Sas4 and Sas5 Are Required for the Histone Acetyltransferase Activity of Sas2 in the SAS Complex

Abstract

The SAS2 gene is involved in transcriptional silencing in Saccharomyces cerevisiae. Based on its primary sequence, the Sas2 protein is predicted to be a member of the MYST family of histone acetyltransferases (HATs). Sas2 forms a complex with Sas4 and Sas5, which are required for its silencing function. Here we show that recombinant Sas2 has HAT activity that absolutely requires Sas4 and is stimulated by Sas5. The recombinant SAS complex acetylates H4 lysine 16 and H3 lysine 14. Furthermore, a purified SAS complex from yeast shows similar activity and specificity. In contrast to other MYST HATs, neither the recombinant nor the native SAS complex acetylated nucleosomal histones under conditions that were optimum for acetylating free histones. Finally, although the SAS subunits interact genetically and physically with Asf1, a histone deposition factor, association of H3 and H4 with Asf1 blocks their acetylation by the SAS complex, raising the possibility that the SAS HAT complex may acetylate free histones prior to their deposition onto DNA by Asf1 or CAF-I. The SAS2 gene is involved in transcriptional silencing in Saccharomyces cerevisiae. Based on its primary sequence, the Sas2 protein is predicted to be a member of the MYST family of histone acetyltransferases (HATs). Sas2 forms a complex with Sas4 and Sas5, which are required for its silencing function. Here we show that recombinant Sas2 has HAT activity that absolutely requires Sas4 and is stimulated by Sas5. The recombinant SAS complex acetylates H4 lysine 16 and H3 lysine 14. Furthermore, a purified SAS complex from yeast shows similar activity and specificity. In contrast to other MYST HATs, neither the recombinant nor the native SAS complex acetylated nucleosomal histones under conditions that were optimum for acetylating free histones. Finally, although the SAS subunits interact genetically and physically with Asf1, a histone deposition factor, association of H3 and H4 with Asf1 blocks their acetylation by the SAS complex, raising the possibility that the SAS HAT complex may acetylate free histones prior to their deposition onto DNA by Asf1 or CAF-I. histone acetyltransferase isopropyl औ-d-thiogalactoside tandem affinity purification calmodulin binding protein In the yeast Saccharomyces cerevisiae, as in higher eukaryotes, transcriptional silencing results from the formation of highly condensed chromatin called heterochromatin. Many factors have been identified that influence the formation and maintenance of heterochromatin. Some of these proteins either physically interact with or modify the histone N termini, while others are required for deposition of histones onto DNA (1Richards E.J. Elgin S.C. Cell. 2002; 108: 489-500Abstract Full Full In cerevisiae, chromatin are the and and Full SAS2 gene identified in for involved in transcriptional silencing are in silencing and in silencing a and that SAS2 in a with other and and that the proteins as a complex in yeast SAS SAS2 a member of the MYST family of histone acetyltransferases which and and and and of these proteins have been to HAT activity activity been for that acetyltransferase activity may be for Sas2 function. and others have that the binding of Sas2 is required for and as in the silencing as of SAS2 Furthermore, a in the histone H4 the of on silencing The of silencing and silencing a to the as of a SAS2 gene with the to in 2002; 2002; have that lysine 16 of histone H4 is in these are with the possibility that histone H4 lysine 16 is a for acetylation by SAS complex has been to the histone deposition proteins and Sas4 in a with Asf1 as and in a with Sas2 as that the SAS complex with Asf1 and in yeast is a of the yeast complex Asf1 and to histones H3 and H4 and are to in for histone deposition and chromatin Full Full Cell. Cell. Full Cell. Full Full Cell. 2002; the on silencing and as have or in silencing have in and silencing although the of these Furthermore, silencing and that are with these that the the SAS complex, and Asf1 may be complex and we show that of and to formation of a SAS complex that acetylates histones. Sas4 is for the acetyltransferase activity of and is The in for the recombinant SAS complex are lysine 16 of histone H4 and lysine of histone In contrast to the and the the SAS complex shows activity or onto DNA under conditions of have purified SAS complex from which and Sas5. native shows the as the recombinant the for free of MYST family proteins identified to as in in the of other factors Sas2 a of to MYST family we and others have been to HAT activity recombinant Sas2 is of a complex with Sas4 and in and or the as of we that Sas2 these subunits for Furthermore, Sas2 is to a has been in of subunits of a complex the of the proteins Full we a for of and in SAS2 for a with gene under of proteins were although a of and the purified by of the on of Sas4 and with that the proteins a complex in complex HAT activity histones as In of recombinant and purified in the in the of Sas4 and activity proteins were required for HAT or the activity from either or were that and either Sas4 or Sas5. In of the Sas4 or proteins with as proteins were in the that Sas4 and to these were in a HAT histones as the complex activity the complex the activity of the complex that of the complex, these of Sas2 and Sas4 proteins and the complex and the complex were to acetylate a that to the of histone that the N of H4 is a for acetylation by the SAS as the activity of the complex that of the complex, and the complex activity results that Sas2 is a HAT in that its HAT activity requires the complex has HAT activity requires the that the binding of Sas2 is for in a of Sas2 in which were to the silencing activity of formation of the SAS complex we a of the protein with Sas4 and Sas5, we that a SAS complex in with similar to that for the complex the complex HAT activity either histones or a H4 the silencing by the with the of HAT binding in Sas2 is required for HAT HAT with histones or H4 as were the purified complex, or a similar complex in which in were to of the purified proteins the of the SAS complex, we the of a HAT on a to the histones. that histone H4 is the for the SAS histone H3 is acetylated by acetylation is similar to that of member of the MYST family of acetyltransferases The complex a similar activity that the which histone are acetylated by which the histone H3 and H4 are acetylated by the SAS complex, we as to the of H4 or the of were acetylated with the SAS complex, with Sas2 and and In were to and the of in that the primary of acetylation on histone H4 lysine 16 and that the primary of acetylation on the H3 lysine the of the HAT the SAS complex were on a protein as as histones H3 and the of Sas2 and the protein the from of Sas2 of for for a histone acetyltransferase that is a member of the MYST family the of Sas2 the of histones were from the Sas2 is or and is for the of the is MYST family to in and interact with chromatin in to acetylate histones In cerevisiae, these a of the complex and the HAT for the complex the and acetylate histones in a nucleosomal we that SAS a similar with a binding as to the of silencing to from histone acetylation histone that Sas2 acetylate histones in the SAS complex acetylates nucleosomal and histones from were as of the SAS we acetylation of conditions that were optimum for acetylating free histones the SAS complex acetylate free histones from these that these histones were acetylated on histone H3 lysine and H4 lysine Furthermore, the complex, which acetylates H3 lysine on activity the as purified from and yeast and as for the SAS The were conditions that are for other HATs, with a from The SAS complex activity on from either In the complex on SAS acetylate histones H3 and H4 in the of onto in the of of the histones although the of histones to the acetylated under free histones H3 and onto are of the recombinant SAS complex in recombinant SAS complex acetylate HAT or histones as were as in the to or purified nor acetylate in are of a complex for and or for the of the recombinant SAS complex to acetylate may a is we purified SAS complex from in which either Sas2 or their a for to native SAS complex of and these purified for HAT activity histones and recombinant yeast histones. as for the recombinant SAS complex, the native yeast complex acetylate free the histones The native complex, the recombinant SAS complex, acetylated histones H3 and with a for H4 for H3 the of the yeast we as a of to of the histone H4 N were either or and in which lysine 16 acetylated were for the native SAS complex under conditions the recombinant and native SAS show similar acetylate free histone H4 lysine and to a histone and neither acetylates histones that are of the native SAS to the histone H4 were as in HAT The SAS complex purified from and in as were either or as of the of for silencing and the Asf1 and SAS subunits we the that SAS acetylates histones that are to In were purified Full Full and as for recombinant SAS in a HAT we that association of histones with Asf1 their acetylation by SAS predicted to be a histone acetyltransferase of its to of the MYST in activity either from recombinant Sas2 or from Sas2 purified from In we show that recombinant Sas2 acetylate with the Sas4 the in of other is by proteins is the in which activity of a histone acetyltransferase absolutely Furthermore, activity of required the may be required to the complex, or to in has to proteins yeast which is a of a of Cell. Full Cell. the of is involved in histone are as in silencing may be for HAT activity in in a in in by and others to SAS complex from yeast were although the that were purified and Sas5. In we show that the tandem affinity purification to the SAS complex results in the with or the purification the the recombinant and yeast native show similar acetylate histone H4 and to a histone Furthermore, acetylate free in that the recombinant and native acetylate lysine 16 of histone that histone H4 lysine 16 to a silencing similar to of and that H4 lysine 16 is in 2002; 2002; that these are a of acetylation of histone H4 lysine 16 by the SAS the recombinant and native acetylate histone and we the recombinant that the lysine 14. has been histone H3 lysine is in for the SAS of H3 lysine is in may be 2002; the histone H4 the in silencing as of SAS2 and may be that acetylation of histone H3 lysine by Sas2 either in or is for HAT to for acetylate free and nucleosomal histones. neither the recombinant nor native SAS complex acetylate histones in the acetylate free histones from these Furthermore, the recombinant activity on purified from and for these is that a required to SAS to nucleosomal histones the purification of the from SAS may on histones that have been in and these histones are in the or yeast we be required histones are of from the the histones were for the SAS acetylation of by SAS may conditions from that we is that in SAS acetylate free histones and in proteins physically interact with subunits of the Asf1 and histone deposition Furthermore, and similar on silencing the and Asf1 and to free histone H3 and and are to in histone SAS may in these to in the deposition we have that recombinant SAS acetylate H3 or H4 are to DNA as or with to the and SAS and Asf1 be that SAS acetylates H3 and H4 and these histones on to Asf1 for deposition onto SAS has been in formation of a and of the 2002; 2002; Asf1 and of SAS in the formation of these by acetylated histones to of In the yeast Saccharomyces cerevisiae, as in higher eukaryotes, transcriptional silencing results from the formation of highly condensed chromatin called heterochromatin. Many factors have been identified that influence the formation and maintenance of heterochromatin. Some of these proteins either physically interact with or modify the histone N termini, while others are required for deposition of histones onto DNA (1Richards E.J. Elgin S.C. Cell. 2002; 108: 489-500Abstract Full Full In cerevisiae, chromatin are the and and Full The SAS2 gene identified in for involved in transcriptional silencing are in silencing and in silencing a and that SAS2 in a with other and and that the proteins as a complex in yeast SAS SAS2 a member of the MYST family of histone acetyltransferases which and and and and of these proteins have been to HAT activity activity been for that acetyltransferase activity may be for Sas2 function. and others have that the binding of Sas2 is required for and as in the silencing as of SAS2 Furthermore, a in the histone H4 the of on silencing The of silencing and silencing a to the as of a SAS2 gene with the to in 2002; 2002; have that lysine 16 of histone H4 is in these are with the possibility that histone H4 lysine 16 is a for acetylation by The SAS complex has been to the histone deposition proteins and Sas4 in a with Asf1 as and in a with Sas2 as that the SAS complex with Asf1 and in yeast is a of the yeast complex Asf1 and to histones H3 and H4 and are to in for histone deposition and chromatin Full Full Cell. Cell. Full Cell. Full Full Cell. 2002; the on silencing and as have or in silencing have in and silencing although the of these Furthermore, silencing and that are with these that the the SAS complex, and Asf1 may be complex and In we show that of and to formation of a SAS complex that acetylates histones. Sas4 is for the acetyltransferase activity of and is The in for the recombinant SAS complex are lysine 16 of histone H4 and lysine of histone In contrast to the and the the SAS complex shows activity or onto DNA under conditions of have purified SAS complex from which and Sas5. native shows the as the recombinant the for free histones. of MYST family proteins identified to as in in the of other factors Sas2 a of to MYST family we and others have been to HAT activity recombinant Sas2 is of a complex with Sas4 and in and or the as of we that Sas2 these subunits for Furthermore, Sas2 is to a has been in of subunits of a complex the of the proteins Full we a for of and in SAS2 for a with gene under of proteins were although a of and the purified by of the on of Sas4 and with that the proteins a complex in complex HAT activity histones as In of recombinant and purified in the in the of Sas4 and activity proteins were required for HAT or the activity from either or were that and either Sas4 or Sas5. In of the Sas4 or proteins with as proteins were in the that Sas4 and to these were in a HAT histones as the complex activity the complex the activity of the complex that of the complex, these of Sas2 and Sas4 proteins and the complex and the complex were to acetylate a that to the of histone that the N of H4 is a for acetylation by the SAS as the activity of the complex that of the complex, and the complex activity results that Sas2 is a HAT in that its HAT activity requires the complex has HAT activity requires the that the binding of Sas2 is for in a of Sas2 in which were to the silencing activity of formation of the SAS complex we a of the protein with Sas4 and Sas5, we that a SAS complex in with similar to that for the complex the complex HAT activity either histones or a H4 the silencing by the with the of HAT the of the SAS complex, we the of a HAT on a to the histones. that histone H4 is the for the SAS histone H3 is acetylated by acetylation is similar to that of member of the MYST family of acetyltransferases The complex a similar activity that the which histone are acetylated by which the histone H3 and H4 are acetylated by the SAS complex, we as to the of H4 or the of were acetylated with the SAS complex, with Sas2 and and In were to and the of in that the primary of acetylation on histone H4 lysine 16 and that the primary of acetylation on the H3 lysine the of the HAT the SAS complex were on a protein as as histones H3 and the of Sas2 and the protein the from of Sas2 of for for a histone acetyltransferase that is a member of the MYST family the of Sas2 the of histones were from the Sas2 is or and is for the of the is MYST family to in and interact with chromatin in to acetylate histones In cerevisiae, these a of the complex and the HAT for the complex the and acetylate histones in a nucleosomal we that SAS a similar with a binding as to the of silencing to from histone acetylation histone that Sas2 acetylate histones in the SAS complex acetylates nucleosomal and histones from were as of the SAS we acetylation of conditions that were optimum for acetylating free histones the SAS complex acetylate free histones from these that these histones were acetylated on histone H3 lysine and H4 lysine Furthermore, the complex, which acetylates H3 lysine on activity the as purified from and yeast and as for the SAS The were conditions that are for other HATs, with a from The SAS complex activity on from either In the complex on SAS acetylate histones H3 and H4 in the of onto in the of of the histones although the of histones to the acetylated under free histones H3 and onto are of the recombinant SAS complex in recombinant SAS complex acetylate HAT or histones as were as in the to or purified nor acetylate in are of a complex for and or for the of the recombinant SAS complex to acetylate may a is we purified SAS complex from in which either Sas2 or their a for to native SAS complex of and these purified for HAT activity histones and recombinant yeast histones. as for the recombinant SAS complex, the native yeast complex acetylate free the histones The native complex, the recombinant SAS complex, acetylated histones H3 and with a for H4 for H3 the of the yeast we as a of to of the histone H4 N were either or and in which lysine 16 acetylated were for the native SAS complex under conditions the recombinant and native SAS show similar acetylate free histone H4 lysine and to a histone and neither acetylates histones that are of the native SAS to the histone H4 were as in HAT The SAS complex purified from and in as were either or as of the of for silencing and the Asf1 and SAS subunits we the that SAS acetylates histones that are to In were purified Full Full and as for recombinant SAS in a HAT we that association of histones with Asf1 their acetylation by SAS of MYST family proteins identified to as in in the of other factors Sas2 a of to MYST family we and others have been to HAT activity recombinant Sas2 is of a complex with Sas4 and in and or the as of we that Sas2 these subunits for Furthermore, Sas2 is to a has been in of subunits of a complex the of the proteins Full we a for of and in SAS2 for a with gene under of proteins were although a of and the purified by of the on of Sas4 and with that the proteins a complex in complex HAT activity histones as In of recombinant and purified in the in the of Sas4 and activity proteins were required for HAT or the activity from either or were that and either Sas4 or Sas5. In of the Sas4 or proteins with as proteins were in the that Sas4 and to these were in a HAT histones as the complex activity the complex the activity of the complex that of the complex, these of Sas2 and Sas4 proteins and the complex and the complex were to acetylate a that to the of histone that the N of H4 is a for acetylation by the SAS as the activity of the complex that of the complex, and the complex activity results that Sas2 is a HAT in that its HAT activity requires the complex has HAT activity requires the that the binding of Sas2 is for in a of Sas2 in which were to the silencing activity of formation of the SAS complex we a of the protein with Sas4 and Sas5, we that a SAS complex in with similar to that for the complex the complex HAT activity either histones or a H4 the silencing by the with the of HAT the of the SAS complex, we the of a HAT on a to the histones. that histone H4 is the for the SAS histone H3 is acetylated by acetylation is similar to that of member of the MYST family of acetyltransferases The complex a similar activity that the which histone are acetylated by which the histone H3 and H4 are acetylated by the SAS complex, we as to the of H4 or the of were acetylated with the SAS complex, with Sas2 and and In were to and the of in that the primary of acetylation on histone H4 lysine 16 and that the primary of acetylation on the H3 lysine the of the HAT the SAS complex were on a protein as as histones H3 and the of Sas2 and the protein the from of Sas2 of for for a histone acetyltransferase that is a member of the MYST family the of Sas2 the of histones were from the Sas2 is or and is for the of the is MYST family to in and interact with chromatin in to acetylate histones In cerevisiae, these a of the complex and the HAT for the complex the and acetylate histones in a nucleosomal we that SAS a similar with a binding as to the of silencing to from histone acetylation histone that Sas2 acetylate histones in the SAS complex acetylates nucleosomal and histones from were as of the SAS we acetylation of conditions that were optimum for acetylating free histones the SAS complex acetylate free histones from these that these histones were acetylated on histone H3 lysine and H4 lysine Furthermore, the complex, which acetylates H3 lysine on activity the as purified from and yeast and as for the SAS The were conditions that are for other HATs, with a from The SAS complex activity on from either In the complex on SAS acetylate histones H3 and H4 in the of onto in the of of the histones although the of histones to the acetylated under free histones H3 and onto are of the recombinant SAS complex in nor acetylate in are of a complex for and or for the of the recombinant SAS complex to acetylate may a is we purified SAS complex from in which either Sas2 or their a for to native SAS complex of and these purified for HAT activity histones and recombinant yeast histones. as for the recombinant SAS complex, the native yeast complex acetylate free the histones The native complex, the recombinant SAS complex, acetylated histones H3 and with a for H4 for H3 the of the yeast we as a of to of the histone H4 N were either or and in which lysine 16 acetylated were for the native SAS complex under conditions the recombinant and native SAS show similar acetylate free histone H4 lysine and to a histone and neither acetylates histones that are of the of for silencing and the Asf1 and SAS subunits we the that SAS acetylates histones that are to In were purified Full Full and as for recombinant SAS in a HAT we that association of histones with Asf1 their acetylation by SAS predicted to be a histone acetyltransferase of its to of the MYST in activity either from recombinant Sas2 or from Sas2 purified from In we show that recombinant Sas2 acetylate with the Sas4 the in of other is by proteins is the in which activity of a histone acetyltransferase absolutely Furthermore, activity of required the may be required to the complex, or to in has to proteins yeast which is a of a of Cell. Full Cell. the of is involved in histone are as in silencing may be for HAT activity in in a in in by and others to SAS complex from yeast were although the that were purified and Sas5. In we show that the tandem affinity purification to the SAS complex results in the with or the purification the the recombinant and yeast native show similar acetylate histone H4 and to a histone Furthermore, acetylate free in that the recombinant and native acetylate lysine 16 of histone that histone H4 lysine 16 to a silencing similar to of and that H4 lysine 16 is in 2002; 2002; that these are a of acetylation of histone H4 lysine 16 by the SAS the recombinant and native acetylate histone and we the recombinant that the lysine 14. has been histone H3 lysine is in for the SAS of H3 lysine is in may be 2002; the histone H4 the in silencing as of SAS2 and may be that acetylation of histone H3 lysine by Sas2 either in or is for HAT to for acetylate free and nucleosomal histones. neither the recombinant nor native SAS complex acetylate histones in the acetylate free histones from these Furthermore, the recombinant activity on purified from and for these is that a required to SAS to nucleosomal histones the purification of the from SAS may on histones that have been in and these histones are in the or yeast we be required histones are of from the the histones were for the SAS acetylation of by SAS may conditions from that we is that in SAS acetylate free histones and in proteins physically interact with subunits of the Asf1 and histone deposition Furthermore, and similar on silencing the and Asf1 and to free histone H3 and and are to in histone SAS may in these to in the deposition we have that recombinant SAS acetylate H3 or H4 are to DNA as or with to the and SAS and Asf1 be that SAS acetylates H3 and H4 and these histones on to Asf1 for deposition onto SAS has been in formation of a and of the 2002; 2002; Asf1 and of SAS in the formation of these by acetylated histones to of Sas2 predicted to be a histone acetyltransferase of its to of the MYST in activity either from recombinant Sas2 or from Sas2 purified from In we show that recombinant Sas2 acetylate with the Sas4 the in of other is by proteins is the in which activity of a histone acetyltransferase absolutely Furthermore, activity of required the may be required to the complex, or to in has to proteins yeast which is a of a of Cell. Full Cell. the of is involved in histone are as in silencing may be for HAT activity in in a in in by and others to SAS complex from yeast were although the that were purified and Sas5. In we show that the tandem affinity purification to the SAS complex results in the with or the purification the the recombinant and yeast native show similar acetylate histone H4 and to a histone Furthermore, acetylate free in that the recombinant and native acetylate lysine 16 of histone that histone H4 lysine 16 to a silencing similar to of and that H4 lysine 16 is in 2002; 2002; that these are a of acetylation of histone H4 lysine 16 by the SAS the recombinant and native acetylate histone and we the recombinant that the lysine 14. has been histone H3 lysine is in for the SAS of H3 lysine is in may be 2002; the histone H4 the in silencing as of SAS2 and may be that acetylation of histone H3 lysine by Sas2 either in or is for HAT to for acetylate free and nucleosomal histones. neither the recombinant nor native SAS complex acetylate histones in the acetylate free histones from these Furthermore, the recombinant activity on purified from and for these is that a required to SAS to nucleosomal histones the purification of the from SAS may on histones that have been in and these histones are in the or yeast we be required histones are of from the the histones were for the SAS acetylation of by SAS may conditions from that we is that in SAS acetylate free histones and in proteins physically interact with subunits of the Asf1 and histone deposition Furthermore, and similar on silencing the and Asf1 and to free histone H3 and and are to in histone SAS may in these to in the deposition we have that recombinant SAS acetylate H3 or H4 are to DNA as or with to the and SAS and Asf1 be that SAS acetylates H3 and H4 and these histones on to Asf1 for deposition onto SAS has been in formation of a and of the 2002; 2002; Asf1 and of SAS in the formation of these by acetylated histones to of for the the for and for in