Award details

Alterations in Sp3 activity as a global determinant of the colonocyte response to butyrate

Reference	BB/D004187/1
Principal Investigator / Supervisor	Professor Bernard Corfe
Co-Investigators / Co-Supervisors	Professor Colin Bingle, Dr Hari Chirakkal, Dr Elizabeth Williams
Institution	University of Sheffield
Department	Human Nutrition Unit
Funding type	Research
Value (£)	253,819
Status	Completed
Type	Research Grant
Start date	01/12/2005
End date	31/05/2009
Duration	42 months

Abstract

Our previous in vitro studies have identified the pro-apoptotic protein Bak as a mediator of the apoptotic response of colon epithelial cells to butyrate. We have analysed the regulation of the Bak promoter region by nested deletion and EMSA and have identified an Sp1 binding site as responsible for gene regulation. This site is bound by both Sp1 and Sp3. Our preliminary data are suggesting acetylation of Sp3 may be responsible for the observed up-regulation of Bak in response to butyrate. A small number of genes have been identified on an ad hoc basis as being regulated in the same way. This project will identify i) how Sp3 activity is regulated by butyrate, ii) determine the numbers of genes responding in the same way and iii) for the first time assess the regulation of genes in response to butyrate in the colon of healthy human subjects. 1 Determination of the regulation of Sp3 by butyrate. We will purify Sp3 bound to the bak promoter using an oligonucleotide capture strategy and analyse Sp3 forms bound in the presence or absence of butyrate using proteomic techniques. Changes in Sp3 isoforms will be identified using Q-TOF mass spec, additionally we will use an immunoprecipitation and western blot strategy to identify post-transcriptional modifications of Sp3 before and after butyrate treatment and reciprocal IP to confirm these observations . We will identify proteins complexing with Sp3 in the presence and absence of butyrate using an oligonucleotide capture protocol. This strategy will be complemented by co-immunoprecipitation and western blotting for candidate HAT and HDACs. 2 Identification of the extent of the butyrate-responding Sp3 regulon A small number of ad hoc studies have identified Sp3 as a mediator of the butyrate response. We aim to determine how many Sp3-regulated genes respond to butyrate using a global strategy. Cell lines will be generated carrying up- or down-regulated Sp3 and up-regulated, non-acetylatable Sp3. By comparing basal gene expression profiles in these cell lines we will establish the potential subset of genes regulated of Sp3. By comparing the responsiveness of these genes to butyrate we will further qualify this gene group to include those responding to butyrate, particularly (owing to the use of non-acetylatable Sp3) through alterations in Sp3 acetylation. We will use the 10 most dysregulated genes from this regulon as barometers of a butyrate response for an in vivo study. 3 Assessment of the in vivo relevance of our in vitro findings Whilst a number of studies have assessed the impact of butyrate upon gene expression in vitro, we are not aware of any studies addressing this question in vivo. We will recruit 10 healthy subjects to a nutritional intervention. Recruits will be asked to increase fibre intake using a panel of options high in fibre and with the support of the co-applicant as nutritionist. After a six week intervention we will analyse increases in faecal butyrate, and alterations in the expression levels of 10 ranked genes identified under objective 2. Gene expression analysis will be by quantitative RT-PCR. Tissue taken before and after the intervention will be a rectal brush biopsy.

Summary

There is considerable evidence from human studies that diets high in fibre and plant products are associated with a low risk of cancer of the bowel (colorectum). Colorectal cancer is now the second biggest cause of cancer-related death in the western world. The prognosis is good if colorectal cancer is diagnosed early, however this is not always achieved. The cost of treating colorectal cancer is also considerable. The disease therefore places a huge burden on the individual and on society as a whole. Prevention of colorectal cancer is a much better option. The government recommends increasing fruit and vegetable consumption, eating a healthy diet and leading an active lifestyle in order to reduce cancer risk. This advocacy is based upon the large number of studies around the world linking peoples diet to health outcomes. Most studies find that high fibre intake is associated with a reduced risk of colorectal cancer, but the mechanisms underlying the preventive effect are unclear. Some studies in rats, and in cells grown Petri dishes, suggest that a compound called butyrate may be very important in the cancer-preventing action of fibre. When fibre reaches the colon, it is fermented by friendly bacteria to form a number of compounds including butyrate. Studies with cells show that butyrate helps cells die when they are damaged, by a process called apoptosis. This may help the colon remove damaged cells and stop them becoming cancers. Butyrate also stops cells growing. This may stop cancers from growing in the colon. It is thought that butyrate exerts these effects by changing the genes expressed in cells. How does butyrate do this? Butyrate inhibits of enzymes called histone deacetylases (HDACs) which in turn regulate protein acetylation. Protein acetylation causes physical changes in the chromosome, altering the access to the DNA by factors that allow the DNA sequence to be copied (transcription). Acetylation is an epigenetic event, that is a change to the chromosome which does not alter the sequence of the DNA, but can alter the expression of genes. It is through such epigenetic events that nutrients and nutrient derived compounds (like butyrate) alter cancer risk by switching on and off cancer related genes (such as those that instruct cancer cells to die). In our previous studies we have found that butyrate causes the acetylation of proteins called histones around which the DNA wraps, this allows access of factors to copy the DNA. We have also found that butyrate increases the binding of one of these transcription factors (Sp3) to the DNA regulating a gene called Bak, that promotes cell death. We think that the binding of Sp3 increases because it is also acetylated. In this study, we aim to ask 1) how changes in Sp3 allow it to increase gene expression, 2) how many genes other than Bak are regulated by Sp3 in cells treated with butyrate, and finally 3) are these findings true in humans. These findings will help us in two ways: we will understand much better how a diet rich in fruit and vegetables will protect us from cancer and will possibly have some useful indicators for whether people are eating enough fruit and veg. Secondly, by understanding what goes wrong in the very earliest steps on cancer formation, we will be able to spot new ways of treating or diagnosing cancer, or new easy to stop cancers growing in the bowel in the first place.

Committee	Closed Committee - Agri-food (AF)
Research Topics	Diet and Health
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

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