Chromatin structure and induction-dependent conformational changes of human interferon-beta genes in a mouse host cell

Multiple copies of a human interferon-beta gene introduced into a mouse host cell line can be activated by induction with double-stranded RNA. Several induction-dependent changes of the chromatin structure could be traced by mapping techniques using four different agents [DNase I, micrococcus nuclease, bromoacetaldehyde and methidiumpropyl-EDTA X iron(II)]. Our data show that all copies of the interferon gene have adopted a very similar conformation in the host cell and respond to the inducing stimulus in a highly synchronous fashion. Detailed induction-specific changes were observed best with the chemical reagents which disclose a specific hypersensitive site within a sequence that has been shown to be required for the induction process (around position -80) and three other regions which, in addition to the transcribed region itself, gain single-strand character by an auxiliary process which can be mimicked by the addition of butyrate to the medium and may therefore involve histone hyperacetylation. Six discrete 'phased' nucleosomes are present upstream from the gene and are modulated by induction. At least four nucleosomes are located downstream. The interferon genes are largely protected from micrococcus nuclease in the inactive state. Gene activation increases access to micrococcus nuclease and DNase I indicating gross conformational changes on a higher level of chromatin structure.     

ADP-ribosylation induced changes in the conformation of the chromatin of the brain of developing rats

Conformational changes in the chromatin of the cerebral hemisphere of 3-, 14- and 30-day old developing rats were studied before and after its ADP-ribosylation using DNase I and micrococcal nuclease (MNase). The rate and extent of digestion of chromatin by DNase I are the highest at 3-day and decline progressively thereafter. The rate and extent of digestion by MNase do not change during development. ADP-ribosylation of chromosomal proteins was carried out by incubating nuclei with NAD+ for 30 min and was followed by endonuclease digestion. Both the rate and extent of digestion by DNase I and MNase were enhanced after ADP-ribosylation which was the maximum for 3-day rats.     

Long-range nucleosome ordering is associated with gene silencing in Drosophila melanogaster pericentric heterochromatin

We have used line HS-2 of Drosophila melanogaster, carrying a silenced transgene in the pericentric heterochromatin, to investigate in detail the chromatin structure imposed by this environment. Digestion of the chromatin with micrococcal nuclease (MNase) shows a nucleosome array with extensive long-range order, indicating regular spacing, and with well-defined MNase cleavage fragments, indicating a smaller MNase target in the linker region. The repeating unit is ca. 10 bp larger than that observed for bulk Drosophila chromatin. The silenced transgene shows both a loss of DNase I-hypersensitive sites and decreased sensitivity to DNase I digestion within an array of nucleosomes lacking such sites; within such an array, sensitivity to digestion by MNase is unchanged. The ordered nucleosome array extends across the regulatory region of the transgene, a shift that could explain the loss of transgene expression in heterochromatin. Highly regular nucleosome arrays are observed over several endogenous heterochromatic sequences, indicating that this is a general feature of heterochromatin. However, genes normally active within heterochromatin (rolled and light) do not show this pattern, suggesting that the altered chromatin structure observed is associated with regions that are silent, rather than being a property of the domain as a whole. The results indicate that long-range nucleosomal ordering is linked with the heterochromatic packaging that imposes gene silencing.     

Chromatin structure of transferred genes in transgenic plants

The chromatin structure of foreign genes in transgenic tobacco plants was investigated by digestion of nuclei with DNase I and micrococcal nuclease, respectively, followed by restriction and Southern analysis of the digestion products. The results were compared to the differential expression of the different transgenes. Two model systems were used: plants harbouring vector DNA derived from the disarmed vector pGV 3850 and plants harbouring the light-regulated and organ-specifically expressed potato ST-LS1 gene and the cotransferred ncpaline synthase (nos) reporter gene. Our results show that transferred genes are located in DNase l-sensitive domains in all transformants. Slight variations of DNase l-sensitivity of the transferred ST-LS1 constructs in different transformants neither reflected the between-transformant variability of expression nor the organ-specific activity of the transgenes. A deletion event was found responsible for silencing the ST-LS1 gene but not the nos gene in one of the transformants. Whereas no DNase l-hypersensitive sites were found within the 3850-T-DNA and the ST-LS1 gene, one prominent site was mapped to the nos promoter within the ST-LS1 construct in all transformants. Digestion of chromatin harbouring 3850-T-DNA with micrococcal nuclease resulted in a blurred nucleosomal pattern as compared to nucleolar and bulk chromatin, the extent of blurring being independent of the expression of transferred genes. The present results favour the “permissive domain” hypothesis which capitalizes on the chromatin surrounding the integration site as the determining factor for the chromatin structure of incoming alien genes. However, between-transformant variability of expression is not reflected by differential sensitivity to DNase I. Hence, other factors than chromatin structure must be involved in creating “position effects”.     

Histone deacetylation is required for the maturation of newly replicated chromatin

The effects of inhibiting histone deacetylation on the maturation of newly replicated chromatin have been examined. HeLa cells were labeled with [3H]thymidine in the presence or absence of sodium butyrate; control experiments demonstrated that butyrate did not significantly inhibit DNA replication for at least 70 min. Like normal nascent chromatin, chromatin labeled for brief periods (0.5-1 min) in the presence of butyrate was more sensitive to digestion with DNase I and micrococcal nuclease than control bulk chromatin. However, chromatin replicated in butyrate did not mature as in normal replication, but instead retained approximately 50% of its heightened sensitivity to DNase I. Incubation of mature chromatin in butyrate for 1 h did not induce DNase I sensitivity: therefore, the presence of sodium butyrate was required during replication to preserve the increased digestibility of nascent chromatin DNA. In contrast, sodium butyrate did not inhibit or retard the maturation of newly replicated chromatin when assayed by micrococcal nuclease digestion, as determined by the following criteria: 1) digestion to acid solubility, 2) rate of conversion to mononucleosomes, 3) repeat length, and 4) presence of non-nucleosomal DNA. Consistent with the properties of chromatin replicated in butyrate, micrococcal nuclease also did not preferentially attack the internucleosomal linkers of chromatin regions acetylated in vivo. The observation of a novel chromatin replication intermediate, which is highly sensitive to DNase I but possesses normal resistance to micrococcal nuclease, suggests that nucleosome assembly and histone deacetylation are not obligatorily coordinated. Thus, while deacetylation is required for chromatin maturation, histone acetylation apparently affects chromatin organization at a level distinct from that of core particle or linker, possibly by altering higher order structure.     

Deoxyribonuclease I sensitivity of the ovomucoid-ovoinhibitor gene complex in oviduct nuclei and relative location of CR1 repetitive sequences

The location of CR1 middle repetitive sequences within or near the boundaries of the ovalbumin DNase I sensitive domain has suggested that CR1 sequences may play a role in defining transition regions of DNase I sensitivity in hen oviduct nuclei. We have examined this apparent relationship of CR1 sequences and transitions of chromatin structure by determining the DNase I sensitivity in oviduct nuclei of a 47-kilobase region that contains five CR1 sequences and the transcribed ovomucoid and ovoinhibitor genes. We find that three of the CR1 sequences occur within a broad transition region of decreasing DNase I sensitivity downstream of the ovomucoid gene. Another CR1 is in a region of decreased DNase I sensitivity within the ovoinhibitor gene. The fifth CR1 sequence is in a DNase I sensitive region between the two genes but which is less sensitive to DNase I digestion than the region immediately upstream from the ovomucoid gene. Thus, the CR1 sequences occur within regions of reduced relative DNase I sensitivity, suggesting that CR1s could facilitate the formation of a chromatin conformation that is less sensitive to DNase I digestion. Unexpectedly, the noncoding strand of sequences within and immediately adjacent to the 5' end of the actively transcribed ovomucoid and ovalbumin genes was less sensitive to DNase I digestion than their respective coding strands.     

Butyrate induces cell apoptosis through activation of JNK MAP kinase pathway in human colon cancer RKO cells

Butyrate has been shown to display anti-cancer activity through the induction of apoptosis in various cancer cells. However, the underlying mechanism involved in butyrate-induced apoptosis is still not fully understood. Here, we investigated the cytotoxicity mechanism of butyrate in human colon cancer RKO cells. The results showed that butyrate induced a strong growth inhibitory effect against RKO cells. Butyrate also effectively induced apoptosis in RKO cells, which was characterized by DNA fragmentation, nuclear staining of DAPI, and the activation of caspase-9 and caspase-3. The expression of anti-apoptotic protein Bcl-2 decreased, whereas the apoptotic protein Bax increased in a dose-dependent manner during butyrate-induced apoptosis. Moreover, treatment of RKO cells with butyrate induced a sustained activation of the phosphorylation of c-jun N-terminal kinase (JNK) in a dose- and time-dependent manner, and the pharmacological inhibition of JNK MAPK by SP600125 significantly abolished the butyrate-induced apoptosis in RKO cells. These results suggest that butyrate acts on RKO cells via the JNK but not the p38 pathway. Butyrate triggered the caspase apoptotic pathway, indicated by an enhanced Bax-to-Bcl-2 expression ratio and caspase cascade reaction, which was blocked by SP600125. Taken together, our data indicate that butyrate induces apoptosis through JNK MAPK activation in colon cancer RKO cells.