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Tumourigenesis within the intestine is potently driven by deregulation of the Wnt pathway, a process epigenetically regulated by the chromatin remodelling factor Brg1. We aimed to investigate this interdependency in an in vivo setting and assess the viability of Brg1 as a potential therapeutic target. Using a range of transgenic approaches, we deleted Brg1 in the context of Wnt-activated murine small intestinal epithelium. Pan-epithelial loss of Brg1 using VillinCreERT2 and AhCreERT transgenes attenuated expression of Wnt target genes, including a subset of stem cell-specific genes and suppressed Wnt-driven tumourigenesis improving animal survival. A similar increase in survival was observed when Wnt activation and Brg1 loss were restricted to the Lgr5 expressing intestinal stem cell population. We propose a mechanism whereby Brg1 function is required for aberrant Wnt signalling and ultimately for the maintenance of the tumour initiating cell compartment, such that loss of Brg1 in an Apc-deficient context suppresses adenoma formation. Our results highlight potential therapeutic value of targeting Brg1 and serve as a proof of concept that targeting the cells of origin of cancer may be of therapeutic relevance.

Recent studies have suggested that C-MYC may be an excellent therapeutic cancer target and a number of new agents targeting C-MYC are in preclinical development. Given most therapeutic regimes would combine C-MYC inhibition with genotoxic damage, it is important to assess the importance of C-MYC function for DNA damage signalling in vivo. In this study, we have conditionally deleted the c-Myc gene in the adult murine intestine and investigated the apoptotic response of intestinal enterocytes to DNA damage. Remarkably, c-Myc deletion completely abrogated the immediate wave of apoptosis following both ionizing irradiation and cisplatin treatment, recapitulating the phenotype of p53 deficiency in the intestine. Consistent with this, c-Myc-deficient intestinal enterocytes did not upregulate p53. Mechanistically, this was linked to an upregulation of the E3 Ubiquitin ligase Mdm2, which targets p53 for degradation in c-Myc-deficient intestinal enterocytes. Further, low level overexpression of c-Myc, which does not impact on basal levels of apoptosis, elicited sustained apoptosis in response to DNA damage, suggesting c-Myc activity acts as a crucial cell survival rheostat following DNA damage. We also identify the importance of MYC during DNA damage-induced apoptosis in several other tissues, including the thymus and spleen, using systemic deletion of c-Myc throughout the adult mouse. Together, we have elucidated for the first time in vivo an essential role for endogenous c-Myc in signalling DNA damage-induced apoptosis through the control of the p53 tumour suppressor protein.

Contributions of null and hypomorphic alleles of Apc in mice produce both developmental and pathophysiological phenotypes. To ascribe the resulting genotype-to-phenotype relationship unambiguously to the Wnt/beta-catenin pathway, we challenged the allele combinations by genetically restricting intracellular beta-catenin expression in the corresponding compound mutant mice. Subsequent evaluation of the extent of resulting Tcf4-reporter activity in mouse embryo fibroblasts enabled genetic measurement of Wnt/beta-catenin signaling in the form of an allelic series of mouse mutants. Different permissive Wnt signaling thresholds appear to be required for the embryonic development of head structures, adult intestinal polyposis, hepatocellular carcinomas, liver zonation, and the development of natural killer cells. Furthermore, we identify a homozygous Apc allele combination with Wnt/beta-catenin signaling capacity similar to that in the germline of the Apc(min) mice, where somatic Apc loss-of-heterozygosity triggers intestinal polyposis, to distinguish whether co-morbidities in Apc(min) mice arise independently of intestinal tumorigenesis. Together, the present genotype-phenotype analysis suggests tissue-specific response levels for the Wnt/beta-catenin pathway that regulate both physiological and pathophysiological conditions.

The characterization of early folding intermediates is key to understanding the protein folding process. Previous studies of the N-domain of phosphoglycerate kinase (PGK) from Bacillus stearothermophilus combined equilibrium amide exchange data with a kinetic model derived from stopped-flow kinetics. Together, these implied the rapid formation of an intermediate with extensive native-like hydrogen bonding. However, there was an absence of protection in the region proximal to the C-domain in the intact protein. We now report data for the intact PGK molecule, which at 394 residues constitutes a major extension to the protein size for which such data can be acquired. The methods utilised to achieve the backbone assignment are described in detail, including a semi-automated protocol based on a simulated annealing Monte Carlo technique. A substantial increase in the stability of the contact region is observed, allowing protection to be inferred on both faces of the beta-sheet in the intermediate. Thus, the entire N-domain acts concertedly in the formation of the kineticrefolding intermediate rather than there existing a distinct local folding nucleus. (C) 2003 Elsevier Ltd. All rights reserved.

The characterization of early folding intermediates is key to understanding the protein folding process. Previous studies of the N-domain of phosphoglycerate kinase (PGK) from Bacillus stearothermophilus combined equilibrium amide exchange data with a kinetic model derived from stopped-flow kinetics. Together, these implied the rapid formation of an intermediate with extensive native-like hydrogen bonding. However, there was an absence of protection in the region proximal to the C-domain in the intact protein. We now report data for the intact PGK molecule, which at 394 residues constitutes a major extension to the protein size for which such data can be acquired. The methods utilised to achieve the backbone assignment are described in detail, including a semi-automated protocol based on a simulated annealing Monte Carlo technique. A substantial increase in the stability of the contact region is observed, allowing protection to be inferred on both faces of the beta-sheet in the intermediate. Thus, the entire N-domain acts concertedly in the formation of the kineticrefolding intermediate rather than there existing a distinct local folding nucleus. (C) 2003 Elsevier Ltd. All rights reserved.

The characterization of early folding intermediates is key to understanding the protein folding process. Previous studies of the N-domain of phosphoglycerate kinase (PGK) from Bacillus stearothermophilus combined equilibrium amide exchange data with a kinetic model derived from stopped-flow kinetics. Together, these implied the rapid formation of an intermediate with extensive native-like hydrogen bonding. However, there was an absence of protection in the region proximal to the C-domain in the intact protein. We now report data for the intact PGK molecule, which at 394 residues constitutes a major extension to the protein size for which such data can be acquired. The methods utilised to achieve the backbone assignment are described in detail, including a semi-automated protocol based on a simulated annealing Monte Carlo technique. A substantial increase in the stability of the contact region is observed, allowing protection to be inferred on both faces of the beta-sheet in the intermediate. Thus, the entire N-domain acts concertedly in the formation of the kineticrefolding intermediate rather than there existing a distinct local folding nucleus. (C) 2003 Elsevier Ltd. All rights reserved.

Sawflies (Perga species) are leaf-damaging pests of the major plantation tree species Eucalyptus globulus ssp. globulus. This work describes the patterns of quantitative genetic variation in susceptibility to attack by Perga affinis ssp. insularis, based on data from a large open-pollinated trial containing genetic material from throughout the geographic range of E. globulus ssp. globulus. Forty three percent of the trees in the trial exhibited damage from sawflies in either their sixth or seventh growth seasons. The incidence of damage was genetically based, with significant variation between geographic races of E. globulus ssp. globulus and highly heritable (h2 = 0.43 +/- 0.05) variation in damage incidence within races was observed. Susceptibility to sawfly damage significantly affects plant fitness with both genetic and environmental correlations demonstrating that that sawfly damage causes slow growth and increased mortality of trees. Mild and severe sawfly damage resulted in 16 % and 31 % reduction in the basal area of surviving trees, and the effect was consistent across races and families. Based on this data, we propose a model predicting relative plantation productivity of races of different susceptibility under different infestation regimes.

Sawflies (Perga species) are leaf-damaging pests of the major plantation tree species Eucalyptus globulus ssp. globulus. This work describes the patterns of quantitative genetic variation in susceptibility to attack by Perga affinis ssp. insularis, based on data from a large open-pollinated trial containing genetic material from throughout the geographic range of E. globulus ssp. globulus. Forty three percent of the trees in the trial exhibited damage from sawflies in either their sixth or seventh growth seasons. The incidence of damage was genetically based, with significant variation between geographic races of E. globulus ssp. globulus and highly heritable (h2 = 0.43 +/- 0.05) variation in damage incidence within races was observed. Susceptibility to sawfly damage significantly affects plant fitness with both genetic and environmental correlations demonstrating that that sawfly damage causes slow growth and increased mortality of trees. Mild and severe sawfly damage resulted in 16 % and 31 % reduction in the basal area of surviving trees, and the effect was consistent across races and families. Based on this data, we propose a model predicting relative plantation productivity of races of different susceptibility under different infestation regimes.