Undermethylation and DNase I hypersensitivity of myeloperoxidase gene in HL-60 cells before and after differentiation.

Methylation and DNase I-hypersensitive sites of the myeloperoxidase gene in human myeloid leukemia HL-60 cells were studied by Southern blot hybridization using the myeloperoxidase gene probes. Digestion of DNA with a methylation-sensitive restriction endonuclease indicated that a CpG in the CCGG sequence located 3.53 kbp upstream of the myeloperoxidase gene was unmethylated in HL-60 cells expressing the gene, whereas it was methylated in K562 cells and human placenta not expressing the gene. The site in HL-60 cells remained unmethylated after retinoic acid- or 12-O-tetradecanoyl-phorbol-13-acetate-induced differentiation that arrests myeloperoxidase synthesis. Digestion of isolated nuclei with various amounts of DNase I indicated that four DNase I-hypersensitive sites were in an upstream region of the myeloperoxidase gene in HL-60 cells and three sites were within the gene. In retinoic acid-induced cells, the bands of the hypersensitive site near the 5' side of the gene and that in the first intron became weak, while that of the site in the fifth intron became strong. The bands of these hypersensitive sites were weak in K562 cells. The implications of these changes in tissue-specific expression and developmental down-regulation of the myeloperoxidase gene are discussed.     

Changes in the conformation of the interferon beta gene during differentiation and induction

DNase I sensitivity was used to investigate the chromatin conformation of the interferon beta gene during differentiation of the mouse teratocarcinoma cell line PC13 . These cells do not produce interferon upon viral induction in their undifferentiated state, but do so on differentiation from stem cells to endoderm. Only in induced differentiated cells were the interferon beta genes digested by DNase I. A similar effect was seen in a line of human cells ( MG63 ) upon induction. We conclude that it is induction of interferon production that brings about the change in the DNase I sensitivity of these genes, rather than differentiation.     

Three-dimensional distribution of DNase I-sensitive chromatin regions in interphase nuclei of embryonal carcinoma cells

In situ nick-translation allows the visualization of nuclease-sensitive chromatin regions in interphase nuclei. We have analyzed the three-dimensional (3-D) distribution of DNase I-sensitive regions of chromatin in nuclei from mouse P19 embryonal carcinoma cells by making optical sections using confocal scanning laser microscopy. In undifferentiated as well as embryonal carcinoma cells differentiated in vitro, DNase I-sensitive regions of chromatin are observed as discrete spots in the nucleus. These spots represent clusters of DNase I-sensitive sites. By optical sectioning, we show that these spots are preferentially, but not exclusively, localized at the nuclear periphery. No differences were observed in the spatial distribution of DNase I-sensitive sites in P19 EC cells or the differentiated P19 END-2 cells. Furthermore, we did not observe differences in the distribution of DNase I-sensitive chromatin regions during the cell cycle. These findings indicate, at least for P19 mouse embryonal carcinoma cells and their differentiated derivative END-2, that the compartmentalization of DNase I-sensitive chromatin regions is a general characteristic of the nucleus, independent of cell cycle stage or differentiation state. Since evidence has been presented that DNase I-sensitive sites are associated with actively transcribed chromatin, our results indicate that active transcribing chromatin is compartmentalized, preferentially in the periphery of the nucleus.     

Fusion of fibroblasts with differentiated and nondifferentiated leukemia cells resulting in blockage of DNA synthesis

We have investigated the regulation of DNA synthesis in the heterokaryons of HL60 human myelomonocytic leukemia cells and NIH3T3 mouse fibroblasts to examine if the differentiated leukemia cells contained a replication inhibiting activity. Cell fusions were performed either by exposing a suspension of mixed cells to an electric pulse or by the polyethylene glycol method. To identify the origin of the nuclei in a heterokaryon, one set of partner cells was prelabeled with [3H]thymidine before fusion. DNA synthetic activity after fusion was then revealed immunohistochemically by bromodeoxyuridine incorporation. DNA synthesis in the nuclei of 3T3 was inhibited in the heterokaryons of 3T3 and in either one of the two differentiated forms of HL60, i.e., the macrophage-like or the granulocyte-like. The result supports that a negative regulator of DNA synthesis exists in the differentiated HL60. Surprisingly, we have also found that DNA synthesis was inhibited in the nuclei of both 3T3 and nondifferentiated, proliferating HL60 when these two cells were fused. When unfused, proliferating cells were eliminated with cytosine arabinoside; these nonreplicating heterokaryons survived for at least 5 days, and 15% of them showed alpha-naphthylacetate esterase activity, a trait of the macrophage differentiation. The blockage of DNA synthesis in both partner nuclei was also observed in the heterokaryons of NIH3T3 cells and nondifferentiated human promonocytic leukemia cells U937, and in nondifferentiated HL60 and human diploid fibroblasts WI38. However, this effect was not found in the heterokaryons of NIH3T3 cells and human B lymphoma WI-729-HF2 cells. This is the first demonstration of the inhibition of DNA synthesis upon fusion of two proliferating cells.     

Chromatin structure of simian virus 40-pBR322 recombinant plasmids in COS-1 cells.

To study the nucleoprotein structure formed by recombinant plasmid DNA in mammalian cells, nuclei were isolated from COS-1 cells after transfection with a recombinant (pJI1) containing pBR322 sequences and a segment of simian virus 40 containing information for a nuclease-sensitive chromatin structure. The nuclei were incubated with DNase I. DNA fragments which were the size of linear pJI1 DNA were isolated, redigested with restriction enzymes, fractionated by electrophoresis, and detected by hybridization with nick-translated segments prepared from the plasmid DNA. Two DNase I-sensitive sites were detected in the simian virus 40 portion of the plasmid at the same sites that were DNase I sensitive in simian virus 40 chromatin prepared late after infection of African green monkey kidney (BSC-1) cells. One site extended from the viral origin of replication to approximately nucleotide 40. The 21-base pair repeated sequences were relatively DNase I resistant. A second site occurred over the single copy of the 72-base pair segment present in this plasmid. These results indicate that the nuclease-sensitive chromatin structure does not depend on the presence of viral structural proteins. In addition, late viral proteins added to pJI1-transfected COS-1 cells by superinfection with simian virus 40 caused no change in the distribution of DNase I-sensitive sites in plasmid chromatin. Analysis of transfected plasmid DNA may provide a general method applicable to the study of the chromatin structure of cloned segments of DNA.     

Changes in inositol transport during DMSO-induced differentiation of HL60 cells towards neutrophils

[3H]Inositol uptake by HL60 cells was measured during DMSO-induced differentiation towards neutrophils. The values for Km (53.2 microM) and Vmax (5.3 pmol/min per 10(6) cells) obtained for control HL60 cells are in good agreement with previously published figures for this cell line. Inositol transport into HL60 cells was an active, saturable and specific process which was unaffected by extracellular glucose concentrations. Inositol transport rates changed during DMSO-induced differentiation of HL60 cells towards neutrophils. An increase in inositol transport rates occurred during the first 4 days of exposure to 0.9% DMSO and was concommitant with the period leading to growth arrest and prior to the acquisition of the differentiated phenotype. These changes preceded the rise in intracellular inositol concentration from 10.9 to 132.7 microM seen between day 1 and day 5. After 4 days exposure to DMSO the rate of inositol transport fell to a value of 3.2 +/- 0.3 pmol/min per 10(6) cells at day 7, this was accompanied by a small reduction in intracellular inositol from a peak value of 132.7 to 112 microM. The inositol transport rate, thus, appears to closely accompany changes in the intracellular concentration of inositol. Inositol transport in human peripheral blood neutrophils was an order of magnitude slower than the value for uninduced HL60 cells, but the Km for inositol transport was similar in both cell types and was unchanged during HL60 differentiation. This suggests that changes in inositol transport rate are achieved by the modulation of a commonly expressed inositol transporter, one consequence of which is the alteration of intracellular inositol concentrations.     

Differential effects of Ca2+ and Mg2+ on endonuclease activation in isolated promyelocytic HL-60 cell nuclei

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Methylation state and DNase I sensitivity of chromatin containing Moloney murine leukemia virus DNA in exogenously infected mouse cells

The nature of Moloney murine leukemia virus (M-MuLV)-specific proviral DNA in exogenously infected mouse cells was studied. M-MuLV clone A9 cells, NIH-3T3 fibroblasts productively infected with M-MuLV, were used. These cells contain 10 to 15 copies of M-MuLV proviral DNA. The state of methylation of M-MuLV proviral DNA was examined by cleaving A9 cell DNA with restriction endonucleases which have the dinucleotide CpG in their cleavage sequences. Analysis with such enzymes, which recognized nine different sites in M-MuLV DNA, indicated that most if not all of the M-MuLV proviruses in A9 cells were completely unmethylated. An individual proviral integration was examined, using as probe adjacent single-copy cellular sequences. These sequences were obtained from a lambda phage recombinant clone containing an M-MuLV provirus from the A9 cells. This individual integration also showed no detectable methylation. In contrast, endogenous MuLV-related sequences present in NIH-3T3 cells before infection were largely methylated. The configuration chromatin containing M-MuLV proviruses was also investigated by digesting A9 nuclei with DNase I, followed by restriction analysis of the remaining DNA. Endogenous MuLV-related DNA was in chromatin relatively resistant to DNase I digestion, whereas the majority of M-MuLV-specific proviruses were in domains of intermediate DNase I sensitivity. Two proviral copies hypersensitive to DNase I digestion were identified. Analogy to the DNase I sensitivity of expressed and nonexpressed globin genes suggested that the proviral copies containing DNase I-hypersensitive sites were transcribed.     

Nuclear chromatin texture and sensitivity to DNase I in human leukaemic CEM cells incubated with nanomolar okadaic acid

It is now known that the analysis of chromatin texture can be used in oncology as a sensitive detection method, either to define diagnostic classifications or to locate a lesion along a defined trend curve. However, the functional significance of these variations in textural features remains sometimes unclear. Several drugs have been shown to be able to modulate chromatin structure. Among them, the phosphatase inhibitor okadaic acid at low concentration can increase accessibility to DNA in chromatin of carcinoma cells. This paper demonstrates that short exposures (0–3h) to a 10-nM dose of okadaic acid induced an increased sensitivity to DNase I digestion in human CEM leukaemic cell nuclei and that this sensitization was associated to variations of nuclear texture characteristics, as evaluated by image cytometry. CEM cells treated with okadaic acid for 0–3h displayed changes in chromatin supraorganization with a more homogeneous and fine chromatin texture, as compared to control cells. This suggests that the appearance of an open configuration of chromatin structure as evaluated by biochemical methods corresponds to a more decondensed texture of nuclei measured by image cytometry. Longer exposures (6–24h) of CEM cells to 10nM okadaic acid lead to apoptosis. As reported previously for camptothecin-treated HL60 cells, okadaic acid-treated CEM cells display biphasic nuclear chromatin texture changes, i.e. a decondensation phase followed by the appearance of typical apoptotic cells with a smaller nuclear area and a highly condensed chromatin. Finally, using the multidrug-resistant CEM-VLB cell line, it was confirmed that these multidrug-resistant cells also display cross-resistance to okadaic acid, as this compound was unable to induce either increased DNase I sensitivity, apoptosis, or altered nuclear texture in this particular cell line.     

50-kb Chromatin Fragmentation in the Absence of Apoptosis

Treatment with ionic detergents of nuclei isolated from various continuously growing cell lines generally yields chromatin samples of high viscosity. Extensive treatment with nuclease-free proteinase K or pronase solubilized the viscous lysates with >90% of the DNA migrating at 50 kb. Freshly prepared human peripheral blood T cells also yield a substantial fraction of their DNA in an 50- to 100-kb band. The cleavage sites may coincide with a class of DNase I-hypersensitive regions, since digestion of chromatin by DNase I at 10 U/ml, without protease, also yields fragments of preferentially 50-kb size. Occasionally, the oligonucleosomal ladder was also detected together with high molecular weight degradation products. Remarkably, all of these fragmentation patterns were seen in healthy, resting or proliferating cells, i.e., in the absence of apoptosis. Tritiated thymidine incorporation could be readily detected in the 50-kb DNA fragments. The effect of an apoptotic intracellular milieu on the integrity of isolated chromatin is apparently imitated by the extensive protease treatment used in our DNA isolation protocol.     

Loss of irreversibility of granulocytic differentiation induced by dimethyl sulfoxide in HL-60 sublines with a homogeneously staining region.

The human HL-60 acute leukemia cell line harbors double minutes (dmins) during early passages. During its continuous culture for a long term, a single marker chromosome with a homogeneously staining region (HSR) replaces the dmins. The both structures harbor amplified c-MYC sequences. Here we ask how the cellular phenotype is altered by the c-MYC integration into a HSR. Treatment with dimethyl sulfoxide induces granulocytic differentiation in the both types of cells. In contrast to HL-60/dmin cells, however, no apoptosis followed differentiation and the differentiation phenotype was reverted upon withdrawal of the drug in HL-60/HSR cells. Terminal differentiation and loss of DNase I hypersensitivity sites at c-MYC P2 promoter appeared to be unlinked in the both types of cells. By comparison with HL-60/dmin cells, we conclude that the integration into a HSR of an extrachromosomal gene(s) but not c-MYC likely leads to the loss of irreversibility of the differentiation phenotype.     

DNA and chromatin structure of the human alpha 1 (I) collagen gene

The human alpha 1 (I) collagen gene and 48 kilobase pairs of flanking DNA have been isolated on two overlapping cosmids. The alpha 1 (I) gene is 18 kilobase pairs long and contains a single repetitive element of the Alu family; at least 15 repetitive elements are present in the flanking DNA. Analysis of chromatin structure in nuclei isolated from cultured fibroblasts demonstrated a single chromatin domain greater than 65 kilobase pairs in length that contained 9 DNase I-hypersensitive sites. The pattern of hypersensitive sites was also determined in nuclei derived from placental tissue. Five of the DNase I-hypersensitive sites were observed in both placental and fibroblast chromatin including one site near the 5' end and another near the 3' end of alpha 1 (I). An additional two sites located near the 3' end of the alpha 1 (I) gene in fibroblast chromatin are associated with the tissue-specific use of different polyadenylation sites. Two DNase I-hypersensitive sites found only in fibroblast chromatin and one site found only in placental chromatin were located more than 10 kilobase pairs away from the alpha 1 (I) gene and may be related to tissue-specific expression of other genes in the domain. However, the only abundant placental mRNAs from the 65-kilobase pair domain were those transcribed from the alpha 1 (I) gene. These findings suggest that physical linkage does not play a predominant role in controlling coordinate expression of collagen genes.     

A phosphatase inhibitor enhances the DNase I sensitivity of active chromatin

Although it is well-known that active domains of chromatin have elevated DNase I sensitivity, it can be difficult to observe preferential sensitivity in many cell types. We show that the DNase I sensitivity of active chromatin is enhanced some 10-fold by treating nuclei with the phosphatase inhibitor p-(chloromercuri)benzenesulfonic acid (CMBS) whereas DNase I sensitivity in inactive domains is only 3-fold higher. We further show that CMBS-enhanced DNase I sensitivity is associated with at least two histone modifications. First, the negatively charged CMBS molecule becomes covalently attached to the thiol groups on histone H3. Second, histone H2A phosphorylation is significantly elevated in treated nuclei. The phosphorylation data along with other results point to the possibility that H2A phosphorylation plays a role in enhancing preferential DNase I sensitivity. Whatever the mechanism, CMBS treatment of nuclei followed by DNase I digestion provides a novel and reproducible assay for probing the chromatin structure of active domains.