Fetal gene reactivation.

Reactivation of silent fetal or embryonic genes could be used for the treatment of genetic diseases caused by mutations of genes normally expressed during the adult stage of development. A paradigm of this approach is the activation of fetal hemoglobin synthesis in adult individuals and its use in the treatment of beta chain hemoglobinopathies. The current understanding of the molecular control of the beta globin locus is reviewed, as are the cellular and molecular basis of induction of fetal hemoglobin in the adult and the approaches used for stimulation of fetal hemoglobin synthesis in patients with beta chain hemoglobinopathies.

Four distinct cyclin-dependent kinases phosphorylate histone H1 at all of its growth-related phosphorylation sites.

In mammalian cells, up to six serines and threonines in histone H1 are phosphorylated in vivo in a cell cycle dependent manner that has long been linked with chromatin condensation. Growth-associated H1 kinases, now known as cyclin-dependent kinases (CDKs), are thought to be the enzymes responsible for this process. This paper describes the phosphorylation of histone H1 by four different purified CDKs. The four CDKs phosphorylate only the cell cycle specific phosphorylation sites of H1, indicating that they belong to the kinase class responsible for growth-related H1 phosphorylation in vivo. All four CDKs phosphorylate all of the interphase and mitotic-specific H1 sites. In addition to the (S/T)PXK consensus phosphorylation sites, these four CDKs also phosphorylate a mitotic-specific in vivo H1 phosphorylation site that lacks this sequence. There is no site selectivity among the growth-related phosphorylation sites by any of the four CDKs because all four CDKs phosphorylate all relevant sites. The results imply that the cell cycle dependent H1 phosphorylations observed in vivo must involve differential accessibility of H1 sites at different stages of the cell cycle.

Phosphorylation of the Oxytricha telomere protein: possible cell cycle regulation.

In the macronucleus of the ciliate Oxytricha nova, telomeres end with single-stranded (T4G4)2 DNA bound to a heterodimeric telomere protein (alpha beta). Both the alpha and beta subunits (alpha-TP and beta-TP) were phosphorylated in asynchronously growing Oxytricha; beta-TP was phosphorylated to a much higher degree. In vitro, mouse cyclin-dependent kinases (Cdks) phosphorylated beta-TP in a lysine-rich domain that is not required for specific DNA binding but is implicated in higher order structure formation of telomeres. Therefore, phosphorylation of beta-TP could modulate a function of the telomere protein that is separate from specific DNA binding. Phosphoamino acid analysis revealed that the mouse Cdks modify predominantly threonine residues in beta-TP, consistent with the observation that beta-TP contains two consensus Cdk recognition sequences containing threonine residues. In Xenopus egg extracts that undergo cell cycling, beta-TP was phosphorylated in M phase and dephosphorylated in interphase. This work provides the first direct evidence of phosphorylation at telomeres in any organism, as well as indirect evidence for cell cycle regulation of telomere phosphorylation. The Cdc2/cyclin A and Cdc2/cyclin B kinases are required for major mitotic events. An attractive model is that phosphorylation of beta-TP by these kinases is required for the breakdown of telomere associations with each other and/or with nuclear structures prior to nuclear division.

Chromosome condensation induced by fostriecin does not require p34cdc2 kinase activity and histone H1 hyperphosphorylation, but is associated with enhanced histone H2A and H3 phosphorylation.

Chromosome condensation at mitosis correlates with the activation of p34cdc2 kinase, the hyperphosphorylation of histone H1 and the phosphorylation of histone H3. Chromosome condensation can also be induced by treating interphase cells with the protein phosphatase 1 and 2A inhibitors okadaic acid and fostriecin. Mouse mammary tumour FT210 cells grow normally at 32 degrees C, but at 39 degrees C they lose p34cdc2 kinase activity and arrest in G2 because of a temperature-sensitive lesion in the cdc2 gene. The treatment of these G2-arrested FT210 cells with fostriecin or okadaic acid resulted in full chromosome condensation in the absence of p34cdc2 kinase activity or histone H1 hyperphosphorylation. However, phosphorylation of histones H2A and H3 was strongly stimulated, partly through inhibition of histone H2A and H3 phosphatases, and cyclins A and B were degraded. The cells were unable to complete mitosis and divide. In the presence of the protein kinase inhibitor starosporine, the addition of fostriecin did not induce histone phosphorylation and chromosome condensation. The results show that chromosome condensation can take place without either the histone H1 hyperphosphorylation or the p34cdc2 kinase activity normally associated with mitosis, although it requires a staurosporine-sensitive protein kinase activity. The results further suggest that protein phosphatases 1 and 2A may be important in regulating chromosome condensation by restricting the level of histone phosphorylation during interphase, thereby preventing premature chromosome condensation.

Inhibition of histone phosphorylation by staurosporine leads to chromosome decondensation.

In mammalian cells, hyperphosphorylation of histone H1 and phosphorylation of histone H3 correlate well with the G2 phase to metaphase condensation of chromosomes, and these phosphorylations most probably have a role in initiating and controlling the entry into mitosis. The protein kinase inhibitor staurosporine has been used to examine the role of H1 and H3 phosphorylations in controlling chromosome condensation in the mouse FM3A cell line. We present evidence that (i) staurosporine inhibits the protein kinases that phosphorylate histone H1 during mitosis, (ii) staurosporine also inhibits the histone H3-specific kinase, (iii) the inhibition of these kinase activities prevent cells from entering mitosis, and (iv) addition of staurosporine to cells already arrested at metaphase by nocodazole causes a rapid dephosphorylation of histones H1 and H3 and the decondensation of the metaphase chromosomes. The results show that the hyperphosphorylation of histone H1 and phosphorylation of histone H3 are required to maintain metaphase chromosomes in their condensed state.

Staurosporine is a potent inhibitor of p34cdc2 and p34cdc2-like kinases.

We previously demonstrated that nontransformed cells arrest in the G1 phase of the cell cycle when treated with low concentrations (21 nM) of staurosporine (1). Both normal and transformed cells are blocked in the G2 phase of the cell cycle when treated with higher concentrations (160 nM) of staurosporine (1,2). In the present study, we show that staurosporine inhibits the activity of fractionated p34cdc2 and p34cdc2-like kinases with IC50 values of 4-5 nM. We propose that the G2 phase arrest in the cell cycle caused by staurosporine is due, at least in part, to the inhibition of the p34cdc2 kinases.

Photoaffinity polyamines: sequence-specific interactions with DNA.

ANB-spermine is a photoaffinity analog of the naturally-occurring polyamine, acetylspermine. ANB-spermine was used to determine its binding sites on naked double stranded DNA, at the nucleotide level, using a modification of the primer extension technique. A total of 1,275 nucleotides was examined in 5 sequences of DNA from Saccharomyces cerevisiae. Binding sites were non-random. The primary determinant of binding was the presence of a thymidine residue. Secondary determinants appeared to depend on the secondary structure of the DNA, with runs of thymidines providing unusually poor binding sites while TA and, especially, TATA providing the strongest binding sites. The 'TATA element' upstream of the URA3 gene from S. cerevisiae was the strongest binding site. The data indicate that ANB-spermine binding to DNA is a probe for DNA secondary structure and suggest a role for polyamines in regulating the structure of chromatin in vivo.

Two new photoaffinity polyamines appear to alter the helical twist of DNA in nucleosome core particles.

Two new photoaffinity derivatives of polyamines have been synthesized by the reaction of spermine or spermidine with methyl 4-azidobenzimidate. The new compounds were purified chromatographically and characterized by several methods including proton magnetic resonance spectroscopy. The spermine derivative is N1-ABA-spermine [(azidobenzamidino)spermine], and the spermidine derivative is a mixture of N1- and N8-ABA-spermidine. ABA-spermine stabilizes nucleosome core particles in thermal denaturation experiments, with similar but not identical effects when compared with the parent polyamine, spermine. In circular dichroism experiments, ABA-spermine was capable of producing a B----Z transition in poly(dG-m5dC) at a concentration of 30 microM, compared with 5 microM required to produce the same effect with spermine. On the other hand, ANB-spermine [(azidonitrobenzoyl)spermine; Morgan, J. E., Calkins, C. C., & Matthews, H. R. (1989) Biochemistry 28, 5095-5106] stabilized the B form of poly(dG-br5dC). ABA-spermine is a potent inhibitor of ornithine decarboxylase from Escherichia coli, giving 50% inhibition at 0.12 mM, while ANB-spermine is a modest inhibitor, comparable to spermine or spermidine. Under conditions of nitrogen-limited growth, yeast take up ABA-spermine and ABA-spermidine at approximately one-third to half the rate of spermidine or spermine. In contrast, ANB-spermine was not significantly taken up. The photoaffinity polyamines were used to photoaffinity label the DNA in nucleosome core particles, and the sites of labeling were determined by exonuclease protection. All photoaffinity reagents showed both nonspecific labeling and specific sites of higher occupancy. However, the positions of the sites varied: the ANB-spermine sites confirmed those previously reported (Morgan et al., 1989); the ABA-spermine and ABA-spermidine sites were spaced at 9.8 bp intervals from the 3' end of each DNA strand. This observation, together with the effect of spermine on the circular dichroism of DNA in nucleosome core particles, implies that polyamines alter the helical twist of DNA in nucleosome core particles. The ABA-polyamines are offered as general-purpose photoaffinity polyamine reagents.

Changes in DNA topology can modulate in vitro transcription of certain RNA polymerase III genes.

The role of DNA supercoiling in eukaryotic gene expression is not fully understood. The objective of this study was to examine the regulation of in vitro chromatin assembly by topological alterations in the DNA template using a cell-free extract from Xenopus laevis oocytes (S-150). The results suggest that input DNA topology may be a determining factor in controlling the transcriptional activity of the Xenopus tRNA and one particular 5S gene. When the input topology is supercoiled, high levels of transcription are observed, whereas input relaxed DNA is transcribed to a much lower extent. Transcription from an input relaxed template is stimulated by the addition of supercoiled nonspecific, vector DNA. Furthermore, in direct competition experiments, supercoiled DNA molecules were shown to be transcriptionally dominant over relaxed DNA molecules. Taken together, these data suggest that the efficiency with which a repressor or activator binding protein interacts with DNA may be significantly influenced by the topological status of its target. We demonstrate that modulation of reaction parameters which alter the normal topological processing events catalyzed by the S-150 can dramatically influence the level of gene expression.

The inhibitory effect of cyclic AMP on phosphatidylinositol kinase is not mediated by the cAMP dependent protein kinase.

Addition of cAMP to cells has been shown to inhibit phosphatidylinositol (PI) metabolism. cAMP has been reported to inhibit an enzyme in this pathway, PI kinase and it has been suggested that this inhibition is due to phosphorylation of PI kinase by the cAMP dependent protein kinase (PKA). In the present study we directly investigated if the inhibitory effect of cAMP was mediated by PKA. In membranes derived from murine hepatocytes we found that cAMP inhibited PI kinase but other adenine derivatives were more potent inhibitors. Moreover, it was found that the effects of the derivatives were unlikely to be due secondarily to the production of cAMP via their interaction with adenosine receptors. Through studies employing an inhibitor of PKA, mutant cells lacking PKA, and addition of purified catalytic subunit of PKA, we found that the inhibitory effect of cAMP was not mediated by PKA. In addition, the inhibitory effect of cAMP and adenosine was retained upon partial purification of PI kinase. Pulse chase experiments affirmed that the inhibitory effect was not due to breakdown of PI but rather to inhibition of its synthesis. We conclude that the inhibitory effect of cAMP and related compounds on PI kinase is not mediated by PKA dependent phosphorylation but rather appears to be a direct effect of these agents.