BackgroundThe polymerase chain reaction (PCR) is commonly used to detect the presence of nucleic acid sequences both in research and diagnostic settings. While high specificity is often achieved, biological requirements sometimes necessitate that primers are placed in suboptimal locations which lead to problems with the formation of primer dimers and/or misamplification of homologous sequences.ResultsPyrococcus abyssi (P.a.) RNase H2 was used to enable PCR to be performed using blocked primers containing a single ribonucleotide residue which are activated via cleavage by the enzyme (rhPCR). Cleavage occurs 5'-to the RNA base following primer hybridization to the target DNA. The requirement of the primer to first hybridize with the target sequence to gain activity eliminates the formation of primer-dimers and greatly reduces misamplification of closely related sequences. Mismatches near the scissile linkage decrease the efficiency of cleavage by RNase H2, further increasing the specificity of the assay. When applied to the detection of single nucleotide polymorphisms (SNPs), rhPCR was found to be far more sensitive than standard allele-specific PCR. In general, the best discrimination occurs when the mismatch is placed at the RNA:DNA base pair.ConclusionrhPCR eliminates the formation of primer dimers and markedly improves the specificity of PCR with respect to off-target amplification. These advantages of the assay should find utility in challenging qPCR applications such as genotyping, high level multiplex assays and rare allele detection.

Modifications of both DNA and chromatin can affect gene expression and lead to gene silencing. Evidence of links between DNA methylation and histone hypoacetylation is accumulating. Several proteins that specifically bind to methylated DNA are associated with complexes that include histone deacetylases (HDACs). In addition, DNA methyltransferases of mammals appear to interact with HDACs. Experiments with animal cells have shown that HDACs are responsible for part of the repressive effect of DNA methylation. Evidence was found in Neurospora that protein acetylation can in some cases affect DNA methylation. The available data suggest that the roles of DNA methylation and histone hypoacetylation, and their relationship with each other, can vary, even within an organism. Some open questions in this emerging field that should be answered in the near future are discussed.

Blue light-induced transcription in Neurospora crassa is regulated by the White Collar-1 (WC-1) photoreceptor. We report that residue K14 of histone H3 associated with the light-inducible albino-3 (al-3) promoter becomes transiently acetylated after photoinduction. This acetylation depends on WC-1. The relevance of this chromatin modification was directly evaluated in vivo by construction of a Neurospora strain with a mutated histone H3 gene (hH3 K14Q ). This strain phenocopies a wc-1 blind mutant and shows a strong reduction of light-induced transcriptional activation of both al-3 and vivid (vvd), another light-inducible gene. We mutated Neurospora GCN Five (ngf-1), which encodes a homologue of the yeast HAT Gcn5p, to generate a strain impaired in H3 K14 acetylation and found that it was defective in photoinduction. Together, our findings reveal a direct link between histone modification and light signaling in Neurospora and contribute to the developing understanding of the molecular mechanisms operating in light-inducible gene activation.

Loss of imprinting (LOI) is an epigenetic alteration involving loss of parental origin-specific expression at normally imprinted genes. A LOI for Igf2, a paracrine growth factor, is important in cancer progression. Epigenetic modifications may be altered by environmental factors. However, is not known whether changes in imprinting occur with aging in prostate and other tissues susceptible to cancer development. We found a LOI for Igf2 occurs specifically in the mouse prostate associated with increased Igf2 expression during aging. In older animals, expression of the chromatin insulator protein CTCF and its binding to the Igf2-H19 imprint control region was reduced. Forced down-regulation of CTCF leads to Igf2 LOI. We further show that Igf2 LOI occurs with aging in histologically normal human prostate tissues and that this epigenetic alteration was more extensive in men with associated cancer. This finding may contribute to a postulated field of cancer susceptibility that occurs with aging. Moreover, Igf2 LOI may serve as a marker for the presence of prostate cancer.

Evolving data support a significant role for epigenetic processes in the development of prostate cancer and benign prostatic hyperplasia. Epigenetic changes can predict tumor behavior and often distinguish between genetically identical tumors. Targeted drugs that alter epigenetic modifications hold promise as a tool for curing and preventing these diseases.

These findings highlight the plasticity of the epigenome in an epithelial organ vulnerable to neoplastic change. They further suggest that chromatin modifications are more susceptible to methyl-deficient diets than DNA methylation at this locus.

Neurospora crassa utilizes DNA methylation to inhibit transcription of heterochromatin. DNA methylation is controlled by the histone methyltransferase DIM-5, which trimethylates histone H3 lysine 9, leading to recruitment of the DNA methyltransferase DIM-2. Previous work demonstrated that the histone deacetylase (HDAC) inhibitor trichostatin A caused a reduction in DNA methylation, suggesting involvement of histone deacetylation in DNA methylation. We therefore created mutants of each of the four classical N. crassa HDAC genes and tested their effect on histone acetylation levels and DNA methylation. Global increases in H3 and H4 acetylation levels were observed in both the hda-3 and the hda-4 mutants. Mutation of two of the genes, hda-1 and hda-3, caused partial loss of DNA methylation. The site-specific loss of DNA methylation in hda-1 correlated with loss of H3 lysine 9 trimethylation and increased H3 acetylation. In addition, an increase in H2B acetylation was observed by two-dimensional gel electrophoresis of histones of the hda-1 mutant. We found a similar increase in the Schizosaccharomyces pombe Clr3 mutant, suggesting that this HDAC has a previously unrecognized substrate and raising the possibility that the acetylation state of H2B may play a role in the regulation of DNA methylation and heterochromatin formation.

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