tightly regulated to ensure supplying them for the newly synthesized DNA and preventing the toxic effect of excess histones. Prior to incorporation into chromatin, newly synthesized histones H3 and H4 are highly acetylated in pre-deposition complex, wherein H4 is di-acetylated at Lys-5 and Lys-12 residues by histone acetyltransferase-1 (Hat1), but their role in histone metabolism is still unclear. Here, using chicken DT 40 cytosolic extracts, we found that histones H3/H4 and their chaperone Asf1, including RbAp48, a regulatory subunit of Hat1 enzyme, were associated with Hat1. Interestingly, in HAT1-deficient cells, cytosolic histones H3/H4 fractions on sucrose gradient centrifugation, having a sedimentation coefficient of 5–6S in DT40 cells, were shifted to lower molecular mass fractions, with Asf1. Further, sucrose gradient fractionation of semi-purified tagged Asf1-complexes showed the presence of Hat1, RbAp48 and histones H3/H4 at 5–6S fractions in the complexes. These findings suggest the possible involvement of Hat1 in regulating cytosolic H3/H4 pool mediated by Asf1-containing cytosolic H3/H4 pre-deposition complex.     

Nucleosomes from normal and regenerating rat liver

Micrococcal-nuclease digestion of rat liver nuclei selectively released mononucleosomes associated with ADP-ribosylated [Caplan, Ord & Stocken (1978) Biochem. J.174, 475-483] histone H1. Two classes of mononucleosome were detected, those that leaked out during digestion and those that were subsequently released by 5mm-sodium phosphate buffer (pH6.8)/0.2mm-NaEDTA. The former, from which histone H1 had been dissociated, contained 140-base-pair-length DNA and core histones;the latter contained core particles and mononucleosomes with histone H1 and 200-base-pair-length DNA. When normal liver nuclei were phosphorylated with [gamma-(32)P]ATP, dissociated histone H1, which could be separated from core particles with Sephadex G-200, showed (32)P uptake. (32)P uptake into histones H2A and poly(ADP-ribosyl)ated H3 was appreciable in core particles, but was less evident in nucleosomes still containing histone H1. When [(3)H]-thymidine was given to partially hepatectomized rats in S-phase, 5-10min pulses in animals of over 300g body wt. showed the presence of high-specific-radioactivity DNA in released core particles and mononucleosomes compared with DNA retained in the nuclear pellets. Mononucleosomes from rat livers in S-phase with new, [(3)H]lysine-containing histones, had higher (32)P incorporation in histones H1 and their core histones, than for di- or tri-nucleosomes. Thermal-denaturation properties of control and phosphorylated mononucleosomes and core particles were very similar; removal of histone H1 and non-histone chromosomal proteins in 0.5m-NaCl markedly increased the proportion of DNA ;melting' below 70 degrees C.     

Effect of inhibition of DNA synthesis on histone synthesis, turnover, and deposition in the rat testis

In testicular seminiferous epithelial cells (SEC) of normal and hypophysectomized rats, 1-beta-D-arabinofuranosylcytosine and hydroxyurea (at concentrations which inhibited DNA synthesis nearly completely) inhibited histone synthesis only partially, and to a different extent for each histone fraction. In the presence of the inhibitors, the extent of synthesis relative to the corresponding control was TH1-x greater than H1 greater than TH2B-x = X2 = H2A greater than H2B = H3 greater than H4, in which synthesis of the H4 fraction was about 50% of control and that of TH1-x was 90-95% of control. The extent of inhibition of synthesis of each histone fraction was similar after hypophysectomy and, therefore, the changing of the relative populations of heterogeneous cells in the SEC did not influence the relative effects of the inhibitors of DNA synthesis on the synthesis of the various histone fractions. After [3H]leucine injection, the molar proportions of labeled histones relative to H4 decreased markedly between 1.5 h and 6-15 days; this finding indicated that there was rapid removal of histones compared to the H4 fraction during this period. When [14C]thymidine was injected 24 h prior to hydroxyurea treatment and [3H]leucine injection, the ratios of specific activities of histone H4 to DNA did not change significantly over an 11-day period. It appears that newly synthesized histone H4 and other somatic histones are associated with existing DNA in the presence of DNA inhibitors.     

Requirement of protein synthesis for the coupling of histone mRNA levels and DNA replication

H1 and core histone mRNA levels have been examined in the presence of protein synthesis inhibitors with different mechanisms of action. Total HeLa cell RNAs were analyzed by Northern Blot hybridization using cloned human histone genes as probes. Inhibition of DNA replication resulted in a rapid decline in histone mRNA levels. However, in the presence of cycloheximide or puromycin, H1 and core mRNAs did not decrease in parallel with DNA synthesis, but were stabilized and accumulated. Inhibition of DNA synthesis with hydroxyurea after the inhibition of protein synthesis did not lead to a decline in histone mRNA levels. These results suggest that synthesis of a protein(s)--perhaps a histone protein(s)--is required for the coordination of DNA synthesis and histone mRNA levels.     

Histone acetylation and histone synthesis in mouse fibroblasts during quiescence and restimulation into S-phase

Mouse NIH 3T3 fibroblasts were starved by serum depletion and subsequently restimulated by addition of serum. Histone acetylation and histone synthesis were studied from the beginning of starvation to the point where most of the cells were in S-phase utilizing electrophoretic and fluorographic techniques. We found that the major part of histone acetylation and histone synthesis occurs during S-phase but that also in the absence of DNA synthesis there are significant changes in the acetylation and synthesis rates of the core histones which occur during the first 6 hours of serum stimulation of quiescent cells, and between 24 and 48 hours of serum starvation.     

Correlation of chromatin composition with metabolic changes in nuclei of primitive erythroid cells from chicken embryos

Changes in levels of biosynthesis of DNA, RNA, and histones were compared with relative proportions of each histone class during primitive erythropoiesis in embryonic chicks. We confirmed that erythrocyte-specific histone 5 (H5) was substantial in the earliest accessible, erythroblast-enriched stage and that it doubled in relative amount between polychromatic and orthochromatic stages to about 1 mol per 2 mol of each nucleosomal histone, still considerable less than in adult definitive erythrocytes. No other histones changed during primitive erythropoiesis, but the molar proportion of histone 1 (H1) always exceeded that of H5 in these cells, unlike definitive erythrocytes. The increase in content of H5 was accompanied by continued decline in synthesis of the other histones and DNA. The accumulation of H5 during development appears to occur in steps corresponding to the maturation of the primitive and definitive erythroid cell lines. Lysine-rich histones were more easily extracted from nuclei of the erythrosynthesis in whole cells and in isolated nuclei.     

Histone Acetyltransferase 1 Promotes Homologous Recombination in DNA Repair by Facilitating Histone Turnover

Faithful repair of DNA double-strand breaks is vital to the maintenance of genome integrity and proper cell functions. Histone modifications, such as reversible acetylation, phosphorylation, methylation, and ubiquitination, which collectively contribute to the establishment of distinct chromatin states, play important roles in the recruitment of repair factors to the sites of double-strand breaks. Here we report that histone acetyltransferase 1 (HAT1), a classical B type histone acetyltransferase responsible for acetylating the N-terminal tail of newly synthesized histone H4 in the cytoplasm, is a key regulator of DNA repair by homologous recombination in the nucleus. We found that HAT1 is required for the incorporation of H4K5/K12-acetylated H3.3 at sites of double-strand breaks through its HIRA-dependent histone turnover activity. Incorporated histones with specific chemical modifications facilitate subsequent recruitment of RAD51, a key repair factor in mammalian cells, to promote efficient homologous recombination. Significantly, depletion of HAT1 sensitized cells to DNA damage compromised the global chromatin structure, inhibited cell proliferation, and induced cell apoptosis. Our experiments uncovered a role for HAT1 in DNA repair in higher eukaryotic organisms and provide a mechanistic insight into the regulation of histone dynamics by HAT1.     

Triiodothyronine nuclear receptor: Role of histones and DNA in hormone binding

The triiodothyronine (T₃) nuclear receptor was previously shown to lose rapidly its high affinity hormone-binding property after a partial purification from the nuclear extract. It was then found that histones + DNA added to the incubation medium with labeled T₃ could restore, at least in part, the high affinity T₃ binding. We now demonstrate that DNA alone increases the high affinity T₃ binding site concentration moderately, and only at low ionic strength where it can bind to the receptor. Total histones and all histone fractions studied (total core histones, F2a, H2B, H3, H4, H1) specifically increase, at low concentrations, the level of T₃ binding; but higher concentrations of some individualized histones, particularly arginine-rich histones, have an inhibitory effect. DNA, or several other polynucleotides, in the presence of histones increase the stimulating histone effect and reverse the inhibitory effect into a true activation. Histones increase the number of T₃ binding sites but decrease the affinity for T₃; addition of DNA restores the high affinity for T₃ and stabilizes the T₃-receptor complexes. Thus, some of the histone molecules could play a role in the maintenance of the T₃ binding site, but multiple interactions between histones or with DNA seem necessary to impair the negative effect exerted by other parts of the histone molecules. Whether these positive and negative effects of histones on the T₃ binding site are of biological relevance in the regulation of T₃ binding to its receptor remains to be determined.