DNA methylation in the promoter of certain genes is associated with transcriptional silencing. Methylation affects gene expression directly by interfering with transcription factor binding and/or indirectly by recruiting histone deacetylases through methyl-DNA-binding proteins. In this study, we demonstrate that the human lung cancer cell line H719 lacks p53-dependent and -independent p21Cip1 expression. p53 response to treatment with gamma irradiation or etoposide is lost due to a mutation at codon 242 of p53 (C3W). Treatment with depsipeptide, an inhibitor of histone deacetylase, was unable to induce p53-independent p21Cip1 expression because the promoter of p21Cip1 in these cells is hypermethylated. Although a strong correlation between promoter methylation and gene silencing has been extensively demonstrated (5,24,35), the molecular mechanisms of this methylation-modulated gene inactivation remains unclear. Two hypotheses have been proposed to explain transcriptional inactivation from promoter methylation. One of them is based on the finding that methyl-CpG-binding proteins (MBPs), such as MeCP2, specifically bind to symmetrically methylated DNA through a methyl-CpG-binding domain (11,41). MBPs then recruit transcriptional repressors such as Sin3, NuRD, and histone deacetylases (HDACs) through its transcriptional-repression domain (25,32,54). Since Sin3 and HDACs are known transcriptional repressors (2, 50), methylated DNA may repress gene expression indirectly through MeCP2 and other MBPs. In addition, deacetylation of histones results in a net increase in positively charged lysines and arginines at the N-terminal tail of the histones (18, 21), thus inducing a tighter noncovalent linkage between the positively charged histones and the negatively charged DNA (3, 47). Consequently, transcription factors have difficulty accessing their DNA-binding sites (4, 29, 47), with a reduction or silencing of gene transcription. This hypothesis, based on the interaction between DNA methylation and histone acetylation status, has been extensively supported by accumulated experimental evidence (7,16,37,40). For example, trichostatin A (TSA), an inhibitor of HDAC, induces a robust reexpression of silenced genes when used with minimal doses of the demethylating agent, 5-aza-2Ј-deoxycytidine (5-azaCdR), although TSA or 5-aza-CdR alone do not lead to gene reexpression (7). Our previous data also show a link between histone acetylation status and DNA methylation, such that 5-aza-CdR significantly enhances acetylation of histones H3 and H4 induced by a HDAC inhibitor, depsipeptide. Related to this, depsipeptide-induced apoptosis is dramatically increased in cells pretreated with 5-aza-CdR (56). In addition, p19 INK4D expression is greatly enhanced when human lung cancer cells are treated with depsipeptide and 5-aza-CdR together compared to treatment with each agent alone (55). These studies support the notion that methylation and histone acetylation work cooperatively to influence gene expression and other biological processes.A...

DNA hypermethylation of CpG islands in the promoter region of genes is associated with transcriptional silencing. Treatment with hypo-methylating agents can lead to expression of these silenced genes. However, whether inhibition of DNA methylation influences the expression of unmethylated genes has not been extensively studied. We analysed the methylation status of CDKN2A and CDKN2D in human lung cancer cell lines and demonstrated that the CDKN2A CpG island is methylated, whereas CDKN2D is unmethylated. Treatment of cells with 5-aza-2'-deoxycytidine (5-Aza-CdR), an inhibitor of DNA methyltransferase 1, induced a dose and duration dependent increased expression of both p16(INK4a) and p19(INK4d), the products of CDKN2A and CDKN2D, respectively. These data indicate that global DNA demethylation not only influences the expression of methylated genes but also of unmethylated genes. Histone acetylation is linked to methylation induced transcriptional silencing. Depsipeptide, an inhibitor of histone deacetylase, acts synergistically with 5-Aza-CdR in inducing expression of p16(INK4a) and p19(INK4d). However, when cells were treated with higher concentrations of 5-Aza-CdR and depsipeptide, p16(INK4a) expression was decreased together with significant suppression of cell growth. Interestingly, p19(INK4d) expression was enhanced even more by the higher concentrations of 5-Aza-CdR and depsipeptide. Our data suggest that p19(INK4d) plays a distinct role from other INK4 family members in response to the cytotoxicity induced by inhibition of DNA methylation and histone deacetylation.

No abstract

No abstract

The histidase structural gene from Streptomyces griseus was expressed from a leaderless, monocistronic transcript. Multiple copies of the DNA located upstream of the hutH transcription initiation site led to a significant level of histidase activity when present in trans in the wild-type strain grown under noninducing conditions.In bacteria that utilize L-histidine as a carbon or nitrogen source, L-histidine is first deaminated by histidine ammonia lyase (histidase [20,24,26]), which is encoded by hutH. The ammonia released by histidase enters pathways for its assimilation. The other product of deamination, urocanate, is catabolized in several steps to form L-glutamate (20, 24). Urocanase, encoded by hutU, is required for the conversion of urocanate to imidazolonepropionate in this pathway (17,20,24).Examination of the regulated synthesis of histidase and urocanase by Bacillus subtilis, Klebsiella aerogenes, and Pseudomonas putida has led to the identification of one (28), two (6, 30), and three (9, 13) contiguous hut operons, respectively, that encode the enzymes required for conversion of L-histidine to L-glutamate. Although the order of hut genes differs among these species, hutH and hutU invariably lie within the same operon. Expression of these hut genes is regulated either by a repressor protein that binds at a site adjacent to the promoter and reduces initiation of transcription (12,34) or by a mechanism that appears to involve transcription antitermination (28). Expression of hut genes in these bacteria is also controlled by the global mechanisms of carbon catabolite repression (27,29,37) and nitrogen regulation (25,31) or by amino acid repression (1).Previous studies have demonstrated that L-histidine is a good nitrogen source for Streptomyces coelicolor (20) and Streptomyces griseus (22). In streptomycetes the enzymes of histidine catabolism are active whenever L-histidine or urocanate is present in the medium, regardless of the presence of other carbon or nitrogen sources (3,20,22). Thus, there is no evidence for carbon or nitrogen regulation of the histidine utilization (Hut) system in streptomycetes. On the basis of enzymological analyses, a Hut Ϫ mutant that we isolated appeared to constitutively synthesize inactive histidase that could be activated in vitro by exposure to an extract prepared from the wild-type strain (23). Earlier studies also indicated that histidase from S. griseus and S. coelicolor is a hysteretic enzyme that undergoes activation during the course of the reaction (19,22). In S. griseus, the hysteretic status of histidase appears to be dependent on the life cycle (22). Because this result suggested the possibility that the metabolic flux of L-histidine is regulated at least in part by the hysteretic activity of histidase, we wished to determine if histidase synthesis is also regulated in S. griseus.The histidase structural gene from the wild-type strain of S. griseus has been cloned, and its nucleotide sequence has been determined (38). Here we describe our analysis of hutH expr...

In transgenic animal models, the conservation of DNA sequences between the transgene and the host wild-type gene can complicate the evaluation of the expression of each gene. The potential for gene silencing may complicate matters further. Here we report the use of RT-PCR heteroduplex analysis to differentiate the expression of a transgene and its homologous wild-type, even when these genes are very similar in their respective DNA sequences. We designed RT-PCR primers to amplify identically sized 243-bp fragments within the DNA binding domain of the p53 gene from both human and mouse mRNA samples. Ten samples from human p53 (273H) transgenic mice and 10 samples from wild-type controls were tested. Heteroduplex bands were formed in all transgenic samples but were absent from all wild-type samples. In addition, RT-PCR heteroduplex analysis was able in one sample to differentiate a silenced transgene from its wild-type allele, without the assistance of sequencing or labeling. In summary, the RT-PCR heteroduplex analysis is easy to use and has the ability to screen a large number of samples in a short time. The RT-PCR heteroduplex analysis is especially useful for the detection of expression when a transgene and the host homologous endogenous allele are too conserved in sequence to design species-specific RT-PCR primers.