Epigenomic Stress Response

Abstract

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 ...

DNA Methyltransferase 1 Knock Down Induces Gene Expression by a Mechanism Independent of DNA Methylation and Histone Deacetylation

“…This allows us to study mainly DNA methylation-independent regulatory functions of DNMT1. We have previously shown that a number of stress response-related genes are activated by DNMT1 knock down (28). In this report we dissect the transcription factor pathway activated by DNMT1 knock down and show that surprisingly DNMT1 regulates gene expression by a new mechanism that does not involve either DNA methylation or chromatin modification.…”

“…Antisense and HDAC Inhibitor TSA Induce p21 and BIK Gene Expression; p21 and BIK Are Regulated by HDAC Activity-DNMT1 knock down was previously shown using a microarray gene expression analysis to induce expression of a cluster of genes involved in stress response (28). The kinetics of induction indicated that some of these genes were induced by a methylation-independent mechanism.…”

“…We have previously shown that DNMT1 expression is regulated with the cell cycle (8,26,27) and that antisense knock down of DNMT1 results in an intra-S-phase arrest of DNA replication (28). This intra-S-phase arrest requires knock down of the DNMT1 protein rather than inhibition of DNA methyl-ation, suggesting that DNMT1 protein plays an important role in the regulatory circuitry independent of its methyltransferase activity (29).…”

“…We and others have previously shown that antisense ODNs directed at DNMT1 exhibit sequence-specific down-regulation of DNMT1 and that they could be utilized to delineate the immediate consequences of knock down of the DNMT1 protein on gene expression and cellular regulation (14,20,28,30,31). In difference from agents such as 5-azacytidine that inhibit the catalytic activity of DNMT1 and, thus, mainly measure the DNA methylation-dependent roles of this protein (32), antisense knock down of DNMT1 causes an immediate arrest of DNA replication (28), resulting in limited passive demethylation. This allows us to study mainly DNA methylation-independent regulatory functions of DNMT1.…”

Induction of apoptosis by thymoquinone in lymphoblastic leukemia Jurkat cells is mediated by a p73-dependent pathway which targets the epigenetic integrator UHRF1

“…Indeed, UHRF1 binds to methylated DNA and recruits DNA methyltransferase 1 (DNMT1) and histone deacetylase 1 (HDAC1) through the SRA (SET and RING finger Associated) domain [13,[16][17][18][19]. These protein-protein interactions precede S phase entry and could be required for cell cycle progression [10,20]. UHRF1 therefore, ensures the crosstalk between DNA methylation and histone modifications, promoting the maintenance of the epigenetic code and its transmission from a mother cell to the descent cells [21].…”