Altered promoter nucleosome positioning is an early event in gene silencing.

Gene silencing in cancer frequently involves hypermethylation and dense nucleosome occupancy across promoter regions. How a promoter transitions to this silent state is unclear. Using colorectal adenomas, we investigated nucleosome positioning, DNA methylation, and gene expression in the early stages of gene silencing. Genome-wide gene expression correlated with highly positioned nucleosomes upstream and downstream of a nucleosome-depleted transcription start site (TSS). Hypermethylated promoters displayed increased nucleosome occupancy, specifically at the TSS. We investigated 2 genes, CDH1 and CDKN2B, which were silenced in adenomas but lacked promoter hypermethylation. Instead, silencing correlated with loss of nucleosomes from the -2 position upstream of the TSS relative to normal mucosa. In contrast, permanent CDH1 silencing in carcinoma cells was characterized by promoter hypermethylation and dense nucleosome occupancy. Our findings suggest that silenced genes transition through an intermediary stage involving altered promoter nucleosome positioning, before permanent silencing by hypermethylation and dense nucleosome occupancy.

FluShuffle and FluResort: new algorithms to identify reassorted strains of the influenza virus by mass spectrometry.

BACKGROUND: Influenza is one of the oldest and deadliest infectious diseases known to man. Reassorted strains of the virus pose the greatest risk to both human and animal health and have been associated with all pandemics of the past century, with the possible exception of the 1918 pandemic, resulting in tens of millions of deaths. We have developed and tested new computer algorithms, FluShuffle and FluResort, which enable reassorted viruses to be identified by the most rapid and direct means possible. These algorithms enable reassorted influenza, and other, viruses to be rapidly identified to allow prevention strategies and treatments to be more efficiently implemented. RESULTS: The FluShuffle and FluResort algorithms were tested with both experimental and simulated mass spectra of whole virus digests. FluShuffle considers different combinations of viral protein identities that match the mass spectral data using a Gibbs sampling algorithm employing a mixed protein Markov chain Monte Carlo (MCMC) method. FluResort utilizes those identities to calculate the weighted distance of each across two or more different phylogenetic trees constructed through viral protein sequence alignments. Each weighted mean distance value is normalized by conversion to a Z-score to establish a reassorted strain. CONCLUSIONS: The new FluShuffle and FluResort algorithms can correctly identify the origins of influenza viral proteins and the number of reassortment events required to produce the strains from the high resolution mass spectral data of whole virus proteolytic digestions. This has been demonstrated in the case of constructed vaccine strains as well as common human seasonal strains of the virus. The algorithms significantly improve the capability of the proteotyping approach to identify reassorted viruses that pose the greatest pandemic risk.