Histone gene expression in early development of Xenopus laevis

Abstract. This study comprises the hybridization analysis of electrophoretically separated histone mRNAs from oocytes and embryos of Xenopus laevis , and analysis of in vitro translation products of these mRNAs on polyacrylamide gels containing sodium dodecyl sulfate (SDS) or Triton X-100. In oocytes and embryos up to the tailbud stage, four types of mRNAs complementary to histone H2B DNA and two complementary to histone H4 DNA can be discriminated by their different electrophoretic mobilities on polyacrylamide gels. Electrophoretic heterogeneity was not detected for messengers for histones H2A and H3.
Histone mRNA, purified by hybridization under stringent conditions with a cloned histone gene cluster, was used to direct histone protein synthesis in a wheat-germ cell free system. The proteins synthesized comigrate with purified marker histones when electrophoresed on SDS-gels or acid-urea gels containing Triton X-100. When hybrid-selected histone mRNAs from oocytes and embryos in different developmental stages are translated, the proteins made by the mRNA from one stage can not be discriminated from those made by the mRNA from another stage after electrophoresis on SDS-gels or acid urea Triton X-100 gels.     

Histone gene expression during sea urchin embryogenesis: isolation and characterization of early and late messenger RNAs of Strongylocentrotus purpuratus by gene-specific hybridization and template activity

We have examined the molecular mechanisms responsible for the shifts in histone protein phenotype during embryogenesis in the sea urchinStrongylocentrotus purpuratus. The H1, H2A, and H2B classes of histone synthesized at the earliest stages of cleavage are heterogeneous: These proteins are replaced at late embryogenesis by a different set of histone-like polypeptides, some of which are also heterogeneous. The H3 and H4 histones appear to be homogeneous classes and remain constant. We have isolated from both early and late embryos the individual messenger RNAs coding for each of the multiple protein subtypes. The RNAs were isolated by hybridization to cloned DNA segments coding for a single histone protein or by elution from polyacrylamide gels. Each RNA was then analyzed and identified by its mobility on polyacrylamide gels and by its template activity in the wheat germ cell-free protein synthesizing system. The mRNAs for each of the five early histone protein classes are heterogeneous in size and differ from the late stage templates. The late mRNAs consist of at least 11 separable types coding for the 5 classes of histones. Each of the 11 has been separated and identified. The late stage proteins were shown to be authentic histones since many of their templates hybridize with histone coding DNA. The early and late stage mRNAs are transcribed from different sets of histone genes since (1) late stage H1 and H2A mRNAs fail to hybridize to cloned early stage histone genes under ideal conditions for detecting homologous early stage hybrids, (2) late stage H2B, H3, and H4 RNA/DNA hybrids melt at 14, 11, and 11°C lower, respectively, than do homologous RNA/DNA hybrids, and (3) purified late stage mRNAs direct the synthesis of the variant histone proteins which are synthesized only during later stages. The time course of synthesis of the late stage mRNAs suggests that they appear many hours before the late histone proteins can be detected, possibly as early as fertilization. In addition, early mRNAs are synthesized in small quantities as late as 40 hr after fertilization, during gastrulation. Thus, the major modulations of histone gene expression are neither abrupt nor an absolute on-off switch, and may represent only a gradual and relative repression of early gene expression. Two histones are detected only transiently during early cleavage. The mRNA for one of them, a subtype of H2A, can be detected in the cytoplasm for as long as 40 hr after fertilization. However, template activity for the other, a subtype of H2B, can be detected only at the blastula stage. Thus, the histone genes represent a complex multigene family that is developmentally modulated.     

Regulation of histone synthesis during early Urechis caupo (Echiura) development

The histones present in mature oocytes and embryos of Urechis caupo and their pattern of synthesis during early development have been characterized. Acid-soluble proteins extracted from mature oocyte germinal vesicles and from embryonic nuclei were analyzed by two-dimensional polyacrylamide gel electrophoresis. Histones are accumulated in the mature oocytes in amounts sufficient to provide for the assembly of chromatin through the 32- to 64-cell stage of embryogenesis. Two H1 histones, which appear to be variants, were found. Germinal vesicles and cleavage-stage nuclei are enriched in H1M (maternal). During late cleavage a faster-migrating H1, H1E (embryonic), appears among the nuclear histones and, as embryogenesis continues, replaces H1M as the predominant H1. No new core histone variants are detected during early development. Examination of [³H]lysine-labeled histones from germinal vesicles and embryonic nuclei reveals stage-specific patterns of histone synthesis. H1M is the major H1 species synthesized in mature oocytes. After fertilization, a switch to the predominant synthesis of H1E occurs. Comparison of the [³H]lysine incorporated into H1E and core histones indicates that H1E synthesis is disproportionately high from midcleavage through the midblastula stage. By the gastrula stage, a balanced synthesis of H1E and each core histone is established. The results indicate that there is noncoordinate regulation of H1 and core histone synthesis during Urechis development.     

Histone synthesis in early amphibian development: histone and DNA syntheses are not co-ordinated

The synthesis of basic proteins has been studied in the oocytes, eggs and embryos of the South African clawed frog, Xenopus laevis. A group of newly synthesized proteins has been identified as histones by the following criteria: solubility properties; incorporation of [³H]lysine and [³H]arginine in the correct proportions, but lack of incorporation of [³H]tryptophan; co-cleotrophoresis with marker histones in various types of polyacrylamide gels, including a type run in two dimensions; peptide analysis of the arginine-rich fraction, F2A1. The four main histone fractions other than F1 were found to be synthesized at all stages of development. F1 histone synthesis was first detected at the late blastula stage.Rates of histone synthesis were estimated for the different stages of development and it was concluded that histone synthesis was not co-ordinated with DNA synthesis either temporally or quantitatively. Histone synthesis was unusual in the following major respects: histones were synthesized in oocytes, and yet in these cells DNA replication had not occurred for several months; histones were synthesized in activated or fertilized eggs at a rate far in excess (about 500 times) of the immediate requirements. We suggest that in order to provide enough histones for the late blastula embryo a store of histone is accumulated during the early cleavage stages and possibly during oogenesis.     

Comparative electrophoretic properties of histones from trout and chicken erythrocytes and calf thymus at different concentrations of EDTA]

An introduction of EDTA into an electrophoretic system was found to cause specific changes in the histone distribution patterns. The electrophoretic mobility of histones H3, H2b and H2a from three evolutionally unrelated sources (trout and chicken erythrocytes and calf thymus) is increased and that for histones H1 and H5 is decreased with respect to histone H4. In general the decrease of electrophoretic mobility of the histones in the presence of EDTA is correlated with the content of basic amino acids in these histones. The effect observed can be used from electrophoretic analysis of histones.     

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.     

Dynamics of histone acetylation in Saccharomyces cerevisiae

Rates of turnover for the posttranslational acetylation of core histones were measured in logarithmically growing yeast cells by radioactive acetate labeling to near steady-state conditions. On average, acetylation half-lives were approximately 15 min for histone H4, 10 min for histone H3, 4 min for histone H2B, and 5 min for histone H2A. These rates were much faster than the several hours that have previously been reported for the rate of general histone acetylation and deacetylation in yeast. The current estimates are in line with changes in histone acetylation detected directly at specific chromatin locations and the speed of changes in gene expression that can be observed. These results emphasize that histone acetylation within chromatin is subject to constant flux. Detailed analysis revealed that the turnover rates for acetylation of histone H3 are the same from mono- through penta-acetylated forms. A large fraction of acetylated histone H3, including possibly all tetra- and penta-acetylated forms, appears subject to acetylation turnover. In contrast, the rate of acetylation turnover for mono- and di-acetylated forms of histones H4 and H2B, and the fraction subject to acetylation turnover, was lower than for multi-acetylated forms of these histones. This difference may reflect the difference in location of these histones within the nucleosome, a difference in the spectrum of histone-specific acetylating and deacetylating enzymes, and a difference in the role of acetylation in different histones.     

Rates of histone synthesis during early development of Rana pipiens

Newly synthesized histones have been extracted from Rana pipiens oocytes or cleaving embryos previously injected with [³H]lysine or [³H]arginine. The radioactive proteins were fractionated by cation-exchange chromatography and electrophoresis on acid/urea or SDS-polyacrylamide gels; histones were identified by coelectrophoresis with authentic markers. From percentage total incorporation in the putative histones, and absolute rates of lysine or arginine incorporation, rates of histone synthesis were estimated. Rates of histone synthesis in two-cell embryos were at least 10-fold higher than in maturing oocytes. Between the two-cell and blastula stages, the rate increased an additional threefold, from about 1200 pg hr^?1 per embryo to about 4500 pg hr^?1 per embryo. While all histone classes are synthesized during cleavage, synthesis of the various classes is not coordinated; histones are not synthesized in the same relative proportions at which they are found in blastula chromatin. The synthesis of histone H4 in particular is barely detectable during cleavage. This, and other observations, suggested the existence of cytoplasmic histone pools. In approaching the possible existence of histone pools, the amount of H4 present in oocytes was determined. Oocytes contain about 74 ng of H4, an amount sufficient to allow development to the blastula stage. These data are compared to those reported by others on histone synthesis during cleavage in Xenopus.     

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.     

Acetylation of histones during spermatogenesis in the rat

Acetate was actively incorporated into rat testis histones when testis cells were prepared by the trypsinization technique in the presence of [³H]acetate. The acetylation was enhanced by 10 mm sodium butyrate. Although histones H3 and H4 were the only histones which incorporated high levels of acetate, the testis-specific histones TH2B and TH3 also appeared to incorporate acetate. This was shown by electrophoresis of the histones on polyacrylamide gels containing Triton X-100. Results, obtained from analysis of histones by two-dimensional gel electrophoresis, confirmed a recent report (P. K. Trostle-Weige, M. L. Meistrich, W. A. Brock, K. Nishioka, and J. W. Bremer, (1982) J. Biol. Chem.257, 5560–5567) that TH2A was a testis-specific histone. The results also confirmed the H2A nature of a testis-enriched histone band, previously designated X2. When histones from populations of cells enriched in specific testis cell types, representing various stages of spermatogenesis, were examined, the patterns of acetylation varied dramatically. Very high levels of acetate were incorporated into multiacetylated species of histone H4 from a population of cells enriched in transition stage spermatids (steps 9–12) compared to the levels of acetate incorporated into H4 from round spermatids (steps 1–8) and earlier stages of spermatogenesis, where acetate was incorporated primarily into the monoacetylated species of H4. Thus, a striking correlation exists between the time of hyperacetylation of histone H4 and the time of removal of histones for their replacement by the basic spermatidal transition proteins designated TP, TP2, and TP4. Hyperacetylation of histone H4 may facilitate the removal of the entire histone complement during the protein transition. In any case, it must be an obligatory step in the dramatic process.     

Hydrolysis of histones by proteinases.

Hydrolysis of histones by proteinases from rat liver, skin and other sources was studied by using a rat thymus histone preparation as the substrate and polyacrylamide-gel electrophoresis and densitometric analysis as the methods to detect histone subtypes and their hydrolysis. The rat mast-cell proteinase I effectively hydrolysed histones except type H4. Thrombin hydrolysed effectively histones H1 and H2A, whereas plasmin hydrolysed all types of histones. Cathepsin D hydrolysed especially histone H2A. Cathepsins B and L hydrolysed all histones more slowly, and cathepsin H hydrolysed them extremely slowly. Epidermal aminoendopeptidase did not hydrolyse histones. Trypsin and chymotrypsin were used as reference enzymes, which hydrolysed all types of histones in very low concentrations. This study suggests that a variety of proteinases could play a role in histone hydrolysis. Hydrolysis of a specific subtype of histones, such as histone H2A at pH 6 by cathepsin D, may be directly involved in regulation of epidermal-cell differentiation.     

Selective synthesis and modification of nuclear proteins during maturation of avian erythroid cells.

The synthesis of the nuclear proteins of duck erythroid cells at different stages of maturation has been investigated. Synthesis of histone fractions H1, H2a, H2b, H3, and H4 is restricted to the erythroblasts, while synthesis of H5 can be detected even at later stages of maturation after DNA synthesis has ceased. The synthesis of nonhistone nuclear proteins (NHNP), on the other hand, occurs in cells at all stages of maturation although their rates of synthesis decline as the cells mature. The same size classes of NHNP appear to be synthesized in erythroblasts and in early- and midpolychromatic erythrocytes. In late polychromatic erythrocytes the synthesis of a new group of NHNP of molecular weights ranging from 54,000 to 130,000 was observed. This group of proteins does not accumulate in the mature erythrocyte, indicating that their relative proportions are very small.Turnover of histone-bound phosphate was found to occur mainly at the erythroblast stage, except for histone H2a which was actively phosphorylated even at more advanced stages of maturation. Phosphorylation of most of the histones appears to be coupled to histone (and coordinate DNA) synthesis.Incorporation of radioactive acetate into histones occurs at all stages, but the rate of acetylation decreases four- to fivefold with maturation. Although the RNA synthetic activity of erythroid cells also decreases with age, experiments involving the use of RNA polymerase inhibitors suggest that the mechanisms that control RNA synthesis and histone acetylation are not tightly coupled.     

Differential expression of histone sequences in Drosophila following heat shock

The expression of the sequences encoding the four nucleosomal histone proteins was examined following heat shock of a variety of Drosophila cells and was found to be highly differential. In Drosophila melanogaster KC-O cells grown in suspension culture, there is a continuation of the synthesis of all four of the nucleosomal histone proteins following heat shock. Analysis of RNA from these cells confirms that histone messengers are transcribed and located on polysomes. This exact same pattern of histone protein synthesis occurs in KC-O cells grown to low density on plates. In contrast, KC-O cells grown to high density on plates exhibit a dramatic elevation of H2b protein synthesis relative to the synthesis of the other core histones. Organs from D melanogaster third instar larvae were examined to ascertain whether histone protein synthesis continues following heat shock in the organism. Different tissue types exhibited differential histone synthesis. Imaginal disks excised from heat-shocked larvae continue to synthesize nucleosomal histones in a variable fashion. In contrast, neither fat bodies, brains, nor salivary glands continues to synthesize core histone proteins at a significant level. D hydei plated cell cultures and larval tissues fail to synthesize histones at any detectable level following a heat shock. Based on these observations, we propose that there is a differential synthesis of nucleosomal proteins in Drosophila that is highly dependent on the state of the cells prior to the heat shock.     

Analysis of dynamic changes in post-translational modifications of human histones during cell cycle by mass spectrometry

The N-terminal tails of the four core histones are subject to several types of covalent post-translational modifications that have specific roles in regulating chromatin structure and function. Here we present an extensive analysis of the core histone modifications occurring through the cell cycle. Our MS experiments characterized the modification patterns of histones from HeLa cells arrested in phase G1, S, and G2/M. For all core histones, the modifications in the G1 and S phases were largely identical but drastically different during mitosis. Modification changes between S and G2/M phases were quantified using the SILAC (stable isotope labeling by amino acids in cell culture) approach. Most striking was the mitotic phosphorylation on histone H3 and H4, whereas phosphorylation on H2A was constant during the cell cycle. A loss of acetylation was observed on all histones in G2/M-arrested cells. The pattern of cycle-dependent methylation was more complex: during G2/M, H3 Lys27 and Lys36 were decreased, whereas H4 Lys20 was increased. Our results show that mitosis was the period of the cell cycle during which many modifications exhibit dynamic changes.     

Histone synthesis and replacement during spermatogenesis in the mouse.

Separation of labelled nuclei by sedimentation velocity at unit gravity (Staput method) was used to study the timing of histone synthesis and replacement by testis-specific basic nuclear protein (TSP) during spermatogenesis in the mouse. Animals were injected (intratesticularly) with 1.25 micronCi per testis 3H-arginine or 2.5 micronCi per testis 3H-lysine, testis nuclei were separated, and the acid extract of each nuclear fraction was analyzed by acrylamide gel electrophoresis. The distribution of labelled histones and TSP in separated nuclei was assessed 2 h after incorporation. Changes in the labelled histone and TSP content of nuclei during subsequent differentiation (1--34 days post-label) was followed in fractions of separated testis cell nuclei and in nuclei of cauda epididymal spermatozoa. Analysis of total histone and (TSP) content indicated quantitative changes during development. Nuclei from primary spermatocytes had relatively larger amounts of histones H1 and H4. Spermatid nuclei showed a relative reduction in histones H1 and H4, coincident with the appearance of TSP in these nuclei. These results suggested that synthesis and/or removal of certain histones must occur in late primary spermatocyte and early spermatid stages of spermatogenesis. Results of labelling experiments indicated several periods of histone synthesis during spermatogenesis: (1) closely associated with the last DNA synthesis(i.e., in early primary spermatocytes), (2) late in meiotic prophase (i.e., in pachytene primary spermatocytes) and (3) simultaneous with TSP synthesis (i.e., in late spermatids). Histone H1 was more heavily labelled toward the end of the primary spermatocyte period. Histone H4 was more heavily labelled in the early primary spermatocyte period, and again at the time of TSP synthesis in spermatids. Histones synthesized before the pachytene primary spermatocyte stage appeared to be replace, but histones synthesized later in spermatogenesis appeared to be at least partially retained in epididymal spermatozoa. These results suggested that repeated specific alterations in the protein complement of the nucleus are an integral part of spermatogenic differentiation in the mouse.     

Declining histone phosphorylation during myogenesis revealed by in vivo and in vitro labeling

To investigate histone phosphate levels during myogenesis, proliferation (d 1), pre-fusion postmitotic (d 2) and myotube (d 3) stage cultured chicken myoblasts were phosphorylated in vivo with [32P]orthophosphate or in vitro by incubating isolated nuclei with 32P-gamma-ATP. Incorporation of radioactive phosphate into histone was assessed by SDS and acid/urea/Triton-X-100 (AUT) gel electrophoresis and radioautography. During proliferation, in vivo labeling with [32P]orthophosphate revealed that all histones except H2b were phosphorylated in the following order of decreasing modification: H1 a greater than H2a greater than H1 b greater than H3 greater than H4. In pre-fusion post-mitotic cells phosphorylation of histones H1 a, H3 and H4 declined, whereas all histones exhibited significantly decreased modification at the myotube stage. It is unlikely that these changes resulted from decreased specific radioactivity of intracellular inorganic phosphate pools, since uptake of [32P]orthophosphate by myotubes increased six-fold, compared with proliferating cells. Isolated nuclei incubated with 32P-gamma-ATP displayed similar decreases during myogenesis; however, 1 a, H1 b and H3 were the only histones modified by in vitro phosphorylation.