Faithful inheritance of the chromatin structure is essential for maintaining the gene expression integrity of a cell. Histone modification by acetylation and deacetylation is a critical control of chromatin structure. In this study, we test the hypothesis that histone deacetylase 1 (HDAC1) is physically associated with a basic component of the DNA replication machinery as a mechanism of coordinating histone deacetylation and DNA synthesis. Proliferating cell nuclear antigen (PCNA) is a sliding clamp that serves as a loading platform for many proteins involved in DNA replication and DNA repair. We show that PCNA interacts with HDAC1 in human cells and in vitro and that a considerable fraction of PCNA and HDAC1 colocalize in the cell nucleus. PCNA associates with histone deacetylase activity that is completely abolished in the presence of the HDAC inhibitor trichostatin A. Trichostatin A treatment arrests cells at the G 2 -M phase of the cell cycle, which is consistent with the hypothesis that the proper formation of the chromatin after DNA replication may be important in signaling the progression through the cell cycle. Our results strengthen the role of PCNA as a factor coordinating DNA replication and epigenetic inheritance.Epigenetic markings play an essential role in regulating the gene expression program of vertebrate cells. One of the fundamental challenges of cell division is therefore coordinating the processes of genetic and epigenetic inheritance. The cell must possess multiple mechanisms to coordinate these processes (1). For example, DNA methylation is coordinated with DNA replication (2) by physical association of maintenance DNA methyltransferase 1 with the DNA replication fork protein PCNA 1 (3). Inhibition of DNA methyltransferase 1 leads to inhibition of initiation of DNA replication (4).Similar to DNA methylation, DNA replication-coupled chromatin assembly is essential for the inheritance of the epigenetic code. The specific targeting of nucleosome assembly to the newly synthesized DNA is achieved by direct interaction of histone chaperone CAF-1 with PCNA (5). PCNA is a homotrimeric protein that forms a sliding clamp around DNA and functions as a DNA polymerase processivity factor during replication and nucleotide excision repair. Through its multiple protein-protein interactions, PCNA coordinates events in replication, epigenetic inheritance, repair, and cell cycle control (6). A recent study in Saccharomyces cerevisiae has shown that several mutations in PCNA decrease silencing at telomeres and at the mating-type HMR locus (7). Furthermore, mutations in the Drosophila PCNA gene mus209 suppress repression in the vicinity of heterochromatin (8). The disruption of epigenetic silencing has been attributed to the inability of some of these mutants to associate with CAF-1. However, synergism of several PCNA mutants with CAF-1 mutants suggested that PCNA may participate in silencing through another factor.During nucleosome assembly, histones H3 and H4 undergo transient acetylation before their deposi...

DNA methyltransferase 1 (DNMT1) catalyzes the postreplication methylation of DNA and is responsible for maintaining the DNA methylation pattern during cell division. A long list of data supports a role for DNMT1 in cellular transformation and inhibitors of DNMT1 were shown to have antitumorigenic effects. It was long believed that DNMT1 promoted tumorigenesis by maintaining the hypermethylated and silenced state of tumor suppressor genes. We have previously shown that DNMT1 knock down by either antisense oligonucleotides directed at DNMT1 or expressed antisense activates a number of genes involved in stress response and cell cycle arrest by a DNA methylation-independent mechanism. In this report we demonstrate that antisense knock down of DNMT1 in human lung carcinoma A549 and embryonal kidney HEK293 cells induces gene expression by a mechanism that does not involve either of the known epigenomic mechanisms, DNA methylation, histone acetylation, or histone methylation. The mechanism of activation of the cell cycle inhibitor p21 and apoptosis inducer BIK by DNMT1 inhibition is independent of the mechanism of activation of the same genes by histone deacetylase inhibition. We determine whether DNMT1 knock down activates one of the nodal transcription activation pathways in the cell and demonstrate that DNMT1 activates Sp1 response elements. This activation of Sp1 response does not involve an increase in either Sp1 or Sp3 protein levels in the cell or the occupancy of the Sp1 elements with these proteins. The methylation-independent regulation of Sp1 elements by DNMT1 unravels a novel function for DNMT1 in gene regulation. DNA methylation was believed to be a mechanism for suppression of CG-rich Sp1-bearing promoters. Our data suggest a fundamentally different and surprising role for DNMT1 regulation of CG-rich genes by a mechanism independent of DNA methylation and histone acetylation. The implications of our data on the biological roles of DNMT1 and the therapeutic potential of DNMT1 inhibitors as anticancer agents are discussed.DNA modification by methylation of cytosines residing at the dinucleotide sequence CG plays an important role in epigenomic programming of gene expression (1). Not all CGs are methylated, and the pattern of distribution of methylated and unmethylated CGs is cell type-specific (2). DNA methylation in regulatory regions of genes plays a role in silencing genes either by directly inhibiting the interaction of transcription factors with their regulatory sequences (3, 4) or by attracting methylated DNA-binding proteins, which in turn recruit histone deacetylases and histone methyltransferases, resulting in an inactive chromatin structure (5, 6). DNA methylation is catalyzed by DNA methyltransferases DNMTs, 1 which transfer the methyl moiety from the methyl donor S-adenosylmethionine to 5th position on the cytosine ring (7). DNMT1 is responsible for maintaining the DNA methylation pattern during embryonal development and cell division (8, 9). DNMT1 deregulation was proposed to play a critical ro...

The DNA methylation pattern is an important component of the epigenome that regulates and maintains gene expression programs. In this paper, we test the hypothesis that vertebrate cells possess mechanisms protecting them from epigenomic stress similar to DNA damage checkpoints. We show that knockdown of DNMT1 (DNA methyltransferase 1) by an antisense oligonucleotide triggers an intra-S-phase arrest of DNA replication that is not observed with control oligonucleotide. The cells are arrested at different positions throughout the S-phase of the cell cycle, suggesting that this response is not specific to distinct classes of origins of replication. The intra-S-phase arrest of DNA replication is proposed to protect the genome from extensive DNA demethylation that could come about by replication in the absence of DNMT1. This protective mechanism is not induced by 5-aza-2 -deoxycytidine, a nucleoside analog that inhibits DNA methylation by trapping DNMT1 in the progressing replication fork, but does not reduce de novo synthesis of DNMT1. Our data therefore suggest that the intra-S-phase arrest is triggered by a reduction in DNMT1 and not by demethylation of DNA. DNMT1 knockdown also leads to an induction of a set of genes that are implicated in genotoxic stress response such as NF-B, JunB, ATF-3, and GADD45␤ (growth arrest DNA damage 45␤ gene). Based on these data, we suggest that this stress response mechanism evolved to guard against buildup of DNA methylation errors and to coordinate inheritance of genomic and epigenomic information.Proper epigenomic regulation of gene expression is essential for the integrity of cell function. One critical component of the epigenome is the pattern of distribution of methylated cytosines in CG dinucleotide sequences in the genome (1). Methylation of CGs marks genes for inactivation by either interfering with the binding of methylated DNA-sensitive transcription factors (2) or by recruiting methylated DNA-binding proteins such as MeCP2, which in turn recruit corepressor complexes and histone deacetylases to the chromatin associated with the gene (3). The methylation pattern can thus determine the chromatin structure and state of activity of genes. Disruption in the proper maintenance of the DNA methylation pattern results in aberrant gene expression, as is observed in tumor suppressor genes that are hypermethylated in cancer (4). Aberrant hypomethylation can also result in improper activation of genes (5).The main enzyme responsible for replicating the DNA methylation pattern is DNMT1 (DNA methyltransferase 1). This enzyme shows preference for hemimethylated DNA and is therefore believed to faithfully copy the DNA methylation pattern (6). Multiple mechanisms have been proposed to coordinate the inheritance of DNA methylation patterns with DNA replication. First, DNMT1 expression is regulated with the cell cycle (7, 8), and it is up-regulated by proto-oncogenes Ras and Jun (9 -11), Fos (12), and T antigen (13). Second, DNMT1 is localized to the replication fork (14) and is associated ...

In murine models of allergic inflammation, IL-12 has been shown to decrease tissue eosinophilia, but the underlying mechanisms are not known. We evaluated the expression of IL-12R and the effect of IL-12 on eosinophil survival. In situ hybridization demonstrated the presence of mRNA and immunoreactivity for IL-12Rβ1 and -β2 subunits in human peripheral blood eosinophils. Surface expression of IL-12Rβ1 and -β2 subunits on freshly isolated human eosinophils was optimally expressed after incubation with PMA. To determine the functional significance of IL-12R studies, we studied cell viability and apoptosis. Morphological analysis and propidium iodide staining for cell cycle demonstrated that recombinant human IL-12 increased in vitro human eosinophil apoptosis in a dose-dependent manner. Addition of IL-5 together with IL-12 abrogated eosinophil apoptosis, suggesting that IL-12 and IL-5 have antagonistic effects. Our findings provide evidence for a novel role for IL-12 in regulating eosinophil function by increasing eosinophil apoptosis.