- Email: [email protected]
Sir proteins as transcriptional silencers
Forum
TRENDS in Biochemical Sciences Vol.26 No.11 November 2001
685
Q&A
Sir proteins as transcriptional silencers What are Sir proteins are why are they important?
The SIR (silent information regulator) genes in the yeast Saccharomyces cerevisiae encode a family of nuclear proteins that localize to specific genomic sequences and mediate the formation of a specialized chromatin structure analogous to heterochromatin in higher eukaryotes. As a consequence, Sir proteins regulate the stability of chromatin structure and repress both transcription and recombination. They are essential for fertility in yeast and have been implicated in regulating longevity, both in yeast and in the nematode Caenorhabditis elegans. Where are Sir proteins found and how do they get there?
Sir proteins associate with three classes of genomic sequence in yeast: telomeres (which serve as the principal reservoirs of the Sir proteins), silenced mating-type loci (i.e. HMR and HML) and rDNA. Sir1, Sir2, Sir3 and Sir4 are required for efficient silencing of the HM loci, whereas Sir2, Sir3 and Sir4 are required for silencing at telomeres. In addition, Sir2 acts to suppress rDNA recombination (a function essential to its lifespanpromoting activity) and to silence transposons integrated within the rDNA. Sir proteins do not bind DNA directly; rather, they are recruited through protein–protein interactions. At telomeres, a protein termed RAP1, bound to terminal C1–3A nucleotide repeats, recruits Sir3 and Sir4. Sir2 is then recruited through its association with Sir4. At the silent mating loci, three proteins – RAP1, ARS-binding factor 1 (ABF1) and the origin recognition complex (ORC) – bind to flanking DNA elements termed silencers (designated E and I; Fig. 1) . These three proteins, together with Sir1, recruit the Sir2/3/4 complex, which then nucleates the assembly of a larger complex that ‘spreads’ to the adjacent chromatin. Such spreading occurs through a network of protein–protein interactions involving the Sir proteins themselves and the highly conserved N-terminal histone tails. http://tibs.trends.com
Formation of the mature Sir2/3/4 complex results in stabilization of the silenced state as well as the spreading of silencing to additional regions of chromatin. At the third type of Sir-regulated genomic sequence, rDNA, Sir2 is recruited via its interaction with the essential nucleolar protein Net1, which decorates the entire rDNA sequence. The protein complexes found at each of the three silenced loci are schematically depicted in Fig. 1. What are the functions of the Sir proteins?
Sir1 plays a crucial role in the establishment, but not in the maintenance, of the repressed state at HML and HMR. It directly interacts with Orc1 (the largest of the six subunits of ORC) and Sir4, thereby acting as a bridge between the silencer-binding proteins and the Sir2/3/4 complex, facilitating recruitment of the latter. Sir2 is a nicotinamide adenine dinucleotide (NAD)-dependent lysine deacetylase, the founding member of an evolutionarily conserved family of proteins termed sirtuins. Sir2 can deacetylate lysines 9 and 14 of histone H3 and lysine 16 of histone H4. Mutation of a highly conserved amino acid within the catalytic domain of Sir2 knocks out histone deacetylase activity in vitro and transcriptional repression
in vivo. Sir3 and Sir4 appear to provide the scaffold upon which Sir2 sits. Sir3/4 interacts with nucleosomes through the N-termini of H3 and H4. Sir3 binds the histone tail domains in such a way that nucleosomal arrays become crosslinked into higher-order structures. It thus might be anticipated that Sir3, and the functionally related Sir4, act stoichiometrically. Consistent with this, the extent to which a robust reporter gene is repressed by Sir2/3/4 linearly correlates with the efficiency with which these proteins are recruited to the promoter. What limits the spread of Sir proteins along the chromatin fiber?
The fact that Sir proteins emanate outwards from both telomeres and silencers raises the question as to what prevents them from extending indefinitely. Two possibilities exist. Either silencing decays stochastically as a function of distance from silencers, or boundary elements exist that actively block the spread of heterochromatin. At telomeres, Sir proteins spread 2–4 kb inwards, and their abundance falls off exponentially with distance, consistent with stochastic decay. At the HML locus, the flanking silencers (Fig. 1) act in an orientation-dependent fashion; this
Silent mating cassettes
RAP1 ORC ABF1 D element Sir1 Sir2/3/4
HML E
I HMR
E
Sir2/Net1/Cdc14 (RENT)
I
Telomere C1–3A repeats
RDN1 (rDNA) locus 35S
5S
35S
5S
35S
5S Ti BS
Fig. 1. Protein complexes associated with silenced chromatin domains in yeast. At each silent mating cassette, two divergently oriented genes (α1 and α2 at HML, and a1 and a2 at HMR, not shown) are regulated by the Sir2/3/4 complex, which is recruited by the silencer-binding proteins RAP1, ORC and ABF1, and the bridging protein Sir1. At each telomere, RAP1 binds to the terminal C1–3A repeats. This structure, ~300-bp long and known as the telosome, folds back onto subtelomeric chromatin via RAP1–Sir, Sir–Sir and Sir–histone interactions. The RDN1 locus comprises ~150 tandem repeats of the 35S and 5S rDNA genes; only three are shown. RDN1 is regulated by a Sir2/Net1/Cdc14-containing complex termed RENT.
0968-0004/01/$ – see front matter © 2001 Elsevier Science Ltd. All rights reserved. PII: S0968-0004(01)01985-5
686
Forum
TRENDS in Biochemical Sciences Vol.26 No.11 November 2001
What’s next?
Pol IIo Act
Act
TBP TATA
P PP Inr
UAS
Ti BS
Fig. 2. Sequence-specific activators and general transcription factors (GTFs) bind promoters silenced by Sir proteins. Illustrated are constitutively bound activators (Act), GTFs (TBP and pol II), the Sir2/3/4 complex (purple) and deacetylated nucleosomes (yellow ovals). Both recruitment-competent pol IIa (not shown) and elongationcompetent, phosphorylated pol IIo have been detected at SIR-repressed promoters. Abbreviations: Inr, initiator sequence; Pol II, RNA polymerase II; TBP, TATA-binding protein; UAS, upstream-activation sequence.
directional character, coupled with stochastic decay, ensures that only the region between the E and I silencers is transcriptionally repressed. At the HMR locus, the robust E silencer works in an orientation-independent fashion; here, boundary elements exist that stop the leftward and rightward spread of Sir proteins. How is silenced chromatin established and maintained?
DNA replication has long been implicated in the establishment of silenced chromatin. Classic experiments performed nearly two decades ago showed that de novo establishment of SIR repression requires passage through S phase, a period during which replication occurs. Further supporting a link between replication and silencing, two of the four HM silencers function as chromosomal origins of replication. However, recent experiments clearly show that, despite the S-phase requirement, HM silencing can be established in the absence of DNA replication. Such repression is dependent on Sir2, Sir3 and Sir4, and is accompanied by structural alterations characteristic of silenced chromatin. Silencers and Sir proteins are also required for the maintenance of silencing at HM loci, even in cells arrested in a specific phase of the cell cycle. Thus, yeast heterochromatin is a dynamic structure with a continuous requirement for Sir recruitment in both dividing and non-dividing cells. How do Sir proteins mediate silencing?
Histones within SIR-regulated chromatin are hypoacetylated, consistent with their http://tibs.trends.com
being part of a compact nucleosomal array. Folding might be further accentuated by the crosslinking activity of Sir3. Indeed, silenced chromatin has been found to be assembled into highly organized domains comprising closely packed pairs of nucleosomes; these domains are inaccessible to exogenous probes, both in vivo and in vitro. It has therefore been suggested that SIR represses transcription by sterically hindering the access of DNA to both regulatory and general transcription factors (GTFs). However, this model has recently been called into question through use of a technique termed chromatin immunoprecipitation. This technique, which permits the identification of proteins associated with specific DNA sequences in living cells, revealed that transcriptional activators, TATA boxbinding protein (TBP) and RNA polymerase II (pol II) do bind the promoters of fully silenced genes. These factors co-occupy the silenced promoters with the Sir2/3/4 complex and deacetylated nucleosomal histones (Fig. 2). Identification of an elongation-competent form of pol II within these promoters has led to the suggestion that SIR silences gene expression not by impeding the access of transcription factors to promoter DNA but rather by pausing a pre-initiated RNA polymerase. Sequence-specific activators and GTFs have likewise been found associated with heterochromatinrepressed promoters and Sir-like repressive protein complexes in the fruitfly Drosophila melanogaster, raising the possibility that the silencing mechanism used by Sir proteins is highly conserved.
The recent and surprising findings that silenced chromatin is both established in the absence of DNA replication and permissive to the binding of activators and GTFs raise several intriguing questions. For example: • What are the S-phase-specific event(s) responsible for establishing SIR repression? • Do Sir proteins work in concert with ATP-dependent chromatin remodeling enzymes to assemble higher-order structures? • As Sir3 and Sir4 preferentially bind hypoacetylated histones, are nucleosomes deacetylated before the spread of Sir proteins, or only subsequent to this spread? • Which step(s) in the transcriptional cascade are targeted by SIR, and how are they affected? Successful reconstitution of SIR heterochromatin from defined components will permit a biochemical approach for dissecting these mechanisms as well as allowing detailed characterization of its higher-order structure. Acknowledgements
I thank Mikhail Erkine and Nisha Patel for assistance with the figures, and the National Science Foundation for funding transcriptional silencing research in my laboratory. Further reading 1 Loo, S. and Rine, J. (1995) Silencing and heritable domains of gene expression. Annu. Rev. Cell Dev. Biol. 11, 519–548 2 Grunstein, M. (1998) Yeast heterochromatin: regulation of its assembly and inheritance by histones. Cell 93, 325–328 3 Gartenberg, M.R. (2000) The Sir proteins of Saccharomyces cerevisiae: mediators of transcriptional silencing and much more. Curr. Opin. Microbiol. 3, 132–137 4 Guarente, L. (2000) Sir2 links chromatin silencing, metabolism, and aging. Genes Dev. 14, 1021–1026 5 Li, Y-C. et al. (2001) Establishment of transcriptional silencing in the absence of DNA replication. Science 291, 650–653 6 Sekinger, E.A. and Gross, D.S. (2001) Silenced chromatin is permissive to activator binding and PIC recruitment. Cell 105, 403–414 7 Breiling, A. et al. (2001) General transcription factors bind promoters repressed by Polycomb group proteins. Nature 412, 651–655
David S. Gross Dept of Biochemistry and Molecular Biology, Louisiana State University Health Sciences Center, Shreveport, LA 71130-3932, USA. e-mail: [email protected]
TRENDS in Biochemical Sciences Vol.26 No.11 November 2001
685
Q&A
Sir proteins as transcriptional silencers What are Sir proteins are why are they important?
The SIR (silent information regulator) genes in the yeast Saccharomyces cerevisiae encode a family of nuclear proteins that localize to specific genomic sequences and mediate the formation of a specialized chromatin structure analogous to heterochromatin in higher eukaryotes. As a consequence, Sir proteins regulate the stability of chromatin structure and repress both transcription and recombination. They are essential for fertility in yeast and have been implicated in regulating longevity, both in yeast and in the nematode Caenorhabditis elegans. Where are Sir proteins found and how do they get there?
Sir proteins associate with three classes of genomic sequence in yeast: telomeres (which serve as the principal reservoirs of the Sir proteins), silenced mating-type loci (i.e. HMR and HML) and rDNA. Sir1, Sir2, Sir3 and Sir4 are required for efficient silencing of the HM loci, whereas Sir2, Sir3 and Sir4 are required for silencing at telomeres. In addition, Sir2 acts to suppress rDNA recombination (a function essential to its lifespanpromoting activity) and to silence transposons integrated within the rDNA. Sir proteins do not bind DNA directly; rather, they are recruited through protein–protein interactions. At telomeres, a protein termed RAP1, bound to terminal C1–3A nucleotide repeats, recruits Sir3 and Sir4. Sir2 is then recruited through its association with Sir4. At the silent mating loci, three proteins – RAP1, ARS-binding factor 1 (ABF1) and the origin recognition complex (ORC) – bind to flanking DNA elements termed silencers (designated E and I; Fig. 1) . These three proteins, together with Sir1, recruit the Sir2/3/4 complex, which then nucleates the assembly of a larger complex that ‘spreads’ to the adjacent chromatin. Such spreading occurs through a network of protein–protein interactions involving the Sir proteins themselves and the highly conserved N-terminal histone tails. http://tibs.trends.com
Formation of the mature Sir2/3/4 complex results in stabilization of the silenced state as well as the spreading of silencing to additional regions of chromatin. At the third type of Sir-regulated genomic sequence, rDNA, Sir2 is recruited via its interaction with the essential nucleolar protein Net1, which decorates the entire rDNA sequence. The protein complexes found at each of the three silenced loci are schematically depicted in Fig. 1. What are the functions of the Sir proteins?
Sir1 plays a crucial role in the establishment, but not in the maintenance, of the repressed state at HML and HMR. It directly interacts with Orc1 (the largest of the six subunits of ORC) and Sir4, thereby acting as a bridge between the silencer-binding proteins and the Sir2/3/4 complex, facilitating recruitment of the latter. Sir2 is a nicotinamide adenine dinucleotide (NAD)-dependent lysine deacetylase, the founding member of an evolutionarily conserved family of proteins termed sirtuins. Sir2 can deacetylate lysines 9 and 14 of histone H3 and lysine 16 of histone H4. Mutation of a highly conserved amino acid within the catalytic domain of Sir2 knocks out histone deacetylase activity in vitro and transcriptional repression
in vivo. Sir3 and Sir4 appear to provide the scaffold upon which Sir2 sits. Sir3/4 interacts with nucleosomes through the N-termini of H3 and H4. Sir3 binds the histone tail domains in such a way that nucleosomal arrays become crosslinked into higher-order structures. It thus might be anticipated that Sir3, and the functionally related Sir4, act stoichiometrically. Consistent with this, the extent to which a robust reporter gene is repressed by Sir2/3/4 linearly correlates with the efficiency with which these proteins are recruited to the promoter. What limits the spread of Sir proteins along the chromatin fiber?
The fact that Sir proteins emanate outwards from both telomeres and silencers raises the question as to what prevents them from extending indefinitely. Two possibilities exist. Either silencing decays stochastically as a function of distance from silencers, or boundary elements exist that actively block the spread of heterochromatin. At telomeres, Sir proteins spread 2–4 kb inwards, and their abundance falls off exponentially with distance, consistent with stochastic decay. At the HML locus, the flanking silencers (Fig. 1) act in an orientation-dependent fashion; this
Silent mating cassettes
RAP1 ORC ABF1 D element Sir1 Sir2/3/4
HML E
I HMR
E
Sir2/Net1/Cdc14 (RENT)
I
Telomere C1–3A repeats
RDN1 (rDNA) locus 35S
5S
35S
5S
35S
5S Ti BS
Fig. 1. Protein complexes associated with silenced chromatin domains in yeast. At each silent mating cassette, two divergently oriented genes (α1 and α2 at HML, and a1 and a2 at HMR, not shown) are regulated by the Sir2/3/4 complex, which is recruited by the silencer-binding proteins RAP1, ORC and ABF1, and the bridging protein Sir1. At each telomere, RAP1 binds to the terminal C1–3A repeats. This structure, ~300-bp long and known as the telosome, folds back onto subtelomeric chromatin via RAP1–Sir, Sir–Sir and Sir–histone interactions. The RDN1 locus comprises ~150 tandem repeats of the 35S and 5S rDNA genes; only three are shown. RDN1 is regulated by a Sir2/Net1/Cdc14-containing complex termed RENT.
0968-0004/01/$ – see front matter © 2001 Elsevier Science Ltd. All rights reserved. PII: S0968-0004(01)01985-5
686
Forum
TRENDS in Biochemical Sciences Vol.26 No.11 November 2001
What’s next?
Pol IIo Act
Act
TBP TATA
P PP Inr
UAS
Ti BS
Fig. 2. Sequence-specific activators and general transcription factors (GTFs) bind promoters silenced by Sir proteins. Illustrated are constitutively bound activators (Act), GTFs (TBP and pol II), the Sir2/3/4 complex (purple) and deacetylated nucleosomes (yellow ovals). Both recruitment-competent pol IIa (not shown) and elongationcompetent, phosphorylated pol IIo have been detected at SIR-repressed promoters. Abbreviations: Inr, initiator sequence; Pol II, RNA polymerase II; TBP, TATA-binding protein; UAS, upstream-activation sequence.
directional character, coupled with stochastic decay, ensures that only the region between the E and I silencers is transcriptionally repressed. At the HMR locus, the robust E silencer works in an orientation-independent fashion; here, boundary elements exist that stop the leftward and rightward spread of Sir proteins. How is silenced chromatin established and maintained?
DNA replication has long been implicated in the establishment of silenced chromatin. Classic experiments performed nearly two decades ago showed that de novo establishment of SIR repression requires passage through S phase, a period during which replication occurs. Further supporting a link between replication and silencing, two of the four HM silencers function as chromosomal origins of replication. However, recent experiments clearly show that, despite the S-phase requirement, HM silencing can be established in the absence of DNA replication. Such repression is dependent on Sir2, Sir3 and Sir4, and is accompanied by structural alterations characteristic of silenced chromatin. Silencers and Sir proteins are also required for the maintenance of silencing at HM loci, even in cells arrested in a specific phase of the cell cycle. Thus, yeast heterochromatin is a dynamic structure with a continuous requirement for Sir recruitment in both dividing and non-dividing cells. How do Sir proteins mediate silencing?
Histones within SIR-regulated chromatin are hypoacetylated, consistent with their http://tibs.trends.com
being part of a compact nucleosomal array. Folding might be further accentuated by the crosslinking activity of Sir3. Indeed, silenced chromatin has been found to be assembled into highly organized domains comprising closely packed pairs of nucleosomes; these domains are inaccessible to exogenous probes, both in vivo and in vitro. It has therefore been suggested that SIR represses transcription by sterically hindering the access of DNA to both regulatory and general transcription factors (GTFs). However, this model has recently been called into question through use of a technique termed chromatin immunoprecipitation. This technique, which permits the identification of proteins associated with specific DNA sequences in living cells, revealed that transcriptional activators, TATA boxbinding protein (TBP) and RNA polymerase II (pol II) do bind the promoters of fully silenced genes. These factors co-occupy the silenced promoters with the Sir2/3/4 complex and deacetylated nucleosomal histones (Fig. 2). Identification of an elongation-competent form of pol II within these promoters has led to the suggestion that SIR silences gene expression not by impeding the access of transcription factors to promoter DNA but rather by pausing a pre-initiated RNA polymerase. Sequence-specific activators and GTFs have likewise been found associated with heterochromatinrepressed promoters and Sir-like repressive protein complexes in the fruitfly Drosophila melanogaster, raising the possibility that the silencing mechanism used by Sir proteins is highly conserved.
The recent and surprising findings that silenced chromatin is both established in the absence of DNA replication and permissive to the binding of activators and GTFs raise several intriguing questions. For example: • What are the S-phase-specific event(s) responsible for establishing SIR repression? • Do Sir proteins work in concert with ATP-dependent chromatin remodeling enzymes to assemble higher-order structures? • As Sir3 and Sir4 preferentially bind hypoacetylated histones, are nucleosomes deacetylated before the spread of Sir proteins, or only subsequent to this spread? • Which step(s) in the transcriptional cascade are targeted by SIR, and how are they affected? Successful reconstitution of SIR heterochromatin from defined components will permit a biochemical approach for dissecting these mechanisms as well as allowing detailed characterization of its higher-order structure. Acknowledgements
I thank Mikhail Erkine and Nisha Patel for assistance with the figures, and the National Science Foundation for funding transcriptional silencing research in my laboratory. Further reading 1 Loo, S. and Rine, J. (1995) Silencing and heritable domains of gene expression. Annu. Rev. Cell Dev. Biol. 11, 519–548 2 Grunstein, M. (1998) Yeast heterochromatin: regulation of its assembly and inheritance by histones. Cell 93, 325–328 3 Gartenberg, M.R. (2000) The Sir proteins of Saccharomyces cerevisiae: mediators of transcriptional silencing and much more. Curr. Opin. Microbiol. 3, 132–137 4 Guarente, L. (2000) Sir2 links chromatin silencing, metabolism, and aging. Genes Dev. 14, 1021–1026 5 Li, Y-C. et al. (2001) Establishment of transcriptional silencing in the absence of DNA replication. Science 291, 650–653 6 Sekinger, E.A. and Gross, D.S. (2001) Silenced chromatin is permissive to activator binding and PIC recruitment. Cell 105, 403–414 7 Breiling, A. et al. (2001) General transcription factors bind promoters repressed by Polycomb group proteins. Nature 412, 651–655
David S. Gross Dept of Biochemistry and Molecular Biology, Louisiana State University Health Sciences Center, Shreveport, LA 71130-3932, USA. e-mail: [email protected]