Summary DNA methylation is a major epigenetic mechanism for gene silencing. While methyltransferases mediate cytosine methylation, it is less clear how unmethylated regions in mammalian genomes are protected from de novo methylation and whether an active demethylating activity is involved. Here we show that either knockout or catalytic inactivation of the DNA repair enzyme Thymine DNA Glycosylase (TDG) leads to embryonic lethality in mice. TDG is necessary for recruiting p300 to retinoic acid (RA)-regulated promoters, protection of CpG islands from hypermethylation, and active demethylation of tissue-specific, developmentally- and hormonally-regulated promoters and enhancers. TDG interacts with the deaminase AID and the damage-response protein GADD45a. These findings highlight a dual role for TDG in promoting proper epigenetic states during development and suggest a two-step mechanism for DNA demethylation in mammals, whereby 5-methylcytosine and 5-hydroxymethylcytosine are first deaminated by AID to thymine and 5-hydroxymethyluracil, respectively, followed by TDG-mediated thymine and 5-hydroxymethyluracil excision repair.

Phosphorylation of p53 at Ser 46 was shown to regulate p53 apoptotic activity. Here we demonstrate that homeodomain-interacting protein kinase-2 (HIPK2), a member of a novel family of nuclear serine/threonine kinases, binds to and activates p53 by directly phosphorylating it at Ser 46. HIPK2 localizes with p53 and PML-3 into the nuclear bodies and is activated after irradiation with ultraviolet. Antisense inhibition of HIPK2 expression reduces the ultraviolet-induced apoptosis. Furthermore, HIPK2 and p53 cooperate in the activation of p53-dependent transcription and apoptotic pathways. These data define a new functional interaction between p53 and HIPK2 that results in the targeted subcellular localization of p53 and initiation of apoptosis.

HBV cccDNA, the template for transcription of all viral mRNAs, accumulates in the nucleus of infected cells as a stable episome organized into minichromosomes by histones and non-histone viral and cellular proteins. Using a cccDNA-specific chromatin immunoprecipitation (ChIP)-based quantitative assay, we have previously shown that transcription of the HBV minichromosome is regulated by epigenetic changes of cccDNA-bound histones and that modulation of the acetylation status of cccDNA-bound H3/H4 histones impacts on HBV replication. We now show that the cellular histone acetyltransferases CBP, p300, and PCAF/GCN5, and the histone deacetylases HDAC1 and hSirt1 are all recruited in vivo onto the cccDNA. We also found that the HBx regulatory protein produced in HBV replicating cells is recruited onto the cccDNA minichromosome, and the kinetics of HBx recruitment on the cccDNA parallels the HBV replication. As expected, an HBV mutant that does not express HBx is impaired in its replication, and exogenously expressed HBx transcomplements the replication defects. p300 recruitment is severely impaired, and cccDNA-bound histones are rapidly hypoacetylated in cells replicating the HBx mutant, whereas the recruitment of the histone deacetylases hSirt1 and HDAC1 is increased and occurs at earlier times. Finally, HBx mutant cccDNA transcribes significantly less pgRNA. Altogether our results further support the existence of a complex network of epigenetic events that influence cccDNA function and HBV replication and identify an epigenetic mechanism (i.e., to prevent cccDNA deacetylation) by which HBx controls HBV replication.histone acetylation ͉ HATs ͉ HDACs H epatitis B virus (HBV) infection is a major health problem, with Ϸ400 million people chronically infected worldwide who are at high risk of developing liver cirrhosis and hepatocellular carcinoma (HCC) (1). The epidemiological evidence linking HBV infection to HCC is very strong, and despite the mechanisms underlying HBV-associated carcinogenesis remain to be fully defined, a growing number of studies support a direct role of HBV in the process (2-5). The HBV-encoded regulatory protein hepatitis B virus X protein (HBx) is thought to contribute to HBV oncogenicity (5, 6). HBx transforms SV40-immortalized murine hepatocytes, induces cell cycle progression within the regenerating liver, causes liver cancer in some transgenic mice, and acts as a cofactor to accelerate cancer development in other mouse models (6-11). HBx is a 154-amino acid protein with an N-terminal negative regulatory domain and C-terminal transactivation or coactivation domain that has been detected both in the cytoplasm and in the nuclei of infected hepatocytes (6,12,13). Studies in transfected cells have shown that HBx expression affects several cellular functions such as cytoplasmic calcium regulation, cell signaling, transcription, cell proliferation, DNA repair, and apoptosis (11, 13-16). To perform its multiple functions, HBx interacts with many cellular partners including the tumor suppressor p53,...

The tumor suppressor p53 and its close relative p73 are activated in response to DNA damage resulting in either cell cycle arrest or apoptosis. Here, we show that DNA damage induces the acetylation of p73 by the acetyltransferase p300. Inhibiting the enzymatic activity of p300 hampers apoptosis in a p53(-/-) background. Furthermore, a nonacetylatable p73 is defective in activating transcription of the proapoptotic p53AIP1 gene but retains an intact ability to regulate other targets such as p21. Finally, p300-mediated acetylation of p73 requires the protooncogene c-abl. Our results suggest that DNA damage-induced acetylation potentiates the apoptotic function of p73 by enhancing the ability of p73 to selectively activate the transcription of proapoptotic target genes.

Che-1 is a RNA polymerase II-binding protein involved in the transcription of E2F target genes and induction of cell proliferation. Here we show that Che-1 contributes to DNA damage response and that its depletion sensitizes cells to anticancer agents. The checkpoint kinases ATM/ATR and Chk2 interact with Che-1 and promote its phosphorylation and accumulation in response to DNA damage. These Che-1 modifications induce a specific recruitment of Che-1 on the TP53 and p21 promoters. Interestingly, it has a profound effect on the basal expression of p53, which is preserved following DNA damage. Notably, Che-1 contributes to the maintenance of the G2/M checkpoint induced by DNA damage. These findings identify a mechanism by which checkpoint kinases regulate responses to DNA damage.