Upregulation of BAK by butyrate in the colon is associated with increased Sp3 binding.

Butyrate is a key bioactive product of dietary fibrefermentation thought to play a key role in cancerprevention.Onecontributorymechanisminthisroleistheregulationofapoptosisbybutyrate.Asbutyrateshowslowlevelsoftoxicity,themechanismsbywhichittriggersor regulates apoptosis are of great interest. We andothershaveshownthattheproapoptoticproteinBAKisupregulatedbybutyrate.Weshowherethatthisobserva-tionisconservedacrossmultiplecelllines,thatitoccursinallcellsinapopulationandisatthetranscriptionallevel.Wehaveusedapromoter-reporterconstructtoidentifytheregulatoryregionsoftheBAKpromoterandfoundthatmuchofthetranscriptionalactivityoccursviaasingleSp1/Sp3bindingsite.WehaveshownthatbothSp1andSp3 bind, but upon butyrate treatment Sp1 bindingdecreasesin favour ofSp3 binding. Wespeculate thatthismaybeanacetylation-mediatedevent.Oncogene(2006)25,7192–7200.doi:10.1038/sj.onc.1209702;publishedonline29May2006Keywords: butyrate;colon;BAK;apoptosis;Sp1;Sp3IntroductionThereissubstantialepidemiologicalevidencetosuggestthatdietaryfibreplaysacriticalroleinthepreventionofcolorectal cancer. A number of studies over the lastthree decades have shown that fibre may suppresscolorectal carcinogenesis (Hill, 1997). These havebeen borne out with publication of data from a largepan-Europeanprospectivecohortstudy(Binghametal.,2003). Although some major intervention studiesfailed to support a role for fibre in adenoma preven-tion (Bonithon-Kopp et al., 2000; Schatzkin et al.,2000),reservationshavebeenmadeaboutthescopeoftheir interpretation (Hill, 2002; Arasaradnam et al.,2004). Studies in rats have shown convincingly thatfibre, specifically fermentable fibre, protects againstcarcinogen-induced tumorigenesis (McIntyre et al.,1993). Of all the fermentation products of fibre,butyrate appears to be the key bioactive agent(McIntyre etal., 1993; Perrin etal., 2001). In additiontoitscancerpreventingfunction,butyrateisthoughttoregulate normal colon physiology and tissue homeo-stasis(Hassetal.,1997)includingbothcelldivisionandapoptosis (Hague etal., 1993). Loss of normal regula-tion of cell division and an increased resistance toapoptosis are both hallmarks of cancer and potentialkey points at which butyrate may exert its antic-arcinogenicfunction.Butyrate is an inhibitor of HDACs at millimolarconcentrations (which are known to be achievable inthe colon and are therefore physiologically relevant).In vitro studies of the cellular response to HDACinhibition (by butyrate or trichostatin A) indicate thatlarge number of genes’ expression are altered, witharound 5% of all genes changed by two-fold or more(Mariadason etal., 2000; Della Ragione etal., 2001).The underpinning mechanisms include increasedaccessoftranscriptionfactorstonucleosomallylocatedbindingsitesthroughdisplacementofhistoneN-terminibyacetylation,movementofnucleosomesandthroughdirect activation or inactivation of cognate regulatorybinding proteins. A number of studies addressing themechanisms by which butyrate drives cell-cycle arresthaveidentifiedtranscriptionalupregulationofp21asakey event. The inhibition of HDAC1 in the trans-cription complex occupying Sp1 binding sites in thep21 promoter seems sufficient to allow upregulationof transcription (Siavoshian et al., 2000; Blottiereetal.,2003).TheBcl2familyofproteinsisdiversebutbroadlyfallsinto three groups – Bcl2-like anti-apoptotic proteins,Bax-like pro-apoptotic proteins, which facilitate cyto-chromecreleasefromthemitochondriaandBH3-onlyproteins which act to signal damage to the other twogroups and repress, or activate their function. TheBax-like group is select having three members, Bax,BAK and Bok. Bax and BAK have widespread tissuedistributionandbothareexpressedinthecolon(Pottenetal.,1997).Theyarethereforecandidateregulatorsofthenormalcolonicresponsetocarcinogens.Determining the mechanism of action of butyrate ininducingapoptosisisintriguingforseveralreasons.Therole for butyrate in cancer prevention may be due, atleast in part, to its function in regulating apoptosis.