Biomedical subjects

R Chalkley

Publications and source records attributed to R Chalkley.

At least 55 records · Page 3Linked to original sources

PubMed1986

Novobiocin precipitates histones at concentrations normally used to inhibit eukaryotic type II topoisomerase.

At concentrations normally used to inhibit eukaryotic type II topoisomerase activity (100-1000 micrograms/ml) novobiocin binds core histones. Approximately 15 moles of novobiocin bind per mole of histone resulting in histone precipitation from solution in either 0.15 M or 2 M NaCl. The interaction between novobiocin and proteins appears to involve arginine residues: histones H3 and H4 (13.5 and 14 mole percent arginine) are precipitated at lower novobiocin concentrations than histones H2A and H2B (9.5 and 6.5 mole percent arginine). Furthermore, polyarginine but not polyornithine competes for novobiocin in histone precipitation. Moreover, histones with arginine residues modified with 1,2-cyclohexanedione are soluble in 1000 micrograms/ml novobiocin. Because novobiocin can remove histones from solution as well as inhibit topoisomerase activity, and because both of these events can alter DNA topology, novobiocin should be used with caution in experiments designed to implicate topoisomerase activity in chromatin dynamics.

Animals↗

PubMed1986

Characterization of actin mRNA levels during BC3H1 cell differentiation.

The expression of a vascular smooth muscle specific alpha-actin isoform can be induced in mouse BC3H1 smooth muscle cells by treating confluent monolayers with serum-free medium (Strauch, A. R., and Rubenstein, P. A. (1984) J. Biol. Chem. 259, 3152-3159; 7224-7229). Using blot hybridization techniques, two size classes of actin RNA were identified in BC3H1 cells with the relative amount of RNA in each size class varying according to the developmental state of the cells; a 2100-nucleotide actin RNA was most abundant in myoblasts, whereas a smaller 1500-nucleotide actin RNA was found predominantly in fully differentiated myocytes. Results of in vitro translation experiments suggested that the 2100-nucleotide actin RNA on blots of myoblast total RNA corresponded to a mixture of similar size transcripts encoding both beta- and gamma-actin, while the 1500-nucleotide actin RNA in myocytes was an alpha-actin mRNA. Cell-cell contact and serum withdrawal initiated a 6-fold increase in the level of alpha-actin mRNA in BC3H1 cells that was followed by a 3-fold decrease in the amount of beta- and gamma-actin mRNA when confluent cells were exposed to serum-free medium for prolonged periods. Vascular smooth muscle alpha-actin was the major alpha-actin isoform synthesized in L-[35S]cysteine-labeled BC3H1 myocytes, indicating that the 1500-nucleotide actin mRNA size class in these cells may be enriched for vascular smooth muscle alpha-actin transcripts.

Actins↗

PubMed1986

Butyrate effects on normal and adapted hepatoma cells: morphological response and implications for vectoral cholesterol transport.

The cell line 4IC6, adapted for growth in 6 mM sodium butyrate from Hepatoma Tissue Culture cells [R. Chalkley, and A. Shires (1985) J. Biol. Chem. 260, 7698-7704], exhibits a fourfold increase in histone acetate turnover. The 4IC6 cells were about 25 times more resistant to butyrate relative to the parental cell line as measured by cloning efficiency. This line also maintains a flatter and more extended morphology when growing in the presence of 6 mM sodium butyrate relative to the parental line. Both cell lines maintain similar intracellular butyrate levels and incorporate [1-14C]butyrate into lipids to similar extents when incubated in medium containing high levels of the fatty acid. These results show that 4IC6 cells have not attained butyrate resistance through acquiring the ability to metabolize butyrate more efficiently or in a significantly different manner when compared with the parental cell line. The membrane lipid composition was nearly identical between the two cell types. Thus the different morphologies exhibited by each cell line were not a consequence of altered membrane lipid composition. The resistant line, 4IC6, maintains about 10-fold higher cholesterol ester levels and half the level of triglycerides found in the parental line. The butyrate-resistant cells also synthesize cholesterol at about a 1.8-fold higher rate than do the parental cells. This difference in de novo synthesis is reflected by a difference of a similar factor in the amount of radioactive cholesterol the two cell lines accumulate over 12 generations. These results are discussed with respect to models for equilibration of serum lipoprotein-derived and newly synthesized cholesterol.

Acetates↗

PubMed1985

Histone synthesis and deposition in the G1 and S phases of hepatoma tissue culture cells.

Hepatoma tissue culture cells were synchronized in G1 and in S phase in order to examine the level of synthesis of different histone types and to determine the rate, timing, and location of their deposition onto DNA. We observe a basal level of synthesis in G1 (5% of that seen in S phase) for H2A.1, H2A.2, H3.2, H2B, and H4. The minor histone variants X and Z are synthesized at 30% of the rate observed in S cells. The rate of synthesis of the ubiquinated histones uH2A.1,2 is not as depressed in G1 cells as seen for H2A.1 and H2A.2. Histones synthesized in G1 are not deposited on the DNA of these cells at equivalent rates. Thus, histones H3.2 and H4 are not deposited significantly until S phase begins, at which time deposition occurs selectively on newly synthesized DNA. The deposition of H2A.1, H2A.2, H2B, X, and Z proceeds in G1; however, it occurs to a 2-4-fold lower extent than seen for the deposition of H1, HMG 14, and HMG 17. The deposition of all histones synthesized in S phase occurs rapidly, but there are variations in the sites of deposition. Thus, newly synthesized H3.1, H3.2, and H4 deposit primarily on newly replicated DNA whereas H2A.1, H2A.2, uH2A.1, 2, and H2B deposit only partially on new DNA (30%) and mostly on old. H1, HMG 14, and HMG 17 are deposited in an apparently fully random manner over the chromatin. To interpret these observations, we propose a model which includes a measure of histone exchange on the chromatin fiber. The model emphasizes the dynamics of histone-histone and histone-DNA interactions in regions of active genes and at replication forks.

Animals↗

PubMed1985

Histone segregation on replicating chromatin.

We have reinvestigated the mode of segregation of preexisting histones onto replicating chromosomes. Since our previous data have indicated that only histones H3 and H4 do not appear to move from their association with the DNA strand with which they are bound until the next round of replication, we have concentrated our attention on these two histones. The strategy we have employed involved density labeling of DNA and radiolabeling of the histones of interest. Subsequently, we followed the association of histones and DNA during further rounds of DNA replication. One can make predictions concerning the nature of the association between specific histones and particular DNA strands depending on the mode of deposition. The results have confirmed our previous findings that histones segregate randomly. The possibility that such a result is a consequence of turnover of radiolabel in non-histone proteins and subsequent reutilization for histone synthesis has been tested directly. This process appears to be occurring to only a very limited extent. The implications of these conclusions for chromatin structure and gene control are discussed.

Animals↗

PubMed1985

The isolation of HTC variant cells which can replicate in butyrate. Changes in histone acetylation and tyrosine aminotransferase induction.

We have obtained a number of variant HTC cells which are capable of vigorous replication in the presence of 6 mM sodium butyrate. These cells show characteristic changes in histone acetylation. H2A/H2B are no longer modified and the turnover of histones H3/H4 acetate is about 4-fold greater than in control HTC cells at the same butyrate concentration. Histone deposition continues successfully even though histones H3/H4 become hyperacetylated upon association with the chromatin. Prompt deacetylation of new histones does not appear to be a prerequisite for successful deposition processes. Initial enzymatic studies indicate that not only do the butyrate-resistant cells show an increased deacetylase activity (on a per cell basis), but also the enzyme is less sensitive to sodium butyrate under in vitro assay conditions. In contrast to control HTC cells in 6 mM butyrate in which dexamethasone induction of tyrosine aminotransferase is inhibited, the butyrate-resistant variant cells are capable of tyrosine aminotransferase induction even in the presence of butyrate. The implications of these observations are discussed.

Acetylation↗

PubMed1985

Exchange of histones H1, H2A, and H2B in vivo.

We have asked whether histones synthesized in the absence of DNA synthesis can exchange into nucleosomal structures. DNA synthesis was inhibited by incubating hepatoma tissue culture cells in medium containing 5.0 mM hydroxyurea for 40 min. During the final 20 min, the cells were pulsed with [3H]lysine to radiolabel the histones (all five histones are substantially labeled under these conditions). By two electrophoretic techniques, we demonstrate that histones H1, H2A, and H2B synthesized in the presence of hydroxyurea do not merely associate with the surface of the chromatin but instead exchange with preexisting histones so that for the latter two histones there is incorporation into nucleosome structures. On the other hand, H3 and H4 synthesized during this same time period appear to be only weakly bound, if at all, to chromatin. These two histones have been isolated from postnuclear washes and purified. Some possible implications of in vivo exchange are discussed.

Animals↗

PubMed1985

Hyperacetylated histones facilitate chromatin assembly in vitro.

We have examined the effect of histone acetylation on the in vitro assembly of nucleosomes with DNA and purified histones at physiological ionic strength in the presence of polyglutamic acid. We have found that hyperacetylated histones assemble nucleosomes with greater efficiency, and to a greater extent, than either control or hypoacetylated histones. Assembly reactions were performed over a range of histone to DNA ratios (0.25 to 3.0, w/w) and polyglutamic acid to histone ratios (0 to 1.6, w/w). Although polyglutamic acid may act as a sink to prevent nonspecific histone-DNA interactions, our data suggest that the polyanion primarily facilitates the assembly of nucleosomes by organizing histones into a form that is amenable to deposition.

Acetylation↗

PubMed1984

Two-stage maturation process for newly replicated chromatin.

HTC cells have been labeled by short exposures to [3H]thymidine in order to identify newly synthesized DNA. By either isolating nuclei directly or isolating them after an extensive fixation with formaldehyde, we have been able to identify two phases in the maturation process of newly replicated chromatin. The first phase which is relatively brief (less than 5 min) is reflected in a diffuse, irregular organization of nucleosomes on new DNA immediately postreplicatively . The second phase which lasts from 5 to 30 min postreplication is characterized by a normal repeat length for the nucleosomes which are nonetheless more weakly bound than bulk nucleosomes. This is reflected in increased sliding during nuclease digestion as well as increased nuclease sensitivity and the presence of easily dissociated histones which has been described by other workers.

Cells, Cultured↗

PubMed1984

In vitro exchange of nucleosomal histones H2a and H2b.

We have asked whether exogenous, radiolabeled histones can exchange with nucleosomal histones in an in vitro system. Using two different electrophoretic techniques, we were able to separate the histones contained in nucleosomes from those histones which were simply bound to the surface of the chromatin. Fluorography was used to determine which of the exogenous histones exchange with the nucleosomal histones. We observed substantial exchange of histones H1, H2a, and H2b when the chromatin and exogenous histones were incubated under approximately physiological conditions. We have also observed a small amount of exchange of H2a and H2b, as well as a substantial exchange of H1, from one chromatin fragment to another. Other conditions affecting the exchange of histones H2a and H2b are also reported.

Carcinoma, Hepatocellular↗

PubMed1983

Inhibition by sodium butyrate of enzyme induction by glucocorticoids and dibutyryl cyclic AMP. A role for the rapid form of histone acetylation.

We have found that butyrate selectively inhibits hormonal induction of a few specific proteins and messenger RNAs in hepatoma cells. The fatty acid salt reversibly abolishes induction of tyrosine aminotransferase by dexamethasone and dibutyryl cyclic AMP in HTC cells by inhibiting the production of tyrosine aminotransferase messenger RNA. Half-maximal inhibition of enzyme induction occurred in 0.9 mM butyrate. This effect is highly specific, since 4 h after the addition of butyrate to induced HTC cells, the relative abundance of only five messenger RNA species out of several hundred observable on two-dimensional gels of translational products is changed. Upon removal of the butyrate from cell cultures pretreated with dexamethasone, tyrosine aminotransferase activity begins to increase more rapidly than if dexamethasone is added to control cultures, indicating that part of the induction process occurs in the presence of butyrate. A dose-dependent reduction of fast histone acetylation by butyrate was demonstrated by treating cells with butyrate followed by a short pulse with [3H]acetate and chase in a high concentration of butyrate. The butyrate concentration test range over which rapid histone acetylation is inhibited is similar to that which inhibits enzyme induction to the same extent. In contrast, the slow form of histone acetylation is unaffected in the concentration range examined. The induction of tyrosine aminotransferase by dexamethasone is delayed in hypoacetylated cells. This lag is consistent with the time required to initiate the recovery of the fast form of histone acetylation after its transient disappearance (Covault, J., Perry, M., and Chalkley, R. (1982) J. Biol. Chem. 257, 13433-13440). We conclude that sodium butyrate interferes with the ability of dexamethasone and dibutyryl cyclic AMP to increase production of several specific species of messenger RNA in hepatoma cells. This effect correlates well with its ability to reduce rapid acetylation of histones in HTC cells; we discuss potential roles of rapid histone acetylation in modulating hormonal stimulation of transcription.

Acetylation↗

PubMed1982

Effects of histone hyperacetylation and hypoacetylation on RNA synthesis in HTC cells.

We have analyzed the RNA synthetic activity in intact HTC cells which have artificially high or low levels of histone acetylation. Rates of total RNA synthesis and the number of nascent transcription complexes have been measured for 20-h butyrate-treated cells and for butyrate-released cells which contain hypoacetylated histones. Exposure of HTC cells to butyrate for 20 h leads to a approximately 40% decrease in the number of nascent RNA molecules and a corresponding approximately 40% decrease in the rate of total RNA synthesis. During the induction of hypoacetylation following the release of cells from butyrate, rates of RNA synthesis and the number of nascent transcripts remain unchanged. Between the fifth and eighth h of release from butyrate, the number of nascent transcripts and the rate of total RNA synthesis increase to control cell levels. This increase in the number of nascent RNA polymerase complexes, while occurring when bulk histone is hypoacetylated, is temporally correlated with an increase in the rate of acetylation for a subset of histones. In contrast, changes in levels and rates of histone acetylation were found to be without effect on the average rate of RNA chain elongation. Additionally, we have found that the complexity of nuclear RNA is unchanged by a 20-h exposure of HTC cells to butyrate, suggesting that the approximately 40% decrease in frequency of nascent polymerase complexes produced by butyrate does not result from the complete loss of transcription of a large number of genes.

Acetylation↗

PubMed1982

Histone acetylation increases the solubility of chromatin and occurs sequentially over most of the chromatin. A novel model for the biological role of histone acetylation.

The effect of histone acetylation on chromatin solubility has been studied. Nucleosome cores are fairly soluble over a range of Mg2+ and Na+ concentrations. Increasing the amount of Na+ relative to a fixed concentration of Mg2+ leads to increased solubility. Although the effect is not large, acetylated cores are more soluble than control cores, all other factors being equal. Acetylated oligonucleosomes are vastly more soluble than control oligonucleosomes with the greatest differential seen in the presence of Mg2+ at low ionic strengths. Solubility of acetylated oligonucleosomes is favored when the majority of the histones in the nucleosomes are all highly acetylated. Contiguous highly acetylated nucleosomes are enriched in the rapid kinetic form of histone acetylation after short term exposure to butyrate. These observations have been exploited in a fractionation scheme for chromatin based on the levels of hyperacetylation attained after varied times of exposure to sodium butyrate. Essentially all DNA sequences in an unsynchronized cell population are associated with rapidly acetylated histones. Since it is highly unlikely that each cell has its own discrete set of sequences bound to acetylated histone and since the amount of rapidly acetylated histone in a single cell is relatively small (approximately 10% of total histone), we surmise that rapid acetylation migrates throughout much of the chromatin, perhaps in a sequential and highly organized manner. We postulate that the biological role of histone acetylation is to provide a means for sequentially exposing the entire chromatin for surveillance for DNA damage and possibly for recognition of different regions of the DNA by specific (regulatory?) proteins.

Acetylation↗

PubMed1982

Histone hypoacetylation following release of HTC cells from butyrate.

Exposure of HTC cells to 6 mM sodium butyrate for 20 h results in significantly reduced amounts of nuclear histone acetyltransferase. While the direct butyrate inhibition of histone deacetylases is rapidly reversed upon removal of cells from butyrate, histone acetyltransferase activity increases only gradually following release of cells from butyrate, requiring several hours to regain control cell levels. As a result, HTC cells released from a 20-h exposure to butyrate display normal kinetics of histone acetate turnover but markedly reduced rates of histone acetylation, the imbalance in these two processes produces histone hypoacetylation.

Acetylation↗

PubMed1981

Only a small fraction of avian erythrocyte histone is involved in ongoing acetylation.

We have studied histone acetylation in chicken erythrocytes. We find that about 30% of the histone in these cells is acetylated, however the majority of these histones are not in a dynamic steady state typical of other chicken cells and of mammalian cells, but rather are frozen in this state of modification. A very small fraction of erythrocyte histones are being modified normally but cannot be detected as shifting to higher levels of acetylation upon treatment with butyrate because the amount of histone so modified is small. Nonetheless, chicken erythrocytes incorporate 3H-acetate into histones about 40% as well as seen in the dynamically active HTC cells. This is most likely due to the formation of very high specific activity Acetyl CoA pools in erythrocytes which have very low levels of coenzyme A. We conclude that these genetically inactive cells are involved in only a minor way with histone acetylation.

Acetates↗

PubMed1981

The sites of deposition of newly synthesized histone.

The chromosomal fragments produced by nuclease digestion of freshly replicated chromatin migrate more rapidly relative to bulk chromatin when analyzed in nucleoprotein gels. The cause of the anomalous migration has been studied and the evidence indicates that rather than reflecting a shorter nucleosomal repeat in vivo that it may be a consequence of nucleosome sliding during the digestion itself. The distinct electrophoretic characteristics of nucleosomal material containing newly replicated DNA have enabled us to examine their histone composition by two dimensional electrophoresis. We find that nucleosomes containing new DNA also contain newly synthesized histones H3 and H4. In contrast more than 50% of newly synthesized H2A and H2B, and essentially all of new H1, are deposited at sites on the bulk chromatin distinct from that material containing newly replicated DNA. In addition we show that newly synthesized histones H3 and H4 are bound unusually weakly when they first become associated with the chromatin.

Animals↗

PubMed1981

A reevaluation of new histone deposition on replicating chromatin.

In this study, we have density-labeled newly replicated DNA in hepatoma tissue culture cells and separated the newly replicated nucleoprotein from bulk material using density gradient centrifugation. These experiments indicate that only newly synthesized histones H3 and H4 deposit specifically on newly replicated DNA. Histones H2A and H2B show a partial preference and histone H1 shows no preference for deposition on new DNA. These experiments also indicate that for a whole cell cycle, the non-H1 histones remain associated with the same DNA upon which they were initially deposited. However, when that region is replicated during the next cell cycle, the histones distribute equally to both daughter strands. An increase or decrease in the level of histone modification (acetylation) induced by sodium butyrate treatment does not alter the in vivo stability of the histone-DNA interactions. Experiments which involved labeling SV40 minichromosomes with [3H]lysine confirm our observations that only newly synthesized histone H3 and H4 are selectively depend on replicated DNA.

Animals↗

PubMed1981

The effect of histone hyperacetylation on the nuclease sensitivity and the solubility of chromatin.

We have examined the effects of histone hyperacetylation upon nuclease digestion of nuclei and subsequent fractionation of chromosomal material in the presence of MgCl2. DNase I shows a maximum sensitivity towards hyperacetylated nuclei at somewhat elevated ionic strengths (150-200 mM NaCl), whereas micrococcal nuclease exhibits no specificity for acetylated nuclei over a broad range of ionic strengths. Fractionation in the presence of MgCl2 of hyperacetylated nuclei digested with micrococcal nuclease results in a substantial increase in the amount of soluble chromatin relative to that obtained with control nuclei. This increased yield of Mg2+-soluble chromatin results from the recruitment into this fraction of oligonucleosomes containing extremely hyperacetylated histones. These results suggest that contiguous nucleosomes containing highly acetylated histones may be altered in their ability to interact with themselves and with other nucleosomes.

Acetylation↗