Aberrant gene silencing in mammalian cells is associated with promoter region methylation, but the sequence of these two events is not clear. This review will consider the possibility that gene silencing is not a single event, but instead a series of events that begins with a dramatic drop in transcription potential and ends with its complete cessation. This transition will be portrayed as a chaotic process that ensues when transcription levels drop and DNA methylation begins spreading haltingly towards the diminished promoter. According to this view, silencing is stabilized when the promoter region is 'captured' by the spread of DNA methylation near or into its transcription factorbinding sites.

Disruption of the BRCA1 tumor suppressor can be caused not only by inherited mutations in familial cancers but also by BRCA1 gene silencing in sporadic cancers. Hypoxia, a key feature of the tumor microenvironment, has been shown to downregulate BRCA1 at the transcriptional level via repressive E2F4/p130 complexes. Here we showed that hypoxia also drives epigenetic modification of the BRCA1 promoter, with decreased H3K4 methylation as a key repressive modification produced by the lysine-specific histone demethylase LSD1. We also observed increased H3K9 methylation coupled with decreased H3K9 acetylation. Similar modifications were seen in the RAD51 promoter, which is also downregulated by hypoxia, whereas exactly opposite changes were seen in the promoter of the hypoxia-inducible gene VEGF. In cells containing the BRCA1 promoter driving a selectable HPRT gene, long-term silencing of the promoter was observed following exposure to hypoxic stress. Clones with silenced BRCA1 promoters were detected at frequencies of 2% or more following hypoxia, but at less than 6 ؋ 10 ؊5 without hypoxia. The silenced clones showed decreased H3K4 methylation and decreased H3K9 acetylation in the BRCA1 promoters, consistent with the acute effects of hypoxic stress. Hypoxia-induced BRCA1 promoter silencing persisted in subsequent normoxic conditions but could be reversed by treatment with a histone deacetylase (HDAC) inhibitor but not with a DNA methylation inhibitor. Interestingly, treatment of cells with inhibitors of poly(ADP-ribose) polymerase (PARP) can cause short-term repression of BRCA1 expression, but such treatment does not produce H3K4 or H3K9 histone modification or BRCA1 promoter silencing. These results suggest that hypoxia is a driving force for long-term silencing of BRCA1, thereby promoting genome instability and tumor progression.Solid tumors constitute a unique tissue type, characterized by hypoxia, low pH, and nutrient deprivation. Previous work has shown that hypoxic stress is a source of genetic instability in tumors (3,5,7,40,58), causing increased point mutations (40), gene amplification (11, 58), and fragile-site induction (12). Our previous work revealed that BRCA1 and RAD51, key genes in the homology-dependent repair (HDR) pathway, and MLH1, a key DNA mismatch (MMR) repair gene, are downregulated at the mRNA and protein levels in response to hypoxia via specific pathways of transcriptional regulation (3-5, 7). Moreover, BRCA1 and MLH1 have been found to be silenced in many sporadic cancers of multiple sites (8,14,16). The silencing of BRCA1 and MLH1 has been attributed primarily to promoter DNA hypermethylation at CpG sites (14). However, recent studies suggest that silenced promoters in cancer cells are also marked by characteristic histone modifications (9, 33, 48), and evidence is emerging that histone methylation may be a mediator of silencing that is independent of DNA methylation (26,29,30).Posttranslational modification of histones is widely recognized as an important epigenetic mechanism in the orga...

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Most accelerator-based space radiation experiments have been performed with single ion beams at fixed energies. However, the space radiation environment consists of a wide variety of ion species with a continuous range of energies. Due to recent developments in beam switching technology implemented at the NASA Space Radiation Laboratory (NSRL) at Brookhaven National Laboratory (BNL), it is now possible to rapidly switch ion species and energies, allowing for the possibility to more realistically simulate the actual radiation environment found in space. The present paper discusses a variety of issues related to implementation of galactic cosmic ray (GCR) simulation at NSRL, especially for experiments in radiobiology. Advantages and disadvantages of different approaches to developing a GCR simulator are presented. In addition, issues common to both GCR simulation and single beam experiments are compared to issues unique to GCR simulation studies. A set of conclusions is presented as well as a discussion of the technical implementation of GCR simulation.

The promoter region of the mouse adenine phosphoribosyltransferase (aprt) gene contains one non-consensus Sp1 binding site at its 5' end followed by three consensus Sp1 binding sites. The two 3'-most binding sites are sufficient for maximal expression of aprt , suggesting that the non-consensus and consensus binding sites at the 5' end are redundant. However, the two 3' sites are not sufficient to block epigenetic inactivation, which led to the hypothesis that the redundant consensus and/or non-consensus 5' Sp1 binding sites are required to block inactivation events. To test this hypothesis, promoter region constructs were made in which the two 5' Sp1 binding sites were mutated alone or in tandem, and then each construct was tested for its ability to withstand epigenetic inactivation. A cis -acting methylation center that is normally located 1.2 kb upstream of the promoter was used to induce inactivation. The results demonstrate that the presence of the redundant consensus Sp1 binding site is required to block methylation-associated gene inactivation. Therefore, the Sp1 binding sites comprising the mouse aprt promoter have evolved two distinct functions, one to promote transcription and the other to block epigenetic inactivation.

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In this report we test the hypothesis that a cis-acting methylation center can induce epigenetic gene inactivation. The cis-acting element used is an 838-base pair fragment that was shown previously to provide a de novo methylation signal (Mummaneni, P., Bishop, P. L., and Turker, M.S. (1993) J. Biol. Chem. 268, 552-558). Its normal location is approximately 1.3 kilobase pairs upstream of the mouse aprt (adenine phosphoribosyltransferase) gene. To determine if the methylation center could induce inactivation of the aprt gene, a plasmid construct was created in which the methylation center was moved next to the aprt promoter. Transfection experiments demonstrated inactivation of the aprt gene on the hybrid construct. The inactivation event was shown with a Southern blot analysis to correlate with hypermethylation and to be reversible by treatment with 2-deoxy-5'-azacytidine, a demethylating agent. Interestingly, gene inactivation induced by the methylation center required truncation of the aprt promoter. The results demonstrate that epigenetic gene inactivation can be induced by a DNA methylation center.

Regulatory regions of the human genome can be modified through epigenetic processes during prenatal life to make an individual more likely to suffer chronic diseases when they reach adulthood. The modification of chromatin and DNA contributes to a larger well-documented process known as “programming” whereby stressors in the womb give rise to adult onset diseases, including cancer. It is now well known that death from ischemic heart disease is related to birth weight; the lower the birth weight, the higher the risk of death from cardiovascular disease as well as type 2 diabetes and osteoporosis. Recent epidemiological data link rapid growth in the womb to metabolic disease and obesity and also to breast and lung cancers. There is increasing evidence that “marked” regions of DNA can become “unmarked” under the influence of dietary nutrients. This gives hope for reversing propensities for cancers and other diseases that were acquired in the womb. For several cancers, the size and shape of the placenta are associated with a person’s cardiovascular and cancer risks as are maternal body mass index and height. The features of placental growth and nutrient transport properties that lead to adult disease have been little studied. In conclusion, several cancers have their origins in the womb, including lung and breast cancer. More research is needed to determine the epigenetic processes that underlie the programming of these diseases.