Replication-independent nucleosome exchange is enhanced by local and specific acetylation of histone H4.

We used a novel single-cell strategy to examine the fate of histones during G(2)-phase. Consistent with previous results, we find that in G(2)-phase, the majority of nuclear histones are assembled into chromatin, whereas a small fraction comprises an unassembled pool. Small increases in the amount of histones within the free pool affect the extent of exchange, suggesting that the free pool is in dynamic equilibrium with chromatin proteins. Unexpectedly, acetylated H4 is preferentially partitioned to the unassembled pool. Although an increase in global histone acetylation did not affect overall nucleosome dynamics, an H4 containing lysine to glutamine substitutions as mimics of acetylation significantly increased the rate of exchange, but did not affect the acetylation state of neighbouring nucleosomes. Interestingly, transcribed regions are particularly predisposed to exchange on incorporation of H4 acetylation mimics compared with surrounding regions. Our results support a model whereby histone acetylation on K8 and K16 specifically marks nucleosomes for eviction, with histones being rapidly deacetylated on reassembly.

Quantitative profiling of CpG island methylation in human stool for colorectal cancer detection.

PURPOSE: The aims of this study were to investigate the use of quantitative CGI methylation data from stool DNA to classify colon cancer patients and to relate stool CGI methylation levels to those found in corresponding tissue samples. METHODS: We applied a quantitative methylation-specific PCR assay to determine CGI methylation levels of six genes, previously shown to be aberrantly methylated during colorectal carcinogenesis. Assays were performed on DNA from biopsies of "normal" mucosa and stool samples from 57 patients classified as disease-free, adenoma, or cancer by endoscopy, and in tumour tissue from cancer patients. Additionally, CGI methylation was analysed in stool DNA from an asymptomatic population of individuals covering a broad age range (mean = 47 ± 24 years) RESULTS: CGI methylation levels in stool DNA were significantly higher than in DNA from macroscopically normal mucosa, and a significant correlation between stool and mucosa was observed for ESR1 only. Multivariate statistical analyses using the methylation levels of each CGI in stool DNA as a continuous variable revealed a highly significant (p = 0.003) classification of cancer vs. non-cancer (adenoma + disease-free) patients (sensitivity = 65 %, specificity = 81 %). CONCLUSION: CGI methylation profiling of stool DNA successfully identified patients with cancer despite the methylation status of CGIs in stool DNA not generally reflecting those in DNA from the colonic mucosa.

Recent advances in understanding the role of diet and obesity in the development of colorectal cancer.

Colorectal cancer (CRC) is a major cause of premature death in the UK and many developed countries. However, the risk of developing CRC is well recognised to be associated not only with diet but also with obesity and lack of exercise. While epidemiological evidence shows an association with factors such as high red meat intake and low intake of vegetables, fibre and fish, the mechanisms underlying these effects are only now being elucidated. CRC develops over many years and is typically characterised by an accumulation of mutations, which may arise as a consequence of inherited polymorphisms in key genes, but more commonly as a result of spontaneously arising mutations affecting genes controlling cell proliferation, differentiation, apoptosis and DNA repair. Epigenetic changes are observed throughout the progress from normal morphology through formation of adenoma, and the subsequent development of carcinoma. The reasons why this accumulation of loss of homoeostatic controls arises are unclear but chronic inflammation has been proposed to play a central role. Obesity is associated with increased plasma levels of chemokines and adipokines characteristic of chronic systemic inflammation, and dietary factors such as fish oils and phytochemicals have been shown to have anti-inflammatory properties as well as modulating established risk factors such as apoptosis and cell proliferation. There is also some evidence that diet can modify epigenetic changes. This paper briefly reviews the current state of knowledge in relation to CRC development and considers evidence for potential mechanisms by which diet may modify risk.

Use of DNA from human stools to detect aberrant CpG island methylation of genes implicated in colorectal cancer.

Hypermethylation of cytosine residues in the CpG islands of tumor suppressor genes is a key mechanism of colorectal carcinogenesis. Detection and quantification of CpG island methylation in human DNA isolated from stools might provide a novel strategy for the detection and investigation of colorectal neoplasia. To explore the feasibility of this approach, colorectal biopsies and fecal samples were obtained from 32 patients attending for colonoscopy or surgery, who were found to have adenomatous polyps, colorectal cancer, or no evidence of neoplasia. A further 18 fecal samples were obtained from healthy volunteers, with no bowel symptoms. Isolated DNA was modified with sodium bisulfite and analyzed by methylation-specific PCR and combined bisulfite restriction analysis for CpG island methylation of ESR1, MGMT, HPP1, p16(INK4a), APC, and MLH1. CpG island methylation was readily detectable in both mucosal and fecal DNA with methylation-specific PCR. Using combined bisulfite restriction analysis, it was established that, in volunteers from whom biopsies were available, the levels of methylation at two CpG sites within ESR1 assayed using fecal DNA were significantly correlated with methylation in DNA from colorectal mucosa. Thus, noninvasive techniques can be used to obtain quantitative information about the level of CpG island methylation in human colorectal mucosa. The methods described here could be applied to a much expanded range of genes and may be valuable both for screening purposes and to provide greater insight into the functional consequences of epigenetic changes in the colorectal mucosa of free-living individuals.

A family 11 xylanase from Penicillium funiculosum is strongly inhibited by three wheat xylanase inhibitors.

Steady-state kinetic approaches were used to investigate the binding of a novel Penicillium funiculosum xylanase, XYNC, with three known xylanase inhibitor proteins from wheat (Triticum aestivum). The xylanase gene (xynC) was cloned from a P. funiculosum genomic library and the deduced amino acid sequence of XYNC exhibited high sequence similarity with fungal family 11 xylanases. xynC was overexpressed in P. funiculosum and the product (XYNC: M(r)=23.6 kDa; pI=3.7) purified and shown to efficiently degrade birchwood xylan [K(m)=0.47% w/v, Vmax=2540 micromol xylose min(-1) (mg protein)(-1) at pH 5.5 and 30 degrees C] and soluble wheat arabinoxylans [K(m)=1.45% w/v, Vmax=7190 micromol xylose min(-1) mg protein)(-1) at pH 5.5 and 30 degrees C]. The xylanase activity of XYNC was inhibited strongly by three xylanase inhibitor proteins from wheat; XIP-I, TAXI I and TAXI II. The inhibition for each was competitive, with very tight binding (K(i)=3.4, 16 and 17 nM, respectively) equivalent to free energy changes (deltaG degrees ) of -49, -45 and -45 kJ mol(-1). This is the first report describing a xylanase that is inhibited by all three wheat xylanase inhibitor proteins described to date.

Functional identification of the cDNA coding for a wheat endo-1,4-beta-D-xylanase inhibitor.

Using expressed sequence tag data, we obtained a full-length cDNA encoding a wheat protein inhibitor of xylanases (XIP-I). The 822 bp open reading frame encoded a protein of 274 amino acids with a molecular mass of 30.2 kDa, in excellent agreement with the native protein. Expression in Escherichia coli confirmed that the cDNA encoded a functional endo-1,4-beta-D-xylanase inhibitor. Its deduced amino acid sequence exhibited highest similarity to sequences classified as class III chitinases, but the inhibitor did not exhibit chitinase activity. This is the first full-length cDNA sequence that encodes a novel class of protein which inhibits the activity of endo-1,4-beta-D-xylanases.