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.     

Stability of histone mRNAs is related to their location in polysomes

Synthesis of histone mRNAs is closely coupled to DNA synthesis. Following inhibition of DNA synthesis in L6 myoblasts with cytosine arabinoside, a coordinate and exaggerated rate of degradation of histone mRNAs occurs while other mRNAs, encoding ribosomal protein L32 and actin, are unaffected. Inhibition of protein synthesis by puromycin, emetine, or cycloheximide stabilizes histone mRNAs and results in their accumulation. When inhibition of DNA synthesis was followed immediately by inhibition of protein synthesis, the exaggerated rate of decay of the existing subspecies of histone H4 mRNAs was prevented and histone mRNA accumulated. If inhibition of protein synthesis was delayed longer than 3 minutes following inhibition of DNA synthesis, the ability to accumulate H4 mRNAs was lost. Furthermore, new protein synthesis was required to activate the mechanism which specifically destabilized histone mRNA. Puromycin was able to prevent the exaggerated rate of degradation of the various subspecies of H4 mRNA when added up to 15 min after inhibition of DNA synthesis, whereas emetine was effective only when added up to 5 min following inhibition of DNA synthesis. These data suggest that histone H4 mRNAs in polysomes are better targets than those released from polysomes for the specific mechanism which destabilizes histone mRNAs upon inhibition of DNA synthesis.     

Differential stimulation of histone and high mobility group chromatin protein synthesis in the rat uterus by estrogen

Uterine tissue isolated from immature rats at different times after estradiol injection was incubated with medium containing [3H]lysine. The acid-extractable protein from the uterine tissue was subjected to electrophoresis on sodium dodecyl sulfate and acid-urea-Triton X-100 polyacrylamide gels, and the rate of chromatin protein synthesis determined by densitometric analysis of the fluorographs of the gels. Synthesis of chromatin proteins (histones and high mobility group chromatin proteins) was stimulated by 3 h after estrogen treatment and reached a peak at 9 h, several hours before DNA synthesis was stimulated. Synthesis of chromatin proteins occurred at the same time as total cellular protein synthesis. Estrogen stimulated the synthesis of histone variants at different rates, but the accumulation of histone proteins remained coordinated such that equivalent amounts of histone proteins were being produced at any one time.     

Kinetics of accumulation and depletion of soluble newly synthesized histone in the reciprocal regulation of histone and DNA synthesis

Procedures are presented which permit the identification and analysis of cellular histone that is not bound to chromatin. This histone, called soluble histone, could be distinguished from that bound to chromatin by the state of H4 modification and the lack of H2A ubiquitination. Changes in the levels of newly synthesized soluble histone were analyzed with respect to the balance between histone and DNA synthesis in hamster ovary cells. Pulse-chase protocols suggested that the chase of newly synthesized histone from the soluble fraction into chromatin may have two kinetic components with half-depletion times of about 1 and 40 min. When protein synthesis was inhibited, the pulse-chase kinetics of newly synthesized histone from the solubl fraction into chromatin were not significantly altered from those of the control. However, in contrast to the control, when protein synthesis was inhibited, DNA synthesis was also inhibited with kinetics similar to those of the chase of newly synthesized histone from the soluble fraction. There was a rapid decrease in the rate of DNA synthesis with a half-deceleration time of 1 min down to about 30% of the control rate, followed by a slower decrease with an approximate half-deceleration time of 40 min. When DNA synthesis was inhibited, newly synthesized histone accumulated in the soluble fraction, but H2A and H2B continued to complex with chromatin at a significant rate. Soluble histone in G1 cells showed the same differential partitioning of H4/H3 and H2A/H2B between the soluble and chromatin-bound fractions as was found in cycling cells with inhibited DNA synthesis. These results support a unified model of reciprocal regulatory mechanisms between histone and DNA synthesis in the assembly of chromatin.     

H1 variant synthesis in proliferating and quiescent human cells

The synthesis of histone H1 isoprotein species in human cells of several different types and in several different physiological states was studied. Up to five H1 and two H1 degrees isoprotein species could be resolved by two-dimensional electrophoresis. All five H1 isoprotein species were synthesized in exponentially growing cultures of IMR-90 human fibroblasts; in quiescent IMR-90 cells the synthesis of three H1 isoprotein species was greatly decreased while the synthesis of two others was much less affected. When DNA synthesis in exponentially growing cultures of IMR-90 was inhibited, the pattern of H1 isoprotein synthesis became similar to that found in quiescent cultures. Other human cells, isolated from blood, yielded similar results. These results suggest that the pattern of H1 synthesis is the same for cells in non-S phases of the cell cycle and in quiescent cells. Thus for histone H1 in human cells the relationship of the variant synthesis pattern to the growth state and DNA replication is similar to that of the core histone H3 but not that of H2A.     

Uncoordinate synthesis of histone H1 in cells arrested in the G1 phase

It has been known for several years that DNA replication and histone synthesis occur concomitantly in cultured mammalian cells. Normally all five classes of histones are synthesized coordinately. However, mouse myeloma cells, synchronized by starvation for isoleucine, synthesize increased amounts of histone H1 relative to the four nucleosomal core histones. This unscheduled synthesis of histone H1 is reduced within 1 h after refeeding isoleucine, and is not a normal component of G1. The synthesis of H1 increases coordinately again with other histones during the S phase. The DNA synthesis inhibitors, cytosine arabinoside and hydroxyurea, block all histone synthesis in S-phase cells. The levels of histone H1 mRNA, relative to the other histone mRNAs, is increased in isoeleucine-starved cells and decreases rapidly after refeeding isoleucine. The increased incorporation of histone H1 is at least partially due to the low isoleucine content of histone H1. Starvation of cells for lysine resulted in a decrease in H1 synthesis relative to core histones. Again the ratio was altered on refeeding the amino acid. 3T3 cells starved for serum also incorporated only H1 histones into chromatin. The ratio of different H1 proteins also changed. The synthesis of the H1⁰ protein was predominant in G₀ cells, and reduced in S-phase cells. These data indicate the metabolism of H1 is independent of the other histones when cell growth is arrested.     

Histone synthesis during infection of monkey kidney cells with Simian Virus 40.

The synthesis of histones during lytic infection of BSC-1 (African Green Monkey kidney) cells with SV40 has been investigated. The synthesis of all five classes of histones was stimulated, and all classes appeared to be stimulated to the same extent. The increase in rate of histone synthesis in response to SV40 infection was detectable several hours before SV40 DNA synthesis was measureable, and the rate of histone synthesis decreased at a time when SV40 DNA synthesis was occuring at a maximal or relatively high rate. In addition, the changes in rates of histone synthesis did not correlate well with the rates of host DNA synthesis during infection. Thus it appears that DNA synthesis and histone synthesis may not be strictly coupled in SV40 infected cells.     

DNA and histone synthesis are uncoupled during germination of maize embryos

The rate of synthesis of DNA and histones was studied in germinating maize embryos as a function of the length of the germination period. To that end excised embryos from seeds germinated for different periods of time were pulse labelled either with [¹⁴C]protein hydrolysate or with [³H]TdR. Specific activities were determined for the total cellular proteins and the total histone fraction obtained by acid-extraction of the cellular homogenate and BioRex70 ion exchange chromatography. The results show that the early germination period is characterized by a lack of coupling between the histone synthesis and that of the nuclear DNA. The early histone synthesis peak might be necessitated by the reprogramming of the embryo genome that takes place during germination.     

Biogenesis of chromatin in animal cells. Stimulation of nuclear protein and DNA synthesis in hepatocytes after brief inhibition of translation by cycloheximide]

A drastic and brief inhibition of protein synthesis (up to 95% for 3--6 hrs) by cycloheximide in the liver of rats starved for 24 hrs results in a recovery and subsequent marked stimulation of non-histone proteins, histone chromosomal proteins and DNA. The stimulation of non-histone protein synthesis was observed after 1 hr (inhibition) 12--24 hrs (recovery and stimulation of protein synthesis) and 48--60 hrs (stimulation of DNA synthesis) following the administration of cycloheximide. Two periods of histone biosynthesis were observed. The first one (24--36 hrs) was not coupled and the second one (48--60 hrs) was coupled with DNA replication. During the recovery and stimulation of protein synthesis acetylation of the histone and non-histone proteins proceeds at an increased rate. Possible applicability of the model in question for investigations of chromatin biogenesis is discussed.     

Differential expression of two clusters of mouse histone genes

The mouse histone mRNAs coded for by three different cloned DNA fragments have been characterized. Two of these cloned DNA fragments, MM221 and MM291, located on chromosome 13, code for H3, H2b and H2a histone mRNAs, which are expressed at low levels in cultured mouse cells and fetal mice. The other DNA fragment, MM614, located on chromosome 3, codes for an H3 and an H2a mRNA, which are expressed at high levels in these cells. The mRNAs for each histone protein share common coding region sequences, while the untranslated regions of all the genes have diverged significantly, as judged by S1 nuclease mapping. Amino acid substitutions in some H3, H2a and H2b proteins are detected as internal cleavages in the S1 nuclease maps. All of these genes code for replication variant histone mRNAs, which are regulated in parallel with DNA synthesis.     

Protein/DNA interactions involving ATF/AP1-, CCAAT-, and HiNF-D-related factors in the human H3-ST519 histone promoter: cross-competition with transcription regulatory sites in cell cycle controlled H4 and H1 histone genes.

Protein/DNA interactions of the H3-ST519 histone gene promoter were analyzed in vitro. Using several assays for sequence specificity, we established binding sites for ATF/AP1-, CCAAT-, and HiNF-D related DNA binding proteins. These binding sites correlate with two genomic protein/DNA interaction domains previously established for this gene. We show that each of these protein/DNA interactions has a counterpart in other histone genes: H3-ST519 and H4-F0108 histone genes interact with ATF- and HiNF-D related binding activities, whereas H3-ST519 and H1-FNC16 histone genes interact with the same CCAAT-box binding activity. These factors may function in regulatory coupling of the expression of different histone gene classes. We discuss these results within the context of established and putative protein/DNA interaction sites in mammalian histone genes. This model suggests that heterogeneous permutations of protein/DNA interaction elements, which involve both general and cell cycle regulated DNA binding proteins, may govern the cellular competency to express and coordinately control multiple distinct histone genes.     

Histone synthesis in the cells of proliferating and nonproliferating tissues during gametogenesis and early embryogenesis

A review of available data on the replication-dependent and replication-independent histone synthesis in the proliferating and nonproliferating (quiescent) cells during gametogenesis and early embryogenesis. In each of the considered models the replication-dependent and replication-independent histone synthesis play different roles in the chromatin organization and metabolism. The transition from replication-dependent to replication-independent histone synthesis during gametogenesis is a regular process directed to the formation of a highly compacted metabolically inert chromatin (males) and to the formation of histone protein pool in order to provide the chromatin nucleosome structure in the sperm nucleus during fertilization, as well as the nuclear chromatin in zygotes and blastomeres (females). A suggestion is put forward that the coupling of histone and DNA syntheses should arise not simultaneously in all cells of the embryo but have a regional pattern, due, possibly, to the asynchrony of cell cycle in the early embryos.     

Replication timing and cell differentiation

Cell differentiation may depend in part upon a type of unbalanced growth in which several cell cycles occur with a reduced level of total protein synthesis. During this period the synthesis of the chromatin protein HMG-I/Y is reduced since its synthesis is correlated with that of total protein. The synthesis of histone H1 shows less reduction since its synthesis is entrained with that of DNA. This greater reduction of HMG-I/Y than of histone H1 is thought to delay or prevent replicon initiations within AT-enriched isochores. This shifts their time of replication from early to late S phase. This may restrict certain pathways of cell differentiation in multipotent progenitor cells and allow one particular type of differentiation.     

Histone synthesis at S- and G2-phases of the mitotic cycle of human diploid fibroblasts

The phase-specific synthesis of the total protein, histones, RNA and DNA in human embryonic fibroblasts synchronized by double thymidine blockade was investigated. It was shown that activation of histone synthesis occurs in three steps, i.e. i) immediately before replication, ii) after DNA synthesis maximum, and, iii) at the G2-phase of the mitotic cycle. During maximal replication of DNA histone synthesis is suppressed. It was assumed that at the pre-replication phase histone synthesis controls decondensation, while and the G2-phase--condensation of chromatin.